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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Development and validation of time‐domain 1 H‐NMR relaxometry correlation for high‐throughput phenotyping method for lipid contents of lignocellulosic feedstocks

Abstract The bioenergy crops such as energycane, miscanthus, and sorghum are being genetically modified using state of the art synthetic biotechnology techniques to accumulate energy‐rich molecules such as triacylglycerides (TAGs) in their vegetative cells to enhance their utility for biofuel production. During the initial genetic developmental phase, many hundreds of transgenic phenotypes are produced. The efficiency of the production pipeline requires early and minimally destructive determination of oil content in individuals. Current screening methods require time‐intensive sample preparation and extraction with chemical solvents for each plant tissue. A rapid screen will also be needed for developing industrial extraction as these crops become available. In the present study, we have devised a proton relaxation nuclear magnetic resonance ( 1 H‐NMR) method for single‐step, non‐invasive, and chemical‐free characterization of in‐situ lipids in untreated and pretreated lignocellulosic biomass. The systematic evaluation of NMR relaxation time distribution provided insight into the proton environment associated with the lipids in the biomass. It resolved two distinct lipid‐associated subpopulations of proton nuclei that characterize total in‐situ lipids into bound and free oil based on their “molecular tumbling” rate. The T1T2 correlation spectra also facilitated the resolution of the influence of various pretreatment procedures on the chemical composition of molecular and local 1 H population in each sample. Furthermore, we show that hydrothermally pretreated biomass is suitable for direct NMR analysis unlike dilute acid and alkaline pretreated biomass which needs an additional step for neutralization.

pretreatment↗
DOE OSTI2023

Elongated galactan side chains mediate cellulose–pectin interactions in engineered Arabidopsis secondary cell walls

SUMMARY The plant secondary cell wall is a thickened matrix of polysaccharides and lignin deposited at the cessation of growth in some cells. It forms the majority of carbon in lignocellulosic biomass, and it is an abundant and renewable source for forage, fiber, materials, fuels, and bioproducts. The complex structure and arrangement of the cell wall polymers mean that the carbon is difficult to access in an economical and sustainable way. One solution is to alter the cell wall polymer structure so that it is more suited to downstream processing. However, it remains difficult to predict what the effects of this engineering will be on the assembly, architecture, and properties of the cell wall. Here, we make use of Arabidopsis plants expressing a suite of genes to increase pectic galactan chain length in the secondary cell wall. Using multi‐dimensional solid‐state nuclear magnetic resonance, we show that increasing galactan chain length enhances pectin–cellulose spatial contacts and increases cellulose crystallinity. We also found that the increased galactan content leads to fewer spatial contacts of cellulose with xyloglucan and the backbone of pectin. Hence, we propose that the elongated galactan side chains compete with xyloglucan and the pectic backbone for cellulose interactions. Due to the galactan topology, this may result in comparatively weak interactions and disrupt the cell wall architecture. Therefore, introduction of this strategy into trees or other bioenergy crops would benefit from cell‐specific expression strategies to avoid negative effects on plant growth.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Xylanolytic metabolism is regulated by coordination of transcription factors XynR and XylR in extremely thermophilic Caldicellulosiruptorales

