Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “cellulosic biomass”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

In Vivo Thermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using 13 C and 2 H Tracers

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic anaerobic bacteria with complementary metabolic capabilities that utilize distinct glycolytic pathways for the conversion of cellulosic sugars to biofuels. We integrated quantitative metabolomics with 2 H and 13 C metabolic flux analysis to investigate the in vivo reversibility and thermodynamics of the central metabolic networks of these two microbes. We found that the glycolytic pathway in C. thermocellum operates remarkably close to thermodynamic equilibrium, with an overall drop in Gibbs free energy 5-fold lower than that of T. saccharolyticum or anaerobically grown Escherichia coli . The limited thermodynamic driving force of glycolysis in C. thermocellum could be attributed in large part to the small free energy of the phosphofructokinase reaction producing fructose bisphosphate. The ethanol fermentation pathway was also substantially more reversible in C. thermocellummthan in T. saccharolyticum. These observations help explain the comparatively low ethanol titers of C. thermocellum and suggest engineering interventions that can be used to increase its ethanol productivity and glycolytic rate. In addition to thermodynamic analysis, we used our isotope tracer data to reconstruct the T. saccharolyticum central metabolic network, revealing exclusive use of the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis, a bifurcated tricarboxylic acid (TCA) cycle, and a sedoheptulose bisphosphate bypass active within the pentose phosphate pathway. Thermodynamics constitutes a key determinant of flux and enzyme efficiency in metabolic networks. Here, we provide new insights into the divergent thermodynamics of the glycolytic pathways of C. thermocellum and T. saccharolyticum, two industrially relevant thermophilic bacteria whose metabolism still is not well understood. We report that while the glycolytic pathway in T. saccharolyticum is as thermodynamically favorable as that found in model organisms, such as E. coli or Saccharomyces cerevisiae , the glycolytic pathway of C. thermocellum operates near equilibrium. The use of a near-equilibrium glycolytic pathway, with potentially increased ATP yield, by this cellulolytic microbe may represent an evolutionary adaptation to growth on cellulose, but it has the drawback of being highly susceptible to product feedback inhibition. The results of this study will facilitate future engineering of high-performance strains capable of transforming cellulosic biomass to biofuels at high yields and titers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Data for Complete and Efficient Conversion of Plant Cell Wall Hemicellulose into High-Value Bioproducts by Engineered Yeast

Plant cell wall hydrolysates contain not only sugars but also substantial amounts of acetate, a fermentation inhibitor that hinders bioconversion of lignocellulose. Despite the toxic and non-consumable nature of acetate during glucose metabolism, we demonstrate that acetate can be rapidly co-consumed with xylose by engineered Saccharomyces cerevisiae . The co-consumption leads to a metabolic re-configuration that boosts the synthesis of acetyl-CoA derived bioproducts, including triacetic acid lactone (TAL) and vitamin A, in engineered strains. Notably, by co-feeding xylose and acetate, an engineered strain produces 23.91 g/L TAL with a productivity of 0.29 g/L/h in bioreactor fermentation. This strain also completely converts a hemicellulose hydrolysate of switchgrass into 3.55 g/L TAL. These findings establish a versatile strategy that not only transforms an inhibitor into a valuable substrate but also expands the capacity of acetyl-CoA supply in S. cerevisiae for efficient bioconversion of cellulosic biomass.

