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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 217 records · Page 12
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Single-Atom Pt Boosting Electrochemical Nonenzymatic Glucose Sensing on Ni(OH) 2 /N-Doped Graphene

Conventional nanomaterials in electrochemical non-enzymatic sensing face huge challenge due to their complex size-, surface- and composition- dependent catalytic properties and low active site density. In this work, we designed a single-atom Pt supported on Ni(OH) 2 nanoplates/nitrogen-doped graphene (Pt 1 /Ni(OH) 2 /NG) as the first example for constructing a single-atom catalyst based electrochemical non-enzymatic glucose sensor. The resulting Pt 1 /Ni(OH) 2 /NG exhibited a low anode peak potential of 0.48 V and high sensitivity of 220.75 μA mM -1 cm -2 towards glucose, which are 45 mV lower and 12 times higher than those of Ni(OH) 2 , respectively. The catalyst also showed excellent selectivity for several important interferences, short response time of 4.6 s, and high stability over 4 weeks. Experimental and density functional theory (DFT) calculated results reveal that the improved performance of Pt 1 /Ni(OH) 2 /NG could be attributed to stronger binding strength of glucose on single-atom Pt active centers and their surrounding Ni atoms, combined with fast electron transfer ability by the adding of the highly conductive NG. Finally, this research sheds light on the applications of SACs in the field of electrochemical non-enzymatic sensing.

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

Copper-dependent halogenase catalyses unactivated C−H bond functionalization

Carbon–hydrogen (C–H) bonds are the foundation of essentially every organic molecule, making them an ideal place to do chemical synthesis. The key challenge is achieving selectivity for one particular C(sp 3 )−H bond. In recent years, metalloenzymes have been found to perform C(sp 3 )−H bond functionalization. Despite substantial progresses in the past two decades, enzymatic halogenation and pseudohalogenation of unactivated C(sp 3 )−H—providing a functional handle for further modification—have been achieved with only non-haem iron/α-ketoglutarate-dependent halogenases, and are therefore limited by the chemistry possible with these enzymes. Here, in this work, we report the discovery and characterization of a previously unknown halogenase ApnU, part of a protein family containing domain of unknown function 3328 (DUF3328). ApnU uses copper in its active site to catalyse iterative chlorinations on multiple unactivated C(sp 3 )−H bonds. By taking advantage of the softer copper centre, we demonstrate that ApnU can catalyse unprecedented enzymatic C(sp 3 )−H bond functionalization such as iodination and thiocyanation. Using biochemical characterization and proteomics analysis, we identified the functional oligomeric state of ApnU as a covalently linked homodimer, which contains three essential pairs—one interchain and two intrachain—of disulfide bonds. The metal-coordination active site in ApnU consists of binuclear type II copper centres, as revealed by electron paramagnetic resonance spectroscopy. This discovery expands the enzymatic capability of C(sp 3 )−H halogenases and provides a foundational understanding of this family of binuclear copper-dependent oxidative enzymes.

biocatalysis↗
DOE OSTI2024

Rubisco activity and activation state dictate photorespiratory plasticity in Betula papyrifera acclimated to future climate conditions

Plant metabolism faces a challenge of investing enough enzymatic capacity to a pathway without overinvestment. As it takes energy and resources to build, operate, and maintain enzymes, there are benefits and drawbacks to accurately matching capacity to the pathway influx. The relationship between functional capacity and physiological load could be explained through symmorphosis, which would quantitatively match enzymatic capacity to pathway influx. Alternatively, plants could maintain excess enzymatic capacity to manage unpredictable pathway influx. In this study, we use photorespiration as a case study to investigate these two hypotheses in Betula papyrifera. This involves altering photorespiratory influx by manipulating the growth environment, via changes in CO 2 concentration and temperature, to determine how photorespiratory capacity acclimates to environmental treatments. Surprisingly, the results from these measurements indicate that there is no plasticity in photorespiratory capacity in B. papyrifera, and that a fixed capacity is maintained under each growth condition. The fixed capacity is likely due to the existence of reserve capacity in the pathway that manages unpredictable photorespiratory influx in dynamic environments. Additionally, we found that B. papyrifera had a constant net carbon assimilation under each growth condition due to an adjustment of functional rubisco activity driven by changes in activation state. These results provide insight into the acclimation ability and limitations of B. papyrifera to future climate scenarios currently predicted in the next century.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Critical Enzyme Reactions in Aromatic Catabolism for Microbial Lignin Conversion