ABSTRACT Global transcription factors (TFs) control metabolic processes in bacteria to efficiently utilize available carbon. The orderCaldicellulosiruptoraleshas drawn interest due to the ability of its members to degrade components of lignocellulosic biomass. Regulatory reconstruction ofAnaerocellum (f. Caldicellulosiruptor) besciiidentified two major global transcription factors for xylan utilization, XynR and XylR, and the corresponding putative transcription factor binding sites. Recombinant versions of XynR (LacI family) and XylR (ROK family) were subjected to fluorescence polarization (FP) and biolayer interferometry (BLI) analysis to confirm the predicted binding sites. Four XynR sites and two XylR sites were validated, accounting for 20 of 26 genes regulated by XynR and six of seven genes regulated by XylR. Bioinformatic analysis of the individual genes controlled by the two regulators showed an inter-dependent scheme for xylan conversion; the transport of xylooligosaccharides (XOS) is dependent on XylR, while enzymes responsible for hydrolysis are controlled by both regulators. For xylose catabolism by the xylose isomerase-xylulose kinase pathway, regulation is also split, with XylR controlling xylose isomerase and XynR controlling xylokinase. The XynR/XylR regulator pair withinA. besciiis conserved in all sequenced species ofCaldicellulosiruptorales, suggesting similarities in regulating linear xylan conversion. In other xylanolytic thermophiles, XylR homologs control xylan degradation, compared to just 6 out of 26 genes forA. bescii. These results show that two separate regulatory schemes (dual repression) are coordinated byA. besciito effectively regulate the hemicellulose inventory and xylan catabolism. IMPORTANCE To take full advantage of extreme thermophiles as platform metabolic engineering microorganisms, the tools for genetic manipulation must be further developed, and strategies that exploit a better understanding of metabolic regulation need to be discerned.Anaerocellum bescii, the most studied of the extremely thermophilic fermentative anaerobic bacteria that can utilize microcrystalline cellulose, can degrade microcrystalline cellulose and hemicellulose and has been metabolically engineered to convert the resulting sugars to products such as ethanol and acetone. For xylan, in particular, two major global transcription factors (TFs), XynR and XylR, play a role in sugar metabolism, although their predicted regulatory interdependence from bioinformatics analysis has not been elucidated experimentally. Here, fluorescence polarization (FP) and biolayer interferometry (BLI) were used to explore this issue to support metabolic engineering efforts aimed at improving carbohydrate processing to industrial chemicals.

Biotechnology & Applied Microbiology↗
DOE OSTI2023

Near-complete genome sequence of Lipomyces tetrasporous NRRL Y-64009, an oleaginous yeast capable of growing on lignocellulosic hydrolysates

ABSTRACT Lipomyces tetrasporous is an oleaginous yeast that can utilize a variety of plant-based sugars. It accumulates lipids during growth on lignocellulosic biomass hydrolysates. We present the annotated genome sequence of L. tetrasporous NRRL Y-64009 to aid in its development as a platform organism for producing lipids and lipid-based bioproducts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Genetic and metabolic drivers of membrane remodeling in Clostridium thermocellum under alcohol stress

Clostridium thermocellum is a leading candidate for consolidated bioprocessing of lignocellulosic biomass into biofuels due to its native cellulolytic capabilities. Beyond ethanol, C. thermocellum is being developed as a platform for producing higher-chain alcohols such as isobutanol and n-butanol. However, its physiological adaptations to alcohol stress remain poorly understood. Here, we investigate how C. thermocellum remodels its membrane lipid composition in response to exogenous ethanol, n-butanol, isobutanol, and butyrate. Exposure to linear alcohols such as n-butanol or to organic acids like butyrate increased the proportion of straight-chain fatty acids in the membrane at the expense of branched-chain species, whereas exposure to the branched alcohol isobutanol produced the opposite effect. Isotope tracer experiments demonstrated that C. thermocellum directly incorporates the carbon backbones of exogenous alcohols and acids into fatty acids, providing a mechanistic basis for these contrasting shifts. We show that the bifunctional aldehyde/alcohol dehydrogenase AdhE is essential for the assimilation of exogenous alcohols into fatty acids, acting through its oxidative activity by first oxidizing alcohols to aldehydes and then converting them to acyl-CoA intermediates. Deletion of the pyruvate:ferredoxin oxidoreductase isozyme pfor4 abolished branched-chain fatty acid synthesis, but supplementation with isobutanol restored production, indicating that Pfor4 substitutes for the canonical branched-chain α-keto acid dehydrogenase complex. These findings reveal two distinct routes for branched-chain fatty acid production in C. thermocellum: a Pfor4-dependent pathway from α-keto acid intermediates derived from amino acid synthesis, and an AdhE-dependent salvage pathway that assimilates exogenous branched-chain alcohols.

