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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 469 records · Page 26
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Carbon‐negative hydrogen from ethanol via catalytic oxidative reforming

Abstract This study evaluated a commercial technology for producing low‐ or negative‐carbon hydrogen through ethanol catalytic oxidative reforming, focusing on the life cycle greenhouse gas emissions, or carbon intensity (CI). Various scenarios were analyzed: (a) comparing corn ethanol (first‐generation or Gen1 ethanol) and cellulosic ethanol (second‐generation or Gen2 ethanol) as feedstocks; (b) assessing carbon capture and sequestration (CCS) for CO 2 from upstream fermentation; and (c) evaluating oxygen sourcing via air separation units vs. on‐site or off‐site water electrolysis using a proton exchange membrane. Findings indicate that the CI for hydrogen production using Gen2 ethanol from corn stover is lower than that of Gen1 corn ethanol. Additionally, using proton exchange membrane‐generated oxygen results in a lower CI than air separation unit‐generated oxygen, regardless of the sourcing method. Implementing CCS for the hydrogen production plant's evolved CO 2 is essential for achieving a net‐negative CI for hydrogen from Gen1 ethanol. All examined scenarios, including both ethanol generations, oxygen sources, and CCS applications, demonstrated a net‐negative carbon intensity, surpassing the life cycle greenhouse gas emissions threshold of 0.45 kg CO 2 e/kg to enable policy credits as outlined in the Inflation Reduction Act §45V. In comparison, the CI for hydrogen from steam methane reforming stands at 3.4 kg CO 2 e/kg with CCS and 9.4 kg CO 2 e/kg without CCS.

08 HYDROGEN↗
DOE OSTI2026

Harnessing plant‐based platform for low‐cost cellulosic sugar recovery from bioenergy crops

The price of pure cellulase enzyme for the recovery of fermentable cellulosic sugars is one of the major challenges that limit the commercialization of second-generation biofuels and bio-based products. This work shows a means to greatly reduce the cost of cellulases. The abundant capacity of plants to synthesize and hyperaccumulate transgenic proteins in their plastids has been demonstrated for two gene constructs using either NPT II or Tet C to regulate the expression of bacterial cellulase Cel6A. Previously, the expression of Cel6A to 20–35% of total soluble leaf protein in tobacco was shown in two consecutive field trials. Moving forward, the key challenge was the extraction of active enzymes from the transgenic tobacco leaves hyperaccumulating bacterial cellulases in a cost-effective manner. The study showed that unpurified crude extracts of NPT II and Tet C transgenic tobacco leaves recovered ~23% and ~29% w/w cellulosic sugars from energycane bagasse, respectively. A supplementation of as low as 25% of purified commercial cellulase improved the glucose recovery by 2.9 times (~85% w/w) compared to 100% crude extract, which is comparable to the glucose recovery obtained by commercial cellulases, thereby suggesting a reduced requirement of commercial cellulases leading to a cost reduction of 75% for biorefineries. Assuming a stable 40% cellulase yield in total soluble protein under field conditions and multiple harvests (one to three) a year, the study estimates that the potential cost for saccharification of 1 t of lignocellulosic biomass can be reduced to 67–200 USD by using crude leaf extracts of transgenic tobacco.

biofuel↗
DOE OSTI2023

Glucose assimilation rate determines the partition of flux at pyruvate between lactic acid and ethanol in Saccharomyces cerevisiae

Engineered Saccharomyces cerevisiae expressing a lactic acid dehydrogenase can metab- olize pyruvate into lactic acid. However, three pyruvate decarboxylase (PDC) isozymes drive most carbon flux toward ethanol rather than lactic acid. Deletion of endoge- nous PDCs will eliminate ethanol production, but the resulting strain suffers from C 2 auxotrophy and struggles to complete a fermentation. Engineered yeast assimilating xylose or cellobiose produce lactic acid rather than ethanol as a major product with- out the deletion of any PDC genes. We report here that sugar flux, but not sensing, contributes to the partition of flux at the pyruvate branch point in S. cerevisiae express- ing the Rhizopus oryzae lactic acid dehydrogenase (LdhA). While the membrane glucose sensors Snf3 and Rgt2 did not play any direct role in the option of predominant product, the sugar assimilation rate was strongly correlated to the partition of flux at pyruvate: fast sugar assimilation favors ethanol production while slow sugar assimilation favors lactic acid. Applying this knowledge, we created an engineered yeast capable of simultaneously converting glucose and xylose into lactic acid, increasing lactic acid production to approximately 17 g L –1 from the 12 g L –1 observed during sequential consumption of sugars. This work elucidates the carbon source-dependent effects on product selection in engineered yeast.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Enzyme selection, optimization, and production toward biodegradation of post–consumer poly(ethylene terephthalate) at scale