The application of microbes to valorize aromatic compounds derived from the abundant plant biopolymer lignin is a rapidly developing area of research that may ultimately enable viable conversion of this recalcitrant and heterogeneous resource to valuable bio-based chemical products. Starting from the three canonical lignin building blocks, which differ in the extent of aromatic ring methoxylation, several common classes of enzymatic reaction occur in the upper pathways of aromatic catabolism to prepare aromatic compounds for assimilation into central carbon metabolism, including aromatic O-demethylation, hydroxylation and decarboxylation. These critical enzymatic steps can often be rate-limiting for efficient biological funnelling of aromatic compounds. Here we review the known enzymatic mechanisms for these reactions that are relevant for aerobic aromatic catabolism of lignin-related monomers, highlighting opportunities at the intersection of biochemistry, enzyme engineering and metabolic engineering for applications in the expanding field of microbial lignin valorization.

aromatic catabolism↗
DOE OSTI2022

Combining chemistry and protein engineering for new-to-nature biocatalysis

Biocatalysis, the application of enzymes to solve synthetic problems of human import, has blossomed into a powerful technology for chemical innovation. In the past decade, a threefold partnership, where nature provides blueprints for enzymatic catalysis, chemists introduce innovative activity modes with abiological substrates, and protein engineers develop new tools and algorithms to tune and improve enzymatic function, has unveiled the frontier of new-to-nature enzyme catalysis. In this Perspective, we highlight examples of interdisciplinary studies, which have helped to expand the scope of biocatalysis, including concepts of enzymatic versatility explored through the lens of biomimicry, to achieve activities and selectivities beyond those currently possible with chemocatalysis. We indicate how modern tools, such as directed evolution, computational protein design and machine learning-based protein engineering methods, have already impacted and will continue to influence enzyme engineering for new abiological transformations. As a result, a sustained collaborative effort across disciplines is anticipated to spur further advances in biocatalysis in the coming years.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Mild organosolv pretreatment of sugarcane bagasse with acetone/phenoxyethanol/water for enhanced sugar production

This study conducted mild organosolv pretreatment of sugarcane bagasse with acetone/phenoxyethanol/water (APW) solutions to improve sugar production in the subsequent enzymatic hydrolysis step. Here, the Box–Behnken design was used to optimize pretreatment conditions based on lignin removal. Further examination of the data revealed that lignin removal was positively correlated (R 2 > 0.95) with the combined severity factor (CSF). Pretreatment under the optimal conditions (125 °C–120 min, 0.17 M H 2 SO 4 , liquid–solid ratio of 15) led to 98.1% lignin removal and 74.5% cellulose digestibility compared to a low digestibility of 9.3% with raw sugarcane bagasse (SCB). Moreover, the APW process showed effective fractionation of pine, corn stalk and bamboo, and lignin removal was over 90%. The recovered lignin was characterized by 2D-HSQC NMR and 31P NMR, suggesting that the pretreatment resulted in breakage of β-O-4, total phenolic OH and H-units increased. However, the enzymatic hydrolysis efficiency (EHE) of samples with a lower lignin content (<4%) varied significantly. Correlations between various substrate-related factors of the pretreated SCB and EHE were analyzed to understand this observation. The results showed that the surface area of cellulose, lateral order index, CrI, and specific surface area were the predominant factors for enzymatic hydrolysis rather than lignin properties.

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

Discovery of hybrid chemical synthesis pathways with DORAnet

Developing efficient tools for discovering novel synthesis pathways is essential to advance chemical production methods that maximize the use of resources and energy. We introduce DORAnet (Designing Optimal Reaction Avenues Network Enumeration Tool), an open-source computational framework that addresses key limitations in current computer-aided synthesis planning (CASP) tools. DORAnet integrates both chemical/chemocatalytic (i.e., non-enzymatic) and enzymatic transformations, enabling the discovery of hybrid synthesis pathways. With 390 expert-curated chemical/chemocatalytic reaction rules and 3606 enzymatic rules derived from MetaCyc, it provides extensive flexibility for synthetic chemists and biotechnologists. The framework features customizable network expansion strategies, advanced filtering, and pathway search, ranking, and visualization tools. Validated against known reaction data, DORAnet successfully identified both established and novel synthesis routes for key industrial chemicals. In a case study involving 51 high-volume targets, DORAnet frequently ranked known commercial pathways among the top three results, demonstrating its practical relevance and ranking accuracy, while also uncovering numerous alternative (hybrid) synthesis pathways that were highly ranked.