Conversion↗
DOE OSTI2021

Improving tolerance of yeast to lignocellulosic-derived feedstocks and products

Combined substrate-product toxicity remains one of the main obstacles hampering the scale-up and cost-effectiveness of bioprocesses harnessing lignocellulosic feedstocks, the most abundant, renewable terrestrial resource. Hydrolytic pretreatments release numerous inhibitors impinging on cell viability: the three most acute to yeast S. cerevisiae (the industry dominant biocatalyst) — and universal to all plant sources — are furfural, hydroxymethylfurfural (HMF), and acetic acid. Likewise, desired fermentation products, such as fuel ethanol or commodity organic acids, are toxic to microbes, typically via unknown biological mechanisms. Here, we have engineered both hydrolysate and end-product tolerance in yeast by combining previously-shown alcohol protective modifications with evolved genetic activities targeting the major pretreatment inhibitors. When tested on a wide sampling of genuine lignocellulosic feedstocks, ethanol production increased by >30% on average to titers >100 g/L, achieving parity with clean-sugar equivalents where conditions permit. Furthermore, we designed the tolerance capability to be fully transferable to pre-existing metabolic chassis strains. As such, we “drop-in” hydrolysate competence into one producing lactic acid, demonstrating the first-ever production of a cellulosic plastic at industrial titers. Our advances thus renew the potential of cellulosic biomass utilization for sustainable fuel and non-fuel products at scale.

09 BIOMASS FUELS↗
DOE OSTI2021

Genome-Wide Transcription Factor DNA Binding Sites and Gene Regulatory Networks in Clostridium thermocellum

Clostridium thermocellum is a thermophilic bacterium recognized for its natural ability to effectively deconstruct cellulosic biomass. While there is a large body of studies on the genetic engineering of this bacterium and its physiology to-date, there is limited knowledge in the transcriptional regulation in this organism and thermophilic bacteria in general. The study herein is the first report of a large-scale application of DNA-affinity purification sequencing (DAP-seq) to transcription factors (TFs) from a bacterium. We applied DAP-seq to > 90 TFs in C. thermocellum and detected genome-wide binding sites for 11 of them. We then compiled and aligned DNA binding sequences from these TFs to deduce the primary DNA-binding sequence motifs for each TF. These binding motifs are further validated with electrophoretic mobility shift assay (EMSA) and are used to identify individual TFs’ regulatory targets in C. thermocellum . Our results led to the discovery of novel, uncharacterized TFs as well as homologues of previously studied TFs including RexA-, LexA-, and LacI-type TFs. We then used these data to reconstruct gene regulatory networks for the 11 TFs individually, which resulted in a global network encompassing the TFs with some interconnections. As gene regulation governs and constrains how bacteria behave, our findings shed light on the roles of TFs delineated by their regulons, and potentially provides a means to enable rational, advanced genetic engineering of C. thermocellum and other organisms alike toward a desired phenotype.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica

Yarrowia lipolytica is known to be capable of metabolizing glucose and accumulating lipids intracellularly; however, it lacks the cellulolytic enzymes needed to break down cellulosic biomass directly. To develop Y. lipolytica as a consolidated bioprocessing (CBP) microorganism, we previously expressed the heterologous CBH I, CBH II, and EG II cellulase enzymes both individually and collectively in this microorganism. We concluded that the coexpression of these cellulases resulted in a metabolic drain on the host cells leading to reduced cell growth and lipid accumulation. The current study aims to build a new cellulase coexpressing platform to overcome these hinderances by (1) knocking out the sucrose non-fermenting 1 ( Snf1 ) gene that represses the energetically expensive lipid and protein biosynthesis processes, and (2) knocking in the cellulase cassette fused with the recyclable selection marker URA3 gene in the background of a lipid-accumulating Y. lipolytica strain overexpressing ATP citrate lyase ( ACL ) and diacylglycerol acyltransferase 1 ( DGA1 ) genes. We have achieved a homologous recombination insertion rate of 58% for integrating the cellulases- URA3 construct at the disrupted Snf1 site in the genome of host cells. Importantly, we observed that the disruption of the Snf1 gene promoted cell growth and lipid accumulation and lowered the cellular saturated fatty acid level and the saturated to unsaturated fatty acid ratio significantly in the transformant YL163t that coexpresses cellulases. The result suggests a lower endoplasmic reticulum stress in YL163t, in comparison with its parent strain Po1g ACL-DGA1. Furthermore, transformant YL163t increased in vitro cellulolytic activity by 30%, whereas the “total in vivo newly formed FAME (fatty acid methyl esters)” increased by 16% in comparison with a random integrative cellulase-expressing Y. lipolytica mutant in the same YNB-Avicel medium. The Snf1 disruption platform demonstrated in this study provides a potent tool for the further development of Y. lipolytica as a robust host for the expression of cellulases and other commercially important proteins.