Acetivibrio thermocellus↗
DOE OSTI2022

Virtual Engineering (VE) (vebio)

The Virtual Engineering (VE, vebio) project provides a set of Python modules to enable users to connect mathematical models of unit operations to predict outcomes for an entire chemical process. This VE approach is currently being developed to support the beginning-to-end simulation of the low-temperature conversion of lignocellulosic biomass to a fuel precursor, but the underlying communication functions can be readily extended to connect models from other domains. The models and features currently included in the VE library include a models/simulations for pretreatment, enzymatic hydrolysis, and aerobic bioreaction.

Young, Ethan↗
DOE OSTI2025

Stage-resolved gene regulatory network analysis reveals developmental reprogramming and genes with robust stem-preferred expression in sorghum

Sorghum bicolor is a deep-rooted, heat- and drought-tolerant crop that thrives on marginal lands and is increasingly valued for its applications in biofuel, bioenergy, and biopolymer production. The sorghum stem, which can reach 4–5 m in length, serves as the primary reservoir of both lignocellulosic biomass and soluble sugars, making it a promising bioenergy feedstock. Although recent advances in genetic, genomic, and transcriptomic resources have improved our understanding of sorghum biology, comprehensive genome-wide analyses of functional dynamics across diverse organ types and developmental stages remain limited. In particular, candidate genes with stem preferred expression pattern or their associated cis-regulatory elements, which may program key stem-related functions and enable organ- or tissue-specific engineering, have not yet been identified.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Prebiotic properties of Bacillus coagulans MA-13: production of galactoside hydrolyzing enzymes and characterization of the transglycosylation properties of a GH42 β-galactosidase

The spore-forming lactic acid bacterium Bacillus coagulans MA-13 has been isolated from canned beans manufacturing and successfully employed for the sustainable production of lactic acid from lignocellulosic biomass. Among lactic acid bacteria, B. coagulans strains are generally recognized as safe (GRAS) for human consumption. Low-cost microbial production of industrially valuable products such as lactic acid and various enzymes devoted to the hydrolysis of oligosaccharides and lactose, is of great importance to the food industry. Specifically, α- and β-galactosidases are attractive for their ability to hydrolyze not-digestible galactosides present in the food matrix as well as in the human gastrointestinal tract. In this work we have explored the potential of B. coagulans MA-13 as a source of metabolites and enzymes to improve the digestibility and the nutritional value of food. A combination of mass spectrometry analysis with conventional biochemical approaches has been employed to unveil the intra- and extra- cellular glycosyl hydrolase (GH) repertoire of B. coagulans MA-13 under diverse growth conditions. The highest enzymatic activity was detected on β-1,4 and α-1,6-glycosidic linkages and the enzymes responsible for these activities were unambiguously identified as β-galactosidase (GH42) and α-galactosidase (GH36), respectively. Whilst the former has been found only in the cytosol, the latter is localized also extracellularly. The export of this enzyme may occur through a not yet identified secretion mechanism, since a typical signal peptide is missing in the α-galactosidase sequence. A full biochemical characterization of the recombinant β-galactosidase has been carried out and the ability of this enzyme to perform homo- and hetero-condensation reactions to produce galacto-oligosaccharides, has been demonstrated. Probiotics which are safe for human use and are capable of producing high levels of both α-galactosidase and β-galactosidase are of great importance to the food industry. In this work we have proven the ability of B. coagulans MA-13 to over-produce these two enzymes thus paving the way for its potential use in treatment of gastrointestinal diseases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Enhancement of polyhydroxyalkanoate production by co-feeding lignin derivatives with glycerol in Pseudomonas putida KT2440