Poly(ethylene terephthalate) (PET) is one of the world's most widely used polyester plastics. Due to its chemical stability, PET is extremely difficult to hydrolyze in a natural environment. Recent discoveries in new polyester hydrolases and breakthroughs in enzyme engineering strategies have inspired enormous research on biorecycling of PET. This study summarizes our research efforts toward large-scale, efficient, and economical biodegradation of post-consumer waste PET, including PET hydrolase selection and optimization, high-yield enzyme production, and high-capacity enzymatic degradation of post-consumer waste PET. First, genes encoding PETase and MHETase from Ideonella sakaiensis and the ICCG variant of leaf-branch compost cutinase (LCC ICCG ) were codon-optimized and expressed in Escherichia coli BL21(DE3) for high-yield production. To further lower the enzyme production cost, a pelB leader sequence was fused to LCC ICCG so that the enzyme can be secreted into the medium to facilitate recovery. To help bind the enzyme on the hydrophobic surface of PET, a substrate-binding module in a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM) was fused to the C-terminus of LCC ICCG . The resulting four different LCC ICCG variants (LCC, PelB-LCC, LCC-PBM, and PelB-LCC-PBM), together with PETase and MHETase, were compared for PET degradation efficiency. A fed-batch fermentation process was developed to produce the target enzymes up to 1.2 g L –1 . Finally, the best enzyme, PelB-LCC, was selected and used for the efficient degradation of 200 g L –1 recycled PET in a well-controlled, stirred-tank reactor. Furthermore, the results will help develop an economical and scalable biorecycling process toward a circular PET economy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Reduced type-A carbohydrate-binding module interactions to cellulose I leads to improved endocellulase activity

Dissociation of nonproductively bound cellulolytic enzymes from cellulose is hypothesized to be a key rate-limiting factor impeding cost-effective biomass conversion to fermentable sugars. However, the role of carbohydrate-binding modules (CBMs) in enabling nonproductive enzyme binding is not well understood. Here, we examine the subtle interplay of CBM binding and cellulose hydrolysis activity for three models type-A CBMs (Families 1, 3a, and 64) tethered to multifunctional endoglucanase (CelE) on two distinct cellulose allomorphs (i.e., cellulose I and III). We generated a small library of mutant CBMs with varying cellulose affinity, as determined by equilibrium binding assays, followed by monitoring cellulose hydrolysis activity of CelE–CBM fusion constructs. Finally, kinetic binding assays using quartz crystal microbalance with dissipation were employed to measure CBM adsorption and desorption rate constants kon and koff, respectively, towards nanocrystalline cellulose derived from both allomorphs. Overall, our results indicate that reduced CBM equilibrium binding affinity towards cellulose I alone, resulting from increased desorption rates (koff) and reduced effective adsorption rates (nkon), is correlated to overall improved endocellulase activity. Future studies could employ similar approaches to unravel the role of CBMs in nonproductive enzyme binding and develop improved cellulolytic enzymes for industrial applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Probing specificities of alcohol acyltransferases for designer ester biosynthesis with a high–throughput microbial screening platform

