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At least 325 records · Page 18
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Analytical Modeling of Biomass Transport and Feeding Systems

The processing of biomass solids in a biorefinery consists of pretreatment, enzyme hydrolysis / concurrent fermentation of sugars to ethanol, product recovery, and drying. Sustainable operation requires a front end that transforms wet solids into a pumpable slurry. Otherwise the biorefinery will suffer unscheduled shut-downs and inefficient operation due to solids that obstruct pumps and other equipment and resist mixing in a bioreactor. Downtime in pioneer biorefineries due to interruptions from materials handling problems has been 50% or more, leading to unsustainable manufacturing processes. This work addresses new technology, predictive computational models, and definition of operational conditions that result in formation of slurries of corn stover at up to 300 g/L using low enzyme loadings (1 to 3 FPU cellulase/g) before the biomass (corn stover) enters the pretreatment step. A team of researchers from Purdue University, Idaho National Laboratory (INL), Forest Concepts, AdvanceBio, Argonne National Laboratory, and DOE BETO have combined their knowledge in agricultural and biological engineering, bioprocess engineering, mechanical engineering, chemical engineering, agricultural economics, materials engineering and enzyme and microbial technology to address the challenge of making lignocellulose flow. This team effort has resulted in the development and validation of conditions that employ low levels of commercial enzyme in an agitated bioreactor to which corn stover pellets are added resulting in formation of slurries at high solids loadings, before pretreatment. This approach overcomes challenges caused by handling of dry, particulate biomass materials at the front end of the biorefinery. The subsequent materials handling issues cause obstruction at pumps, pipes and valves. Formation of high loadings slurries with low yield stress, as reported here, significantly decreases the potential for process interruption and enhances plant operability. Key advances in the knowledge of how slurry formation occurs is reported here and in recently published journal papers. We found that pellets are needed to achieve high solids loading, and that commercial enzymes are effective in forming slurries of corn stover particles from pellets that have not been pretreated. Our work has resulted in models that predict solids behavior for formation of compressed solids and pellets that in turn facilitate slurries made of high concentrations of corn stover particles. A computational model was developed that gives mechanistic insights into properties of particles and mixing process that gives the slurry rheology needed to facilitate pumping. Hence, the corn stover may be pumped into a pretreatment reactor in place of auguring in solids against high pressure which is a root cause of interruptions at the front end of a biorefinery. Subsequent mixing in enzyme and microbial bioreactors results in conversion of lignocellulose to sugars in a biorefinery in agitated bioreactors, with flows in and out of the vessels being less likely to be interrupted due to plugging or materials handling problems. The obtained data coupled to process models, techno-economic assessment (TEA) and Life Cycle Analysis (LCA) were used to assess whether this approach is practical. These results are based on a foundation of laboratory characterization and pilot runs. The NREL biochemical sugar model was utilized to carry out techno-economic analysis of enzyme catalyzed liquefaction followed by enzyme hydrolysis. The minimum sugar selling price was between 17.5 and 18.3 ¢/pound or about the same as calculated by the NREL model for dilute acid pretreatment followed by enzyme hydrolysis. Life cycle analysis (LCA) based on Argonne’s Greet Model showed the enzyme catalyzed route had the lowest greenhouse gas emissions of the three combinations studied (i.e., enzyme, enzyme mimetic, and enzyme + mimetic combined). GHG emissions for enzyme-based corn stover liquefaction step, alone, were about 21 g CO 2 -equivalent/kg of liquefied slurry. We believe this approach will further enhance operability of a pioneer biorefinery, and bring large-scale conversion of lignocellulosic biomass to low carbon footprint biofuels closer to implementation.

09 BIOMASS FUELS↗
DOE OSTI2022

Value proposition of coatings or new alloys on hammer wear

The goal of this Case Study was to elucidate the value of improving of the life of parts that wear within a system in terms of the additional material cost that it takes to reach the level of improvement. We recognize that the failure limits and system performance are representative of a single system that may or may not exist in the real world, however, our goal of this project was not to provide an answer for a specific system or to provide a full understanding the economics of all potential systems. The analysis was performed using relative changes from the base alloy cost and considered relative improvements in part life in order to generalize the comparison without referencing specific alloys or coatings that might be employed. Ultimately, this work provides a first check of the potential for improving wear characteristics of grinder hammers to provide a meaningful and impactful benefit to biomass preprocessing and conversion systems. Key takeaways include: • Increasing the life of hammers by 3× provides a delivered feedstock cost benefit of approximately $\$2.25$/dry ton assuming that the relative cost of the hammer material of construction is not increased. • The feasible area of relative hammer cost increase ranges from 110% to 122% of the relative life increase, i.e., a 3× life increase can cost 3.66× the cost of the original hammers • Beyond a 3× increase in relative hammer life, little economic benefit is realized • Even when moisture impacts are included, the relationship between relative hammer cost and relative hammer life was reduced only slightly to a range of 108% to 116% • From the simulation we would expect delivered feedstock costs to be within ±$1.10/dry ton, based on the uncertainty analysis