Abstract Background Efficient utilization of all available carbons from lignocellulosic biomass is critical for economic efficiency of a bioconversion process to produce renewable bioproducts. However, the metabolic responses that enable Pseudomonas putida to utilize mixed carbon sources to generate reducing power and polyhydroxyalkanoate (PHA) remain unclear. Previous research has mainly focused on different fermentation strategies, including the sequential feeding of xylose as the growth stage substrate and octanoic acid as the PHA-producing substrate, feeding glycerol as the sole carbon substrate, and co-feeding of lignin and glucose. This study developed a new strategy—co-feeding glycerol and lignin derivatives such as benzoate, vanillin, and vanillic acid in Pseudomonas putida KT2440—for the first time, which simultaneously improved both cell biomass and PHA production. Results Co-feeding lignin derivatives (i.e. benzoate, vanillin, and vanillic acid) and glycerol to P. putida KT2440 was shown for the first time to simultaneously increase cell dry weight (CDW) by 9.4–16.1% and PHA content by 29.0–63.2%, respectively, compared with feeding glycerol alone. GC–MS results revealed that the addition of lignin derivatives to glycerol decreased the distribution of long-chain monomers (C10 and C12) by 0.4–4.4% and increased the distribution of short-chain monomers (C6 and C8) by 0.8–3.5%. The 1 H– 13 C HMBC, 1 H– 13 C HSQC, and 1 H– 1 H COSY NMR analysis confirmed that the PHA monomers (C6–C14) were produced when glycerol was fed to the bacteria alone or together with lignin derivatives. Moreover, investigation of the glycerol/benzoate/nitrogen ratios showed that benzoate acted as an independent factor in PHA synthesis. Furthermore, 1 H, 13 C and 31 P NMR metabolite analysis and mass spectrometry-based quantitative proteomics measurements suggested that the addition of benzoate stimulated oxidative-stress responses, enhanced glycerol consumption, and altered the intracellular NAD + /NADH and NADPH/NADP + ratios by up-regulating the proteins involved in energy generation and storage processes, including the Entner–Doudoroff (ED) pathway, the reductive TCA route, trehalose degradation, fatty acid β -oxidation, and PHA biosynthesis. Conclusions This work demonstrated an effective co-carbon feeding strategy to improve PHA content/yield and convert lignin derivatives into value-added products in P. putida KT2440. Co-feeding lignin break-down products with other carbon sources, such as glycerol, has been demonstrated as an efficient way to utilize biomass to increase PHA production in P. putida KT2440. Moreover, the involvement of aromatic degradation favours further lignin utilization, and the combination of proteomics and metabolomics with NMR sheds light on the metabolic and regulatory mechanisms for cellular redox balance and potential genetic targets for a higher biomass carbon conversion efficiency.

09 BIOMASS FUELS↗
DOE OSTI2021

Synthesis, antibiotic structure–activity relationships, and cellulose dissolution studies of new room-temperature ionic liquids derived from lignin

Ionic liquids (ILs) are promising pretreatment solvents for lignocellulosic biomass, but are largely prepared from petroleum precursors. Benzaldehydes from depolymerized lignin, such as vanillin, syringaldehyde, and 4-methoxy benzaldehyde, represent renewable feedstocks for the synthesis of ionic liquids. We herein report syntheses of novel lignin-derived ionic liquids, with extended N -alkyl chains, and examine their melting points, cellulose dissolution capacities, and toxicity profiles against Daphnia magna and E. coli strain 1A1. The latter organism has been engineered to produce isoprenol, a drop-in biofuel and precursor for commodity chemicals. The new N,N -diethyl and N,N -dipropyl methyl benzylammonium ILs were liquids at room temperature, showing 75–100 °C decreased melting points as compared to their N,N,N -trimethyl benzylammonium analog. Extension of N -alkyl chains also increased antibacterial activity threefold, while ionic liquids prepared from vanillin showed 2- to 4-fold lower toxicity as compared to those prepared from syringaldehyde and 4-methoxybenzaldehyde. The trend of antibacterial activity for anions of lignin-derived ILs was found to be methanesulfonate < acetate < hydroxide. Microcrystalline cellulose dissolution, from 2 to 4 wt% after 20 min at 100 °C, was observed in all new ILs using light microscopy and IR spectroscopy. Ionic liquids prepared from H-, S- and G-lignin oxidation products provided differential cytotoxic activity against E. coli and D. magna , suggesting these compounds could be tailored for application specificity within a biorefinery.