Alcohol acyltransferases (AATs) enables microbial biosynthesis of a large space of esters by condensing an alcohol and an acyl-CoA. However, substrate promiscuity of AATs prevents microbial biosynthesis of designer esters with high selectivity. Here, we developed a high-throughput microbial screening platform that facilitates rapid identification of AATs for designer ester biosynthesis. First, we established a microplate-based culturing technique with in situ fermentation and extraction of esters. We validated its capability in rapid profiling of the alcohol substrate specificity of 20 chloramphenicol acetyltransferase variants derived from Staphylococcus aureus (CAT Sa ) for microbial biosynthesis of acetate esters with various exogeneous alcohol supply. By coupling the microplate-based culturing technique with a previously established colorimetric assay, we developed a high-throughput microbial screening platform for AATs. We demonstrated that this platform could not only probe the alcohol substrate specificity of both native and engineered AATs but also identify the beneficial mutations in engineered AATs for enhanced ester synthesis. Here, we anticipate the high-throughput microbial screening platform provides a useful tool to identify novel wildtype and engineered AATs that have important roles in nature and industrial biocatalysis for designer bioester production.

2-phenhylethyl acetate↗
DOE OSTI2022

Metabolic engineering of oleaginous yeast Rhodotorula toruloides for overproduction of triacetic acid lactone

Abstract The plant‐sourced polyketide triacetic acid lactone (TAL) has been recognized as a promising platform chemical for the biorefinery industry. However, its practical application was rather limited due to low natural abundance and inefficient cell factories for biosynthesis. Here, we report the metabolic engineering of oleaginous yeast Rhodotorula toruloides for TAL overproduction. We first introduced a 2‐pyrone synthase gene from Gerbera hybrida ( GhPS ) into R. toruloides and investigated the effects of different carbon sources on TAL production. We then systematically employed a variety of metabolic engineering strategies to increase the flux of acetyl‐CoA by enhancing its biosynthetic pathways and disrupting its competing pathways. We found that overexpression of ATP‐citrate lyase (ACL1) improved TAL production by 45% compared to the GhPS overexpressing strain, and additional overexpression of acetyl‐CoA carboxylase (ACC1) further increased TAL production by 29%. Finally, we characterized the resulting strain I12‐ ACL1‐ACC1 using fed‐batch bioreactor fermentation in glucose or oilcane juice medium with acetate supplementation and achieved a titer of 28 or 23 g/L TAL, respectively. This study demonstrates that R. toruloides is a promising host for the production of TAL and other acetyl‐CoA‐derived polyketides from low‐cost carbon sources.

2‐pyrone synthase↗
DOE OSTI2024

Metabolic Engineering of Nonmodel Yeast Issatchenkia orientalis SD108 for 5–Aminolevulinic Acid Production

Biological production of 5-aminolevulinic acid (5-ALA) has received growing attention over the years. However, there is the tradeoff between 5-ALA biosynthesis and cell growth because the fermentation broth will become acidic due to the production of 5-ALA. To address this limitation, we engineered an acid-tolerant yeast, Issatchenkia orientalis SD108, for 5-ALA production. We first discovered that the cell growth rate of I. orientalis SD108 was boosted by 5-ALA and its endogenous ALA synthetase (ALAS) showed higher activity than those homologs from other yeasts. The titer of 5-ALA was improved from 28 mg/L to 120-, 150-, and 300 mg/L, by optimizing plasmid design, overexpressing a transporter, and increasing gene copy number, respectively. After redirecting the metabolic flux using the pyruvate decarboxylase (PDC) knockout strain (SD108ΔPDC) and culturing with urea, we increased the titer of 5-ALA to 510 mg/L, a 13-fold enhancement, proving the importance of the newly identified IoALAS with higher activity and the strategic selection of nitrogen sources for knockout strains. This study demonstrates the acid-tolerant I. orientalis SD108ΔPDC has a high potential for 5-ALA production at a large scale in the future.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Quantitative Analysis of The High‐Yield Hydrolysis of Kelp by Laminarinase and Alginate Lyase

Abstract Kelp is an abundant, farmable biomass‐containing laminarin and alginate as major polysaccharides, providing an excellent model substrate to study their deconstruction by simple enzyme mixtures. Our previous study showed strong reactivity of the glycoside hydrolase family 55 during hydrolysis of purified laminarin, raising the question of its reactivity with intact kelp. In this study, we determined that a combination of a single glycoside hydrolase family 55 β‐1,3‐exoglucanase with a broad‐specificity alginate lyase from the polysaccharide lyase family 18 gives efficient hydrolysis of untreated kelp to a mixture of simple sugars, that is, glucose, gentiobiose, mannitol‐end glucose, and mannuronic and guluronic acids and their soluble oligomers. Quantitative assignments from nanostructure initiator mass spectrometry (NIMS) and 2D HSQC NMR spectroscopy and analysis of the reaction time‐course are provided. The data suggest that binary combinations of enzymes targeted to the unique polysaccharide composition of marine biomass are sufficient to deconstruct kelp into soluble sugars for microbial fermentation.