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

Factors modulating the hydrolysis of Nylon-6,6 by a nylon hydrolase enzyme

The enzymatic hydrolysis of polyamides offers a promising approach to reduce the environmental impact of chemical recycling by enabling lower reaction temperatures, eliminating toxic organic solvents, and enhancing product selectivity. Achieving this goal will require increasing the low overall yield of enzymatic hydrolysis. In this work, we studied the mechanism of hydrolysis of commercial Nylon-6,6 polymer with a thermostable Nylon hydrolyzing enzyme and identified the substrate characteristics that influence the efficiency and deconstruction product yield. These results will guide the development of effective substrate pre-treatment methods to improve the yield of valuable oligoamide building blocks via enzymatic hydrolysis.

Bocharova, Vera [Oak Ridge National Laboratory (OR↗
DOE OSTI2026

Selective designer hydrolysates from Miscanthus bioenergy crop for sustainable utilization

Designer enzymatic hydrolysates deliver targeted, optimized fermentable sugars while reducing the formation of inhibitory by-products. Herein, five distinct enzymatic hydrolysates (EH) were generated from Miscanthus biomass using alkaline-ethanol fractionation and hydrothermal-mechanical refining (HMR) pretreatment approaches. Noticeably, alkali-ethanol fractionated hemicellulose A (HMA) and hemicellulose B (HMB) hydrolysis resulted in xylose-rich enzymatic hydrolysates with 104.8 ± 5.6 g L −1 and 173.4 ± 4.6 g L −1 xylose, respectively. All five hydrolysates encompassing varied carbon to nitrogen ratios were evaluated for lipid production using an engineered oleaginous yeast, R. toruloides 880-ADS. Microbial fermentation of xylose-rich hydrolysates, viz. HMR-EH2, HMA-EH, and HMB-EH, resulted in lipid yields ranging from 0.05 to 0.12 g g −1 sugar. Compositional and 2D NMR analyses revealed that HMA-EH residues are highly lignin-enriched while preserving the non-condensed structure conducive to valorization. Overall, the findings highlighted a novel, selective trade-off approach for maximizing lipid titers from designer hydrolysates, corroborating complete resource recovery for establishing integrated biorefineries.

Singh, Shuchi [University of Illinois at Urbana-Ch↗
DOE OSTI2020

Genomic Analysis Enlightens Agaricales Lifestyle Evolution and Increasing Peroxidase Diversity

Abstract As actors of global carbon cycle, Agaricomycetes (Basidiomycota) have developed complex enzymatic machineries that allow them to decompose all plant polymers, including lignin. Among them, saprotrophic Agaricales are characterized by an unparalleled diversity of habitats and lifestyles. Comparative analysis of 52 Agaricomycetes genomes (14 of them sequenced de novo) reveals that Agaricales possess a large diversity of hydrolytic and oxidative enzymes for lignocellulose decay. Based on the gene families with the predicted highest evolutionary rates—namely cellulose-binding CBM1, glycoside hydrolase GH43, lytic polysaccharide monooxygenase AA9, class-II peroxidases, glucose–methanol–choline oxidase/dehydrogenases, laccases, and unspecific peroxygenases—we reconstructed the lifestyles of the ancestors that led to the extant lignocellulose-decomposing Agaricomycetes. The changes in the enzymatic toolkit of ancestral Agaricales are correlated with the evolution of their ability to grow not only on wood but also on leaf litter and decayed wood, with grass-litter decomposers as the most recent eco-physiological group. In this context, the above families were analyzed in detail in connection with lifestyle diversity. Peroxidases appear as a central component of the enzymatic toolkit of saprotrophic Agaricomycetes, consistent with their essential role in lignin degradation and high evolutionary rates. This includes not only expansions/losses in peroxidase genes common to other basidiomycetes but also the widespread presence in Agaricales (and Russulales) of new peroxidases types not found in wood-rotting Polyporales, and other Agaricomycetes orders. Therefore, we analyzed the peroxidase evolution in Agaricomycetes by ancestral-sequence reconstruction revealing several major evolutionary pathways and mapped the appearance of the different enzyme types in a time-calibrated species tree.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes

Abstract CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The dimeric form of bacterial l ‐asparaginase YpAI is fully active

l ‐asparaginases from mesophilic bacteria (ASNases), including two enzymes very successfully used in the treatment of leukaemia, have been consistently described as homotetramers. On the contrary, structural studies show that homodimers of these enzymes should be sufficient to carry out the catalytic reaction. In this report, we investigated whether the type I Yersinia pestis asparaginase (YpAI) is active in a dimeric form or whether the tetrameric quaternary structure is critical for its activity. Using multiple biophysical techniques that investigate enzymatic properties and quaternary structure at either high or low protein concentration, we found that dimeric YpAI is fully active, suggesting that the tetrameric form of this subfamily of enzymes does not bear significant enzymatic relevance. In this process, we extensively characterized YpAI, showing that it is a cooperative enzyme, although the mechanism of allostery is still not definitely established. We showed that, like most type I ASNases, the substrate affinity of YpAI is low and this enzyme is very similar in terms of both the structure and enzymatic properties to homologous type I ASNase from Escherichia coli (EcAI). We extended these studies to more medically relevant type II ASNases, used as anti‐leukaemia drugs. We confirmed that type II ASNases are not allosteric, and that they might also be functional in a dimeric form. However, the determination of the accurate tetramer⇆dimer dissociation constants of these enzymes that most likely lie in the picomolar range is not possible with currently available biophysical techniques.

Biochemistry & Molecular Biology↗
DOE OSTI2026

Transgenic Mixed‐Linkage‐Glucan Enhancement Affects Root Characteristics and Decomposition in Soils of Contrasting Vegetation History

ABSTRACT Development of transgenic bioenergy sorghum [ Sorghum bicolor (L.) Moench] with increased contents of mixed‐linkage (1,3;1,4)‐β‐glucan (MLG) is an important step towards enhancing quality of bioenergy feedstocks. Since MLG‐enhancement leads to greater biomass digestibility, our overarching hypothesis is that root residues of MLG‐enhanced plants may be more readily decomposed in the soil, potentially creating new opportunities for optimizing soil carbon (C) sequestration, nutrient cycling, and overall agricultural sustainability. The study examined morphological, chemical, and enzymatic characteristics of fine and coarse roots of four bioenergy sorghum genotypes. Then, we incubated the roots within soils with contrasting vegetation histories while measuring C mineralization, microbial biomass C (MBC), and activity of hydrolytic enzymes and calculating vector length and vector angle enzymatic stoichiometry parameters. The results indicated that MLG‐enhancing transformations increased root total nitrogen (N) contents, decreased C/N ratios, and were associated with higher MLG concentrations in fine than in coarse roots. Incubations with transgenic roots led to 16%–38% higher MBC and 19%–41% lower microbial metabolic quotient (qCO 2 ). While enzyme activity differed markedly among the studied genotypes, it did not directly respond to MLG levels in root tissues. The increase in MBC without concurrent increases in C mineralization or hydrolytic enzyme activities in transgenic genotypes suggests that MLG enhancement promoted microbial anabolic retention of root‐derived C rather than stimulating catabolic decomposition. Enzymatic vector results indicated that these parameters reflect a variety of drivers behind microbial enzyme production, including availability of specific substrates, such as MLG here, and/or deficiency in specific nutrients, such as phosphorus (P). The study confirms the positive impacts from the roots of engineered MLG‐enhanced bioenergy plants on soil microbial activity and highlights the interactive influences on the MLG‐enhancement effects from root size and inherent soil properties.

Mahmoodabadi, Majid [Department of Plant, Soil, an↗
DOE OSTI2024

Data for Pilot-Scale Processing of Miscanthus x giganteus for Recovery of Anthocyanins Integrated with Production of Microbial Lipids and Lignin-Rich Residue

Chemical-free hydrothermal pretreatment of Miscanthus x giganteus (Mxg) at the lab scale using high liquid-to-solid ratios resulted in the recovery of anthocyanins and enhanced enzymatic digestibility of residual biomass. In this study, the process is scaled up by using a continuous hydrothermal pretreatment reactor operated at a low liquid-to-solid ratio (50 % w/w solids) as an important step towards commercialization. Anthocyanin yield was 70 % w/w at the pilot scale (50 kg of Mxg), compared to the 94 % w/w yield achieved at the lab scale (0.5 g of Mxg). The pretreated biomass was subsequently refined mechanically using a disc mill to increase the accessibility of cellulose by cellulases. Enzymatic saccharification of the pretreated and disc-milled residue yielded 238 g/L sugar concentration by operating in fed-batch mode at 50 % w/v solids content. Two strains of Rhodosporidium toruloides were evaluated for converting the hydrolysate sugars into microbial lipids, and strain Y-6987 had the highest lipid titer (11.0 g/L). Further, the residue left after enzymatic saccharification was determined to be enriched 1.7-fold in the lignin content. This lignin-rich residue has value as a feedstock for the production of sustainable aviation fuel precursors and other high-value lignin-based chemicals. Hence the proposed biorefinery based on Mxg creates an opportunity for generating revenue from multiple high-value products. As the demand for biofuels and biobased products is rising, the biorefinery products from Mxg would create a niche in the industrial sector.