09 BIOMASS FUELS↗
DOE OSTI2021

Genome-wide Transcription Factor DNA Binding Sites and Gene Regulatory Networks in Clostridium thermocellum

Clostridium thermocellum is a thermophilic bacterium recognized for its natural ability to effectively deconstruct cellulosic biomass. While there is a large body of studies on the genetic engineering of this bacterium and its physiology to-date, there is limited knowledge in the transcriptional regulation in this organism and thermophilic bacteria in general. The study herein is the first report of a high-throughput application of DNA-affinity purification sequencing (DAP-seq) to transcription factors (TFs) from a thermophile. We applied DAP-seq to >90 TFs in C. thermocellum and detected genome-wide binding sites for 11 of them. We then compiled and aligned DNA binding sequences from these TFs to deduce the primary DNA-binding sequence motifs for each TF. These binding motifs are further validated with electrophoretic mobility shift assay (EMSA) and are used to identify individual TFs’ regulatory targets in C. thermocellum. Our results led to the discovery of novel, uncharacterized TFs as well as homologues of previously studied TFs including RexA-, LexA- and LacI-type TFs. We then used these data to reconstruct gene regulatory networks for the 11 TFs individually, which resulted in a global network encompassing the TFs with some interconnections. As gene regulation governs and constrains how bacteria behave, our findings shed light on the roles of TFs delineated by their regulons, and potentially provides a means to enable rational, advanced genetic engineering of C. thermocellum and other organisms alike towards a desired phenotype.

09 BIOMASS FUELS↗
DOE OSTI2020

Chimeric polypeptides having xylose isomerase activity

There is provided chimeric polypeptides capable of converting xylose to xylulose, engineered host cells that express the chimeric polypeptides, methods of creating chimeric polypeptides, and methods of fermenting cellulosic biomass to produce biofuels, including ethanol.

09 BIOMASS FUELS↗
DOE OSTI2021

Chimeric polypeptides having xylose isomerase activity

There is provided chimeric polypeptides capable of converting xylose to xylulose, engineered host cells that express the chimeric polypeptides, methods of creating chimeric polypeptides, and methods of fermenting cellulosic biomass to produce biofuels, including ethanol.

Froehlich, Allan↗
DOE OSTI2021

A Novel Platform for Algal Biomass Production Using Cellulosic Mixotrophy (CeMix) (Final Technical Report)

This novel project, branded CeMIX for cellulosic mixotrophy, targets solutions for several technological barriers limiting the deployment of algae cultivation in sunny, arid regions (specifically the southwest United States). Opening up the region for algae cultivation could increase DOE’s resource assessments and biofuel potential for the U.S. Mixotrophic metabolism in support of liquid fuel production is completely dependent on the availability of waste organic carbon in order to avoid diversion of food resources for fuel production. The use of cellulosic sugar hydrolysate (CSH) produced via the NREL process provided a standardized carbon source for study of the underlying biochemistry and metabolic adjustments to mixotrophy. Mixotrophic metabolism uses both photosynthetic CO 2 fixation and sugar oxidation to improve biomass productivity and harvest yield. The project was high-risk, high-reward in the sense that adding organic carbon to algal cultivation is an invitation for rapid-growth, heterotrophic contaminants to overtake the culture and consume mineral nutrients needed for algal growth. To help evade this outcome the project focused on acidophilic red algae that require low pH conditions with 40-48°C optimal temperature profiles that define them as low-range thermophiles. This is the only project in the BETO portfolio to utilize extreme conditions of low pH and high operating temperatures to control pathogens and competitors in mass culture. The project yielded five major outcomes. 1) Harvest densities of 5-10 g ash-free dry weight are easily achieved, providing a 10-fold reduction in water required and dewatering demand. 2) Catabolic repression of photosynthesis in G. sulphuraria is conditionally repressed by low oxygen. 3) Mixotrophic growth on cellulosic hydrolysate consumes all C6 and C5 sugars concomitant with increases in floridean starch with little change in lipid content. Protein content can be manipulated between 35 and 54 wt% by varying the C:N molar ratio from 10:1 to 20:1, respectively. 4) Mixotrophic cultures with up to 50 mM total sugar are remarkably stable at pH 2.5 and daily average reactor temperatures of 40°C. 5) Capital costs for glass tubular photobioreactors are prohibitive for liquid fuel production using a hydrothermal liquefaction processing pathway. The economic outlook for covered raceway ponds is better with a project minimum fuel selling price at $3.32 dm 3 GE -1 .