2D HSQC NMR↗
DOE OSTI2021

Engineered Sorghum Bagasse Enables a Sustainable Biorefinery with p-Hydroxybenzoic Acid-Based Deep Eutectic Solvent

Integrating multidisciplinary research in plant genetic engineering and renewable deep eutectic solvents (DESs) can facilitate a sustainable and economic biorefinery. In this work, we leveraged a plant genetic engineering approach to specifically incorporate C6C1 monomers into the lignin structure. By expressing the bacterial ubiC gene in sorghum, p-hydroxybenzoic acid (PB)-rich lignin was incorporated into the plant cell wall while this monomer was completely absent in the lignin of the wild-type (WT) biomass. A DES was synthesized with choline chloride (ChCl) and PB and applied to the pretreatment of the PB-rich mutant biomass for a sustainable biorefinery. The release of fermentable sugars was significantly enhanced (~190 % increase) compared to untreated biomass by the DES pretreatment. In particular, the glucose released from the pretreated mutant biomass was up to 12 % higher than that from the pretreated WT biomass. Lignin was effectively removed from the biomass with the preservation of more than half of the β-Ο-4 linkages without condensed aromatic structures. Hydrogenolysis of the fractionated lignin was conducted to demonstrate the potential of phenolic compound production. In addition, a simple hydrothermal treatment could selectively extract PB from the same engineered lignin, showing a possible circular biorefinery. These results suggest that the combination of PB-based DES and engineered PB-rich biomass is a promising strategy to achieve a sustainable closed-loop biorefinery.

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

Genetically engineered poplar wood effectively enhances the efficiency of deep eutectic solvent-mediated one-pot processing

Although lignocellulosic biomass is a renewable resource with the potential to replace fossil-derived fuels and chemicals, its recalcitrance, largely due to lignin, limits its utilization. Recent advancements in genetic engineering have produced transgenic trees with reduced lignin content and/or modified lignin structure without compromising growth traits. Here, three engineered poplar varieties are evaluated as feedstocks using a biocompatible one-pot deep eutectic solvent-mediated process that integrates biomass fractionation and enzymatic saccharification within a single reactor, eliminating water washing and reconditioning. All transgenic poplars exhibit higher fermentable sugar yields than wild-type (WT) trees. Notably, QsuB poplar, incorporating 3,4-dihydroxybenzoate in lignin, achieves the highest glucose conversion yield of 91.3% (vs. 73.0% from WT). AT5 and MdCHS3 poplars, incorporating ferulate esters and naringenin, also demonstrate improved glucose yields (86.7 and 84.7%, respectively), confirming reduced biomass recalcitrance. Additionally, residual lignins are valorized via hydrogenolysis into phenolic compounds, with comparable alkylphenol production across all lines. These findings demonstrate that the transgenic poplar lines not only serve as superior feedstocks for sugar conversion but also provide a rich resource for phenolic compound production, enhancing the operational and economic viability of integrated biorefinery processes.

biomasses↗
DOE OSTI2022

The Brazilian Amazonian rainforest harbors a high diversity of yeasts associated with rotting wood, including many candidates for new yeast species