09 BIOMASS FUELS↗
DOE OSTI2023

Valorization of Hemp-Based Packaging Waste with One-Pot Ionic Liquid Technology

The range of applications for industrial hemp has consistently increased in various sectors over the years. For example, hemp hurd can be used as a resource to produce biodegradable packaging materials when incorporated into a fungal mycelium composite, a process that has been commercialized. Although these packaging materials can be composted after usage, they may present an opportunity for valorization in a biorefinery setting. Here, we demonstrate the potential of using this type of discarded packaging composite as a feedstock for biofuel production. A one-pot ionic liquid-based biomass deconstruction and conversion process was implemented, and the results from the packaging material were compared with those obtained from untreated hemp hurd. At a 120 °C reaction temperature, 7.5% ionic liquid loading, and 2 h reaction time, the packaging materials showed a higher lignocellulosic sugar yield and sugar concentrations than hemp hurd. Hydrolysates prepared from packaging materials also promoted production of higher titers (1400 mg/L) of the jet-fuel precursor bisabolene when used to cultivate an engineered strain of the yeast Rhodosporidium toruloides. Box–Behnken experiments revealed that pretreatment parameters affected the hemp hurd and packaging materials differently, evidencing different degrees of recalcitrance. This study demonstrated that a hemp hurd-based packaging material can be valorized a second time once it reaches the end of its primary use by supplying it as a feedstock to produce biofuels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Dynamic ecosystem assembly and escaping the “fire trap” in the tropics: insights from FATES_15.0.0

Abstract. Fire is a fundamental part of the Earth system, with impacts on vegetation structure, biomass, and community composition, the latter mediated in part via key fire-tolerance traits, such as bark thickness. Due to anthropogenic climate change and land use pressure, fire regimes are changing across the world, and fire risk has already increased across much of the tropics. Projecting the impacts of these changes at global scales requires that we capture the selective force of fire on vegetation distribution through vegetation functional traits and size structure. We have adapted the fire behavior and effects module, SPITFIRE (SPread and InTensity of FIRE), for use with the Functionally Assembled Terrestrial Ecosystem Simulator (FATES), a size-structured vegetation demographic model. We test how climate, fire regime, and fire-tolerance plant traits interact to determine the biogeography of tropical forests and grasslands. We assign different fire-tolerance strategies based on crown, leaf, and bark characteristics, which are key observed fire-tolerance traits across woody plants. For these simulations, three types of vegetation compete for resources: a fire-vulnerable tree with thin bark, a vulnerable deep crown, and fire-intolerant foliage; a fire-tolerant tree with thick bark, a thin crown, and fire-tolerant foliage; and a fire-promoting C4 grass. We explore the model sensitivity to a critical parameter governing fuel moisture and show that drier fuels promote increased burning, an expansion of area for grass and fire-tolerant trees, and a reduction of area for fire-vulnerable trees. This conversion to lower biomass or grass areas with increased fuel drying results in increased fire-burned area and its effects, which could feed back to local climate variables. Simulated size-based fire mortality for trees less than 20 cm in diameter and those with fire-vulnerable traits is higher than that for larger and/or fire-tolerant trees, in agreement with observations. Fire-disturbed forests demonstrate reasonable productivity and capture observed patterns of aboveground biomass in areas dominated by natural vegetation for the recent historical period but have a large bias in less disturbed areas. Though the model predicts a greater extent of burned fraction than observed in areas with grass dominance, the resulting biogeography of fire-tolerant, thick-bark trees and fire-vulnerable, thin-bark trees corresponds to observations across the tropics. In areas with more than 2500 mm of precipitation, simulated fire frequency and burned area are low, with fire intensities below 150 kW m−1, consistent with observed understory fire behavior across the Amazon. Areas drier than this demonstrate fire intensities consistent with those measured in savannas and grasslands, with high values up to 4000 kW m−1. The results support a positive grass–fire feedback across the region and suggest that forests which have existed without frequent burning may be vulnerable at higher fire intensities, which is of greater concern under intensifying climate and land use pressures. The ability of FATES to capture the connection between fire disturbance and plant fire-tolerance strategies in determining biogeography provides a useful tool for assessing the vulnerability and resilience of these critical carbon storage areas under changing conditions across the tropics.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production