Conversion↗
DOE OSTI2024

Data for Process Strategies for Recovery of Sugars, Lipids, and Lignin from Oilcane Bagasse Using Natural Deep Eutectic Solvents (NADES)

Sugarcane is being enhanced as a bioenergy crop by engineering it to accumulate and store lipids along with polymeric sugars in vegetative tissues. However, there is no existing process that allows for processing this new crop to recover both lipid and cellulosic sugars from the oilcane bagasse. Therefore, a comprehensive investigation of two pretreatment methods—natural deep eutectic solvents (NADES) and chemical-free hydrothermal pretreatment (HT) was conducted to judge their suitability for recovering fermentable sugars, lipids, and lignin from bagasse. Two NADES, i.e., choline chloride: lactic acid (ChCl:LA) and betaine: lactic acid (BT:LA) were prepared using a 1:2 M ratio and were evaluated for pretreatment of oilcane bagasse at 10, 20, and 50 % (w/w) solids, followed by enzymatic hydrolysis at 10 % (w/w) solids. Notably, ChCl:LA NADES treatment at 10 % (w/w) solids at 140 °C for 2 h, solubilized 78.8 % of lignin and 80.4 % of hemicellulose and allowed 82.7 % enzymatic conversion of glucans to glucose. In contrast, HT pretreatment removed approximately 87.6 % of the hemicellulose and provided an enzymatic glucose yield of 69.7 %. Furthermore, ChCl:LA operated at 50 % solids loading the enriched lipids 2.6-fold (9.2 wt%) in recovered solids compared to HT (6.4 %) and BT:LA (5.1 %) pretreatment processes. NMR-HSQC and GPC analysis showed that ChCl:LA also cleaved the most lignin β–O–4 linkages and demonstrated lower molecular weight compared to HT. This study demonstrates that NADES pretreatment is an effective green processing method for recovering lipids, sugars, and lignin from bioenergy crops at high solid loading (50 % w/w) within the context of an integrated biorefinery.

Conversion↗
DOE OSTI2020

Data from High Solids Loading Biorefinery for the Production of Cellulosic Sugars from Bioenergy Sorghum

A novel process applying high solids loading in chemical-free pretreatment and enzymatic hydrolysis was developed to produce sugars from bioenergy sorghum. Hydrothermal pretreatment with 50% solids loading was performed in a pilot scale continuous reactor followed by disc refining. Sugars were extracted from the enzymatic hydrolysis at 10% to 50% solids content using fed-batch operations. Three surfactants (Tween 80, PEG 4000, and PEG 6000) were evaluated to increase sugar yields. Hydrolysis using 2% PEG 4000 had the highest sugar yields. Glucose concentrations of 105, 130, and 147 g/L were obtained from the reaction at 30%, 40%, and 50% solids content, respectively. The maximum sugar concentration of the hydrolysate, including glucose and xylose, obtained was 232 g/L. Additionally, the glucose recovery (73.14%) was increased compared to that of the batch reaction (52.74%) by using two-stage enzymatic hydrolysis combined with fed-batch operation at 50% w/v solids content.

Conversion↗
DOE OSTI2023

Development and implementation of an in situ high-resolution isotopic microscope for measuring metabolic interactions in soil mesocosms (Final Report)