This study investigated the diversity of yeast species associated with rotting wood in Brazilian Amazonian rainforests. Herein, a total of 569 yeast strains were isolated from rotting wood samples collected in three Amazonian areas (Universidade Federal do Amazonas-Universidade Federal do Amazonas [UFAM], Piquiá, and Carú) in the municipality of Itacoatiara, Amazon state. The samples were cultured in yeast nitrogen base (YNB)-d-xylose, YNB-xylan, and sugarcane bagasse and corncob hemicellulosic hydrolysates (undiluted and diluted 1:2 and 1:5). Sugiyamaella was the most prevalent genus identified in this work, followed by Kazachstania. The most frequently isolated yeast species were Schwanniomyces polymorphus, Scheffersomyces amazonensis, and Wickerhamomyces sp., respectively. The alpha diversity analyses showed that the dryland forest of UFAM was the most diverse area, while the floodplain forest of Carú was the least. Additionally, the difference in diversity between UFAM and Carú was the highest among the comparisons. Thirty candidates for new yeast species were obtained, representing 36% of the species identified and totaling 101 isolates. Among them were species belonging to the clades Spathaspora, Scheffersomyces, and Sugiyamaella, which are recognized as genera with natural xylose-fermenting yeasts that are often studied for biotechnological and ecological purposes. The results of this work showed that rotting wood collected from the Amazonian rainforest is a tremendous source of diverse yeasts, including candidates for new species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Taxogenomic Analysis of a Novel Yeast Species, Lachancea rosae Sp. Nov. F.A., Isolated From the Wild Rose Rosa californica

A novel Saccharomycotina yeast strain, yHQL494, was isolated from the rose hip of the wild rose Rosa californica from Castle Crags State Park, California, USA. Phylogenetic analyses of both whole genome data and the sequences from the D1/D2 region of the large ribosomal subunit (LSU) rRNA gene placed strain yHQL494 within the genus Lachancea and grouped it into a clade with Lachancea lanzarotensis and Lachancea meyersii. Taxogenomic analyses were conducted on publicly available genome sequences to gain a deeper insight into the carbon and nitrogen gene-trait associations across the Lachancea clade. The results of these analyses were found to be consistent across Lachancea species. Growth assays and microscopic analyses were conducted to determine the physiological characteristics of strain yHQL494, including the presence of hyphae or pseudohyphae, ascospore formation, fermentation abilities, and assimilation of carbon and nitrogen compounds. Based on the phenotypic and genomic characteristics of the strain yHQL494 T (=NRRL Y-64858 T , =CBS 18,574 T ), we propose a new species, Lachancea rosae sp. nov. f.a.

Bajgain, Yakendra [Villanova Univ., PA (United Sta↗
DOE OSTI2020

Chapter 2: Genetics of Unstudied Thermophiles for Industry

Thermophilic organisms hold great potential for industry due to their numerous advantages in biotechnological applications such as higher reaction rate, higher substrate loading, decreased susceptibility to reaction contamination, energy savings in industrial fermentations, and ability to express thermostable proteins that can be utilized in many important industrial processes. Bioprospecting for thermophiles will continue to reveal new enzymatic and metabolic paradigms with industrial applicability. In order to translate these paradigms to production scale, routine methods for microbial genetic engineering are needed, yet remain to be developed in many newly isolated thermophiles. Major challenges and recent developments in the establishment of reliable genetic systems in thermophiles are discussed. Here, we use a hyperthermophilic, cellulolytic bacterium, Caldicellulosiruptor bescii, as a case study to demonstrate the development of a genetic system for an industrially useful thermophile, describing in detail methods for transformation, genetic tool utilization, and chromosomal modification using targeted gene deletion and insertion techniques.

93 SC - Biological and Environmental Research (BER↗
DOE OSTI2021

CRISPR-Cas Genome Editing in the Cellulolytic Bacterium Clostridium thermocellum (C. thermocellum)

Clostridium thermocellum is an anaerobic thermophile that can efficiently degrade lignocellulosic biomass and directly convert it into value-added products such as ethanol. The cellulolytic and ethanologenic capabilities of C. thermocellum make it an excellent candidate for consolidated bioprocessing (CBP) for industrial production, where biomass degradation and fermentation occur simultaneously. In this organism, strain development for effective CBP has traditionally been hindered by the lack of genetic tools. Here, we detail our efficient two-step CRISPR-Cas genome editing protocol for C. thermocellum, using both the native Type I-B CRISPR-Cas system and an exogenous Type-II CRISPR system from Geobacillus stearothermophilus. As recombination is limiting in C. thermocellum genome engineering, we highlight effective thermophilic recombinases necessary to improve genome editing in these systems. We additionally provide design rules for the repair template and synthetic guide RNA (gRNA) for each system. Using these newly developed CRISPR and recombineering tools, targeted C. thermocellum engineering will substantiate efforts toward CBP strain development in industrial applications.