09 BIOMASS FUELS↗
DOE OSTI2020

Fabrication of lignocellulosic biomass paper containing nanofibrillated biomass

Fibrillated cellulose has been frequently used for making nanopapers and thin films. However, limited work has been carried out in the construction of such materials using native lignocellulosic biomass. Making papers from fibrillated biomass allows complete utilization of whole plant material and may reduce chemical and energy consumption. Ultra-friction grinding was used to directly fibrillate knife-milled poplar into micro- to nano-sized biomass fibers. Papers were made using the fibrillated biomass containing nanofibrillated biomass and their mechanical properties were tested. Biomass papers made via press-drying had higher tensile strength than papers made by air-drying. A higher press-drying temperature of 180 °C produced stronger papers than at 150 °C. Guar gum substantially increased the strength of the press-dried papers in comparison to cationic starch. Press-drying increased the thermogravimetric peak decomposition temperature by 13 °C in comparison to air-drying.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis

Biomass deconstruction remains integral for enabling second-generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active cellulosome system that exhibited long-term stability at 75 degrees C. The resulting glycosylated designer cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer cellulosomes. These results indicate that glycosylation can be used as an essential engineering tool to improve the properties of designer cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism.

09 BIOMASS FUELS↗
DOE OSTI2020

A biomass pretreatment using cellulose-derived solvent Cyrene

Despite only recently becoming available in the quantities required for solvent usage, the cellulose-derived solvent, named Cyrene, has gained significant attention in green chemistry in recent years. To fulfill the sustainability criteria of future biorefineries, a novel renewable biomass pretreatment using Cyrene and water was developed for the first time. Results showed that Cyrene has high potential as a green pretreatment solvent in terms of lignin fractionation/recovery and sugar release in the follow-up enzymatic hydrolysis. The mechanism of this pretreatment was revealed by investigating the structural characteristics of pretreated biomass, and the recovered lignin was also fully characterized to assess its valorization potential. Results indicated that Cyrene pretreatment could be performed at a mild condition (120 °C) to reduce the lignin condensation and the cleavage of β-O-4 linkages without compromising lignin removal and the following sugar platform. The successful utilization of this cellulose-derived solvent in pretreatment will further contribute to the realization of a “closed-loop” biorefinery process.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1996

A generalized Biomass Pyrolysis Model Based on Superimposed Cellulose, Hemicellulose and Lignin Kinetics

The pyrolysis of general biomass materials is modeled via a superposition of cellulose, hemicellulose an lingnin kinetics. All three of the primary biomass components are modeled with multi-step kinetics involving both competetive primary pyrolysis and secodary tar decomposition reactions. Only typical (untreated) feedstocks are considered at atmospheric pyrolysis pressures.

biomass↗
DOE OSTI2023

Designing Iowa Agricultural Landscapes to Improve Environmental Co-Benefits of Bioenergy Production