09 BIOMASS FUELS↗
DOE OSTI2022

Water-soluble saponins accumulate in drought-stressed switchgrass and may inhibit yeast growth during bioethanol production

Abstract Background Developing economically viable pathways to produce renewable energy has become an important research theme in recent years. Lignocellulosic biomass is a promising feedstock that can be converted into second-generation biofuels and bioproducts. Global warming has adversely affected climate change causing many environmental changes that have impacted earth surface temperature and rainfall patterns. Recent research has shown that environmental growth conditions altered the composition of drought-stressed switchgrass and directly influenced the extent of biomass conversion to fuels by completely inhibiting yeast growth during fermentation. Our goal in this project was to find a way to overcome the microbial inhibition and characterize specific compounds that led to this inhibition. Additionally, we also determined if these microbial inhibitors were plant-generated compounds, by-products of the pretreatment process, or a combination of both. Results Switchgrass harvested in drought (2012) and non-drought (2010) years were pretreated using Ammonia Fiber Expansion (AFEX). Untreated and AFEX processed samples were then extracted using solvents (i.e., water, ethanol, and ethyl acetate) to selectively remove potential inhibitory compounds and determine whether pretreatment affects the inhibition. High solids loading enzymatic hydrolysis was performed on all samples, followed by fermentation using engineered Saccharomyces cerevisiae . Fermentation rate, cell growth, sugar consumption, and ethanol production were used to evaluate fermentation performance. We found that water extraction of drought-year switchgrass before AFEX pretreatment reduced the inhibition of yeast fermentation. The extracts were analyzed using liquid chromatography–mass spectrometry (LC–MS) to detect compounds enriched in the extracted fractions. Saponins, a class of plant-generated triterpene or steroidal glycosides, were found to be significantly more abundant in the water extracts from drought-year (inhibitory) switchgrass. The inhibitory nature of the saponins in switchgrass hydrolysate was validated by spiking commercially available saponin standard (protodioscin) in non-inhibitory switchgrass hydrolysate harvested in normal year. Conclusions Adding a water extraction step prior to AFEX-pretreatment of drought-stressed switchgrass effectively overcame inhibition of yeast growth during bioethanol production. Saponins appear to be generated by the plant as a response to drought as they were significantly more abundant in the drought-stressed switchgrass water extracts and may contribute toward yeast inhibition in drought-stressed switchgrass hydrolysates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Engineering isoprenoids production in metabolically versatile microbial host Pseudomonas putida

Abstract With the increasing need for microbial bioproduction to replace petrochemicals, it is critical to develop a new industrial microbial workhorse that improves the conversion of lignocellulosic carbon to biofuels and bioproducts in an economically feasible manner. Pseudomonas putida KT2440 is a promising microbial host due to its capability to grow on a broad range of carbon sources and its high tolerance to xenobiotics. In this study, we engineered P. putida KT2440 to produce isoprenoids, a vast category of compounds that provide routes to many petrochemical replacements. A heterologous mevalonate (MVA) pathway was engineered to produce potential biofuels isoprenol (C 5 ) and epi-isozizaene (C 15 ) for the first time in P. putida . We compared the difference between three different isoprenoid pathways in P. putida on isoprenol production and achieved 104 mg/L of isoprenol production in a batch flask experiment through optimization of the strain. As P. putida can natively consume isoprenol, we investigated how to prevent this self-consumption. We discovered that supplementing l -glutamate in the medium can effectively prevent isoprenol consumption in P. putida and metabolomics analysis showed an insufficient energy availability and an imbalanced redox status during isoprenol degradation. We also showed that the engineered P. putida strain can produce isoprenol using aromatic substrates such as p -coumarate as the sole carbon source, and this result demonstrates that P. putida is a valuable microbial chassis for isoprenoids to achieve sustainable biofuel production from lignocellulosic biomass. Graphical Abstract