Many of our planet’s ecosystems rely on the activities of soil microbial communities. These microbes have significant and wide-ranging effects: they metabolize carbon and other nutrients, interact with plants and fungi, and perform other processes important to soil health. However, our ability to directly observe the enzymatic and metabolic activities of microbes within soil is currently limited, not only by the complexity of soil microbial communities themselves, but also by the lack of experimental tools to study them and their molecular interactions in situ. These challenges hinder our understanding of the life-sustaining processes of biomass decomposition and the manner in which it contributes to the movement of freed carbon within soil ecosystems. Here we aimed to develop a novel ultrahigh-resolution isotopic microscope that combines complementary imaging modalities to gain insights into metabolic cycling in soil. This final report covers both the portion of this work that was initially completed at the University of North Carolina at Chapel Hill from 2018 to 2019 (as award DE-SC0019012) and then from 2020 to 2022 (2023 in NCE) at the University of Massachusetts Chan Medical School. This goal was to build an integrated platform consisting of fluorescence microscopy, Raman microspectroscopy, and nanospray desorption electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (nanoDESI-FTICR-MS) to directly investigate microbial activities and molecular transformations occurring in soil by exploiting the use of both fluorescent labels and stable isotope probing. Specifically, our instrument was conceived as integrating: (A) fluorescence detection to localize soil microbes, identify bacteria taking up polysaccharides, and monitor gene expression of enzymes involved in decomposition; (B) Raman microspectroscopy to determine which microbes incorporate decomposition products into their biomass, as well as which decomposers are metabolically active; and (C) nanoDESI-FTICR-MS imaging to spatially probe, in real-time, the metabolites in the surrounding area, which we expect will reveal the distributions of the products of the enzymatic breakdown of polysaccharides, as well as specialized metabolites acting as cell-cell signals between decomposers. To enable these measurements, we exploited fluorescence-based probes to map the microbes that are enzymatically active (per A) and utilized stable isotope-labeled substrates to visualize both the microbial and molecular fate of decomposed biomass (B and C, respectively). To accomplish our goal, we combined the expertise of a multidisciplinary group of scientists to pursue the construction of this microscope and to investigate scientific questions that would be facilitated by such a capability. We aimed to employ this spatially informative, high-resolution isotopic microscope to visualize the critical steps of biomass degradation and the molecular fate of other environmentally relevant substrates within soil mesocosms. Accordingly, this technology will enhance our understanding of the microbial and metabolic interactions occurring within soil communities that are relevant to carbon degradation and other soil processes. The instrument has been developed and housed at EMSL, where it will be available to the entire EMSL User Base, enabling a variety of related DOE-relevant systems to be interrogated in the future by diverse scientific research groups.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Optimizing enzymes for plastic upcycling using machine learning design and high throughput experiments

Plastic use is ubiquitous in the modern world, and polyethylene terephthalate (PET) is one of the most abundantly produced plastics (and the most highly produced polyester), with ~65 million metric tons manufactured annually. To the consumer, PET is likely most recognizable as the plastic used to make beverage bottles. Like many plastics, traditional mechanical or chemical means of PET deconstruction and upcycling are costly and inefficient. Because of these challenges, recycled plastic is generally of lower quality and is more expensive to produce than virgin plastic derived from petroleum. Ultimately, this results in most plastic ending up as waste. We view plastic waste as an underutilized resource which, with the development of more efficient and high-quality recycling processes, could (1) generate significant economic value while (2) decreasing petroleum usage and greenhouse gas emissions, as well as (3) minimizing its negative environmental and health impacts. Biocatalytic recycling, or biomanufacturing the basic building blocks of new plastic from plastic waste, is a promising approach to plastic reuse that complements existing recycling technologies. Recently, biological enzymes capable of breaking down PET have garnered significant attention as an attractive means of dealing with the plastic problem. These enzymes are currently undergoing pilot studies for implementation in industrial-scale enzyme-based recycling. However, there are significant limitations to current enzymes, including the need to perform costly pre-processing of the plastic waste before the enzymes are able to work. Further optimization of these enzymes is necessary to make these technologies competitive, and ultimately incentivise industry-wide adoption of this biology-based green recycling technology. n this work we demonstrate a means to design and generate performant biological enzymes, capable of efficiently deconstructing plastic waste. Specifically, we applied recent advances in artificial intelligence, machine learning, and statistical analysis to design new versions and discover natural enzymes capable of breaking down PET. We focused on optimizing key properties that are important for industrial-scale enzymatic recycling such as pH and thermotolerance. Normal testing of enzymatic plastic-deconstruction is extremely labor intensive and so through this work we also developed a robotic-assisted experimental pipeline capable of characterizing thousands of candidate enzymes. The results of this iterative, AI-guided, multi-discipline approach have led to increases in enzymatic breakdown of over 150X over starting enzymes. This work supports the rapidly developing and transformative field of biocatalytic solutions to environmental problems beyond the discovery and predictive understanding of enzymes for polymer recycling, and has wide implications for tackling numerous energy problems such as carbon capture and fixation (e.g., engineering carbon monoxide dehydrogenase and the rubisco-pathway), biomining (e.g., design of lanthanide-binding proteins) and biomanufacturing (e.g., lignin-deconstruction enzymes).

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