Biofuels and value-added biochemicals derived from renewable biomass via biochemical conversion have attracted considerable attention to meet global sustainable energy and environmental goals. Isobutanol is a four-carbon alcohol with many advantages that make it attractive as a fossil-fuel alternative. Zymomonas mobilis is a highly efficient, anaerobic, ethanologenic bacterium making it a promising industrial platform for use in a biorefinery. In this study, the effect of isobutanol on Z. mobilis was investigated, and various isobutanol-producing recombinant strains were constructed. The results showed that the Z. mobilis parental strain was able to grow in the presence of isobutanol below 12 g/L while concentrations greater than 16 g/L inhibited cell growth. Integration of the heterologous gene encoding 2-ketoisovalerate decarboxylase such as kdcA from Lactococcus lactis is required for isobutanol production in Z. mobilis. Moreover, isobutanol production increased from nearly zero to 100-150 mg/L in recombinant strains containing the kdcA gene driven by the tetracycline-inducible promoter Ptet. In addition, we determined that overexpression of a heterologous als gene and two native genes (ilvC and ilvD) involved in valine metabolism in a recombinant Z. mobilis strain expressing kdcA can divert pyruvate from ethanol production to isobutanol biosynthesis. This engineering improved isobutanol production to above 1 g/L. Finally, recombinant strains containing both a synthetic operon, als-ilvC-ilvD, driven by Ptet and the kdcA gene driven by the constitutive strong promoter, Pgap, were determined to greatly enhance isobutanol production with a maximum titer about 4.0 g/L. Finally, isobutanol production was negatively affected by aeration with more isobutanol being produced in more poorly aerated flasks. This study demonstrated that overexpression of kdcA in combination with a synthetic heterologous operon, als-ilvC-ilvD, is crucial for diverting pyruvate from ethanol production for enhanced isobutanol biosynthesis. Moreover, this study also provides a strategy for harnessing the valine metabolic pathway for future production of other pyruvate-derived biochemicals in Z. mobilis.

09 BIOMASS FUELS↗
DOE OSTI2020

Xylose Assimilation for the Efficient Production of Biofuels and Chemicals by Engineered Saccharomyces cerevisiae

Microbial conversion of plant biomass into fuels and chemicals offers a practical solution to global concerns over limited natural resources, environmental pollution, and climate change. Pursuant to these goals, researchers have put tremendous efforts and resources towards engineering the yeast Saccharomyces cerevisiae to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into various fuels and chemicals. Here, we summarize recent advances in metabolic engineering of yeast to address bottlenecks on xylose assimilation, and to enable simultaneous co-utilization of xylose and other carbon sources in lignocellulosic hydrolysates. We also highlight distinct characteristics of xylose metabolism that can be harnessed to produce advanced biofuels and chemicals. Although many challenges remain, recent research investments have facilitated efficient fermentation of xylose and simultaneous co-consumption of xylose and glucose. In particular, understanding of xylose-induced metabolic rewiring in engineered yeast has encouraged use of xylose as a carbon source for producing various non-ethanol bioproducts. Furthermore, to boost the lignocellulosic biomass-based bioeconomy, much attention is expected to promote xylose-utilizing efficiency via reprogramming cellular regulatory networks, to attain robust co-fermentation of xylose and other cellulosic carbon sources under industrial conditions, and to exploit the advantageous traits of yeast xylose metabolism for producing diverse fuels and chemicals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Recent progress in the synthesis of advanced biofuel and bioproducts

Energy is one of the most complex fields of study and an issue that influences nearly every aspect of modern life. Over the past century, combustion of fossil fuels, particularly in the transportation sector, has been the dominant form of energy release. Refining of petroleum and natural gas into liquid transportation fuels is also the centerpiece of the modern chemical industry used to produce materials, solvents, and other consumer goods. In the face of global climate change, the world is searching for alternative, sustainable means of producing energy carriers and chemical building blocks. The use of biofuels in engines predates modern refinery optimization and today represents a small but significant fraction of liquid transportation fuels burnt each year. Similarly, white biotechnology has been used to produce many natural products through fermentation. The evolution of recombinant DNA technology into modern synthetic biology has expanded the scope of biofuels and bioproducts that can be made by biocatalysts. Furthermore, this opinion examines the current trends in this research space, highlighting the substantial growth in computational tools and the growing influence of renewable electricity in the design of metabolic engineering strategies. In short, advanced biofuel and bioproduct synthesis remains a vibrant and critically important field of study whose focus is shifting away from the conversion of lignocellulosic biomass toward a broader consideration of how to reduce carbon dioxide to fuels and chemical products.

09 BIOMASS FUELS↗
DOE OSTI2023

Milligrams to kilograms: making microbes work at scale

Energy is one of the most complex fields of study and an issue that influences nearly every aspect of modern life. Over the past century, combustion of fossil fuels, particularly in the transportation sector, has been the dominant form of energy release. Refining of petroleum and natural gas into liquid transportation fuels is also the centerpiece of the modern chemical industry used to produce materials, solvents, and other consumer goods. In the face of global climate change, the world is searching for alternative, sustainable means of producing energy carriers and chemical building blocks. The use of biofuels in