09 BIOMASS FUELS↗
DOE OSTI2022

Data for The Effects of Sequential Hydrothermal-Mechanical Refining Pretreatment on Cellulose Structure Changes and Sugar Recoveries

The recalcitrance of lignocellulosic biomass necessitates an efficient pretreatment protocol for operating a successful cellulosic biorefinery. It is critical to improve cellulose accessibility for hydrolysis and fermentation by altering the plant cell wall’s physical structure and chemical composition. Sequential hydrothermal-mechanical refining pretreatment (HMR) allows efficient recovery of cellulosic sugars without utilizing any hazardous chemicals. HMR has been successfully applied to Liberty switchgrass, a bioenergy cultivar released by the USDA, and now it is being applied to oilcane, a recently developed transgenic sugarcane variety engineered to accumulate lipids in its vegetative tissues. Sugar yields of oilcane bagasse (OCB) and switchgrass (SG) treated with HMR are 96.4% and 75.4%, respectively. This study sought to correlate cellulosic sugar yields with structural changes within the cell wall caused by HMR on two distinct bioenergy crops. Simon’s staining technique for the specific surface area analysis showed that HMR increased the specific surface area of pretreated biomass residues by 80-112%. In addition, ATR-FTIR was performed to determine the effects of HMR on physical structures based on the total crystallinity index (TCI) and hydrogen bonding intensity (HBI). Irrespective of biomass type, HMR decreased the initial crystalline cellulose contents of untreated biomass residues by 3.5% and reduced TCI and HBI by 7-13%. The study found that sugar yields were negatively correlated to reducing values of hydrogen bonding intensity, crystalline cellulose content, and total crystallinity index.

Biomass Analytics↗
DOE OSTI2020

Data for High-Level β-Carotene Production from Xylose by Engineered Saccharomyces cerevisiae without Overexpression of a Truncated HMG1 (t HMG1 )

β‐Carotene is a natural pigment and health‐promoting metabolite, and has been widely used in the nutraceutical, feed, and cosmetic industries. Here, we engineered a GRAS yeast Saccharomyces cerevisiae to produce β‐carotene from xylose, the second most abundant and inedible sugar component of lignocellulose biomass. Specifically, a β‐carotene biosynthetic pathway containing crtYB, crtI, and crtE from Xanthophyllomyces dendrorhous was introduced into a xylose‐fermenting S. cerevisiae . The resulting strain produced β‐carotene from xylose at a titer threefold higher than from glucose. Interestingly, overexpression of t HMG1 , which has been reported as a critical genetic perturbation to enhance metabolic fluxes in the mevalonate pathway and β‐carotene production in yeast when glucose is used, did not further improve the production of β‐carotene from xylose. Through fermentation profiling, metabolites analysis, and transcriptional studies, we found the advantages of using xylose as a carbon source, instead of glucose, for β‐carotene production to be a more respiratory feature of xylose consumption, a larger cytosolic acetyl‐CoA pool, and an upregulated expression level of rate‐limiting genes in the β‐carotene‐producing pathway, including ACS1 and HMG1 . As a result, 772.8 mg/L of β‐carotene was obtained in a fed‐batch bioreactor culture with xylose feeding. Considering the inevitable large scale production of xylose when cellulosic biomass‐based bioeconomy is implemented, our results suggest xylose utilization is a promising strategy for overproduction of carotenoids and other isoprenoids in engineered S. cerevisiae .