Cellulosic bioenergy feedstocks are needed to improve carbon (C) management while provisioning biomass for bioproducts and biofuel. The transition to increased cellulosic biomass production can be guided by land management plans designed to improve economic, environmental, and ecological performance. We constructed a sustainability model to compare landscape designs for biofuel production from corn (Zea mays L.) stover and switchgrass (Panicum virgatum L.) in central Iowa, USA. We used the model to compare environmental and socioeconomic outcomes associated with four landscape management strategies, with and without cellulosic biomass markets. We evaluated (1) a fuelshed area containing over 1.2 million ha (3 million acres) of corn and soybean (Glycine max (L.) Merr.) within 80 km (50 miles) of a commercial-scale cellulosic biorefinery in Nevada, Iowa, and (2) the South Fork watershed containing over 72,000 ha (178,000 acres) of these row crops within eight north central Iowa HUC-12 (hydrologic unit code) watersheds. At both landscape scales, we found that it is possible to achieve multiple environmental and socioeconomic benefits concomitantly with cellulosic biomass production by strategically collecting corn stover and converting the 10% of the lowest-profitability row crop land to perennial switchgrass. Potential benefits from landscape design include increased biodiversity, soil and water quality improvements, increased soil carbon sequestration for climate change mitigation, and reduced fertilizer use and cost. Our model results showed that increasing benefits can accrue when complementary conservation practices (e.g., reduced tillage, use of a rye cover crop) are combined and integrated throughout a fuelshed or watershed area. We conclude that ecologically based landscape designs offer valuable insights about costs and benefits of land management alternatives, with relevance for achieving stakeholder goals.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Spatially Varying Costs of GHG Abatement with Alternative Cellulosic Feedstocks for Sustainable Aviation Fuels

Cellulosic biomass-based sustainable aviation fuels (SAFs) can be produced from various feedstocks. The breakeven price and carbon intensity of these feedstock-to-SAF pathways are likely to differ across feedstocks and across spatial locations due to differences in feedstock attributes, productivity, opportunity costs of land for feedstock production, soil carbon effects, and feedstock composition. We integrate feedstock to fuel supply chain economics and life-cycle carbon accounting using the same system boundary to quantify and compare the spatially varying greenhouse gas (GHG) intensities and costs of GHG abatement with SAFs derived from four feedstocks (switchgrass, miscanthus, energy sorghum, and corn stover) at 4 km resolution across the U.S. rainfed region. We show that the optimal feedstock for each location differs depending on whether the incentive is to lower breakeven price, carbon intensity, or cost of carbon abatement with biomass or to have high biomass production per unit land. The cost of abating GHG emissions with SAF ranges from $\$181$ Mg –1 CO 2 e to more than $\$444$ Mg –1 CO 2 e and is lowest with miscanthus in the Midwest, switchgrass in the south, and energy sorghum in a relatively small region in the Great Plains. While corn stover-based SAF has the lowest breakeven price per gallon, it has the highest cost of abatement due to its relatively high GHG intensity. Furthermore, our findings imply that different types of policies, such as volumetric targets, tax credits, and low carbon fuel standards, will differ in the mix of feedstocks they incentivize and locations where they are produced in the U.S. rainfed region.

09 BIOMASS FUELS↗
DOE OSTI2020

Solid-State NMR Studies of Solvent-Mediated, Acid-Catalyzed Woody Biomass Pretreatment for Enzymatic Conversion of Residual Cellulose

Enzymes selectively hydrolyze the carbohydrate fractions of lignocellulosic biomass into corresponding sugars, but these processes are limited by low yields and slow catalytic turnovers. Under certain conditions, the rates and yields of enzymatic sugar production can be increased by pretreating biomass using solvents, heat, and dilute acid catalysts. However, the mechanistic details underlying this behavior are not fully elucidated, and designing effective pretreatment strategies remains an empirical challenge. Herein, using a combination of solid-state and high-resolution magic-angle-spinning NMR, infrared spectroscopy, and X-ray diffractometry, we show that the extent to which cellulase enzymes are able to hydrolyze solvent-pretreated biomass can be understood in terms of the ability of the solvent to break the chemical linkages between cellulose and noncellulosic materials in the cell wall. This finding is of general significance to enzymatic biomass conversion research, and implications for designing improved biomass conversion strategies are discussed. Furthermore, these findings demonstrate the utility of solid-state NMR as a tool to elucidate the key chemical and physical changes that occur during the liquid-phase conversion of real biomass.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