anaerobe↗
DOE OSTI2021

High-Biomass Sorghums as a Feedstock for Renewable Fuels and Chemicals

High-biomass sorghums are intended for use in biorefineries that convert vegetative biomass into renewable fuels and chemicals. The majority of plant biomass consists of cell walls, a complex matrix of cellulose, hemicellulosic polysaccharides and lignin. In the biorefinery, the biomass is ground and then subjected to a thermo-chemical pretreatment (elevated temperature, high pressure, chemical catalysts) that disrupts the cell wall matrix and solubilizes some of the cell wall polymers, followed by enzymatic saccharification of the cellulose, which produces D-glucose. The glucose and sometimes also the xylose derived from the hydrolysis of the hemicellulosic polysaccharides, are subsequently converted to fuels or other useful chemicals by microbial biocatalysts. The genetic improvement of high-biomass sorghums has as its ultimate goals to maximize the yield of fermentable sugars on a per-hectare basis, to minimize the inputs of fertilizer, irrigation, fungicides and pesticides and to reduce the environmental footprint. Breeding strategies thus need to target biomass yield, biomass composition, maturity, pest and disease resistance and nutrient use efficiency. Furthermore, this chapter reviews the genetic basis of these traits and their potential application in breeding programs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Bacterial α-diglucoside metabolism: perspectives and potential for biotechnology and biomedicine

In a competitive microbial environment nutrient acquisition is a major contributor to the survival of any individual bacterial species, and the ability to access uncommon energy sources can provide a fitness advantage. One set of soluble carbohydrates that have attracted increased attention for use in biotechnology and biomedicine are the α-diglucosides. Maltose is the most well studied member of this class, however the remaining four less common α-diglucosides (trehalose, kojibiose, nigerose, and isomaltose) are increasingly used in processed food and fermented beverages. The consumption of trehalose has recently been shown to be a contributing factor in gut microbiome disease as certain pathogens are using α-diglucosides to outcompete native gut flora. Kojibiose and nigerose have also been examined potential prebiotics and alternative sweeteners for a variety of foods. Compared to the study of maltose metabolism our understanding of the synthesis and degradation of uncommon α-diglucosides is lacking and several fundamental questions that remain unanswered, particularly in regards to the regulation of bacterial metabolism for α-diglucosides. Therefore this minireview attempts to provide a focused analysis of uncommon α-diglucoside metabolism in bacteria, and suggests some future directions for this research area that could potentially accelerate biotechnology and biomedicine developments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Investigating the role of the transcriptional regulator Ure2 on the metabolism of Saccharomyces cerevisiae: a multi-omics approach

Ure2 regulates nitrogen catabolite repression in Saccharomyces cerevisiae. Deletion of URE2 induces a physiological state mimicking the nitrogen starvation and autophagic responses. Previous work has shown that deletion of URE2 increases the fermentation rate of some wine-producing strains of S. cerevisiae. In this work, we investigated the effect of URE2 deletion (ΔURE2) on the metabolism of S. cerevisiae. During growth on glucose, the ΔURE2 mutant grew less well than the wild type; however, it produced ethanol at higher rates. To better under the behavior of this mutant, we performed transcriptomics and metabolomics. Analysis of the RNA sequencing results and metabolite levels indicate that the mutant strain exhibited characteristics of both nitrogen starvation and autophagy. In addition, many pyruvate decarboxylase and alcohol dehydrogenase isoforms were expressed at higher rates than the wild type. The mutant also accumulated less trehalose and glycogen, and produced more lipids. These results suggest that URE2 may be a promising target for metabolic engineering in S. cerevisiae and potentially other strains of yeast as well for the production of lipid-based fuels and chemicals.

59 BASIC BIOLOGICAL SCIENCES↗