Conversion↗
DOE OSTI2024

Data for Sustainable Co-Production of Plant Lipids and Cellulosic Sugars from Transgenic Energycane at an Industrially Relevant Scale: A Proof of Concept for Alternative Feedstocks

Development of sustainable and scalable technologies to convert lignocellulosic biomass to biofuels is critical to achieving carbon neutrality. The potential of transgenic bioenergy crops as a renewable source of sugars and lipids has been demonstrated at bench-scale. However, scaling up these processes is important for holistic analysis. Here proof-of-concept for chemical-free hydrothermal pretreatment of transgenic energycane-oilcane line L13 at an industrially relevant scale to recover vegetative lipids along with cellulosic sugars is presented. Pilot-scale processing of 97 kg of transgenic energycane-oilcane L13 stems and high solids pretreatment of bagasse enhanced the recovery of cellulosic glucose and xylose by 5-fold as compared to untreated bagasse and helped in the enrichment of vegetative lipids in the biomass residues which allowed its recovery at the end of the bioprocess. Palmitic and oleic acids were the predominant fatty acids (FAs) extracted from stems and leaves. The processing did not affect lipid composition. The efficiency of lipid recovery from untreated biomass was 75.9% which improved to 88.7% upon pretreatment. The vegetative tissues of transgenic energycane-oilcane L13 contained 0.42 metric tons/hectare of lipids. Processing vegetative tissues yielded 0.38 metric tons/hectare of lipids. This approaches an oil yield similar to soybean (global average 0.44 metric tons/hectare) and is almost twice as high as the oil yield from sugarcane engineered to hyperaccumulate lipids (0.20 metric tons/hectare). The study suggests that further optimization by state-of-the-art metabolic engineering and biomass processing can establish transgenic bioenergy crops for commercial drop-in fuel production.

Biomass Analytics↗
DOE OSTI2025

Data for Metabolic Engineering of β-Oxidation to Leverage Thioesterases for Production of 2-Heptanone, 2-Nonanone, and 2-Undecanone

Medium-chain length methyl ketones are potential blending fuels due to their cetane numbers and low melting temperatures. Biomanufacturing offers the potential to produce these molecules from renewable resources such as lignocellulosic biomass. In this work, we designed and tested metabolic pathways in Escherichia coli to specifically produce 2-heptanone, 2-nonanone and 2-undecanone. We achieved substantial production of each ketone by introducing chain-length specific acyl-ACP thioesterases, blocking the β-oxidation cycle at an advantageous reaction, and introducing active β-ketoacyl-CoA thioesterases. Using a bioprospecting approach, we identified 15 homologs of E. coli β-ketoacyl-CoA thioesterase (FadM) and evaluated the in vivo activity of each against various chain length substrates. The FadM variant from Providencia sneebia produced the most 2-heptanone, 2-nonanone, and 2-undecanone, suggesting it has the highest activity on the corresponding β-ketoacyl-CoA substrates. We tested enzyme variants, including acyl-CoA oxidases, thiolases, and bi-functional 3-hydroxyacyl-CoA dehydratases to maximize conversion of fatty acids to β-keto acyl-CoAs for 2-heptanone, 2-nonanone, and 2-undecanone production. In order to address the issue of product loss during fermentation, we applied a 20% (v/v) dodecane layer in the bioreactor and built an external water cooling condenser connecting to the bioreactor heat-transferring condenser coupling to the condenser. Using these modifications, we were able to generate up to 4.4 g/L total medium-chain length methyl ketones.

Metabolic Engineering↗
DOE OSTI2020

Woody Feedstocks 2019 State of Technology Report

The U.S. Department of Energy promotes production of advanced liquid transportation fuels from lignocellulosic biomass by funding fundamental and applied research that advances the state of technology (SOT). As part of its involvement with this overall mission, Idaho National Laboratory completes annual SOT reports for nth-plant biomass feedstock logistics. The purpose of the SOTs is to provide the status of feedstock supply system technology development for biomass to biofuels, based on actual data and experimental results relative to technical targets and cost goals from specific design cases. The 2019 Woody Feedstock SOT presents the State of Technology for feedstock supply to three individual thermochemical conversion pathways that utilize woody feedstocks: Indirect Liquefaction (IDL), Catalytic Fast Pyrolysis (CFP), and Algal-blend Hydrothermal liquefaction (AHTL). The 2019 reactor throat delivered feedstock costs were found to be $63.54/dry ton, $70.15/dry ton and $70.31/dry ton, respectively (2016$).

09 BIOMASS FUELS↗
DOE OSTI2020

Herbaceous Feedstock 2019 State of Technology Report

The U.S. Department of Energy (DOE) promotes the production of advanced liquid transportation fuels from lignocellulosic biomass by funding fundamental and applied research that advances the State of Technology (SOT). As part of its involvement with this mission, Idaho National Laboratory (INL) completes an annual SOT report for biomass feedstock logistics. This report summarizes supply system impacts of Bioenergy Technologies Office (BETO)-funded research and development efforts at INL and elsewhere (such as the High-Tonnage Feedstock Logistics projects (Webb et al. 2013a, Webb et al. 2013b, Webb et al. 2013c, Webb and Sokhansanj 2014, Sokhansanj et al. 2014)) that lead to improvements in feedstock supply systems. These include improvements to and observed performance of innovative harvest and collection methods, storage technologies, transportation and handling approaches, and advanced preprocessing technologies. Biomass quality and variability, and the interface between feedstock quality and conversion performance are key drivers in addition to delivered feedstock cost. In this report, we estimate the benefits of R&D improvements to individual supply system unit operations and present the status of feedstock logistics technology development for converting biomass into biofuels. These analyses are supported by experimental data where possible and help to align the SOT relative to the cost goals defined in the Multi-Year Program Plan. The 2019 Herbaceous SOT incorporates several technology changes in feedstock preprocessing and introduces opportunities from the integrated landscape management (ILM) strategy and increased grower participation to reduce biomass access costs, while maintaining or improving grower profitability. During FY18 uneven flow from the horizontal bale grinder was identified as a significant issue limiting preprocessing system throughput. Based on FSL-funded research at INL, the 2019 Herbaceous SOT replaces the horizontal bale grinder used in the first stage size reduction with a bale processor. The improved uniformity of biomass flow entering the PDU eliminated slugging flow from the first stage size reduction and improved the throughput of downstream operations. In order to achieve moisture reduction through frictional heating during grinding (which allowed elimination of the costly rotary drum dryer in previous SOTs), the second stage grinder was changed from a rotary shear, which does not remove moisture, back to a hammer mill. Finally, the 2019 Herbaceous SOT introduces modified three-pass and two-pass corn stover supply curves derived from the BT16 resource assessment, based on FY19 modeling results (WBS 4.2.1.20) quantifying economic benefits of ILM in the supply area, together with modeling results (WBS 1.2.1.5) identifying ILM strategies to increase grower participation. The 2019 Herbaceous SOT report documents the current modeled cost of an herbaceous feedstock supply system from harvest to the pretreatment reactor throat for hydrocarbon fuel production via biochemical conversion, based on equipment and processes now available or potentially available in the near term. The modeled cost also considers both the required quality and the availability of the biomass resources. The 2019 Herbaceous SOT predicts a modeled delivered feedstock cost of $81.37 /dry ton (2016$); this is a $2.30/dry ton (2016$) decrease from the 2018 Herbaceous SOT. Technology improvements that contributed to this modeled cost reduction include reduced cost for the new preprocessing design and quantification of the opportunities of the integrated landscape management (ILM) strategy and an increased grower participation rate to reduce the grower payment portion of biomass access costs, while maintaining or improving grower profitability. A greenhouse gas emissions (GHG) assessment was completed by Argonne National Laboratory using the 2019 Greenhouse Gases, Regulated Emissions, and Energy use in Transportation model, estimating an increase of 14.89 kg CO2e/ton from the 2018 SOT (69.27 kg CO2e/ton in 2018 to 84.16 kg CO2e/ton in 2019). The increase of energy consumption during preprocessing along with higher transportation distance to access low cost biomass from further distance contributed to the increase of GHG emissions in the 2019 Herbaceous SOT. The reason for the increased transportation distances was the cost tradeoff of going farther from the biorefinery to access the cheaper ILM-derived counties (the cheaper price outweighed the cost of increased supply radius).

09 BIOMASS FUELS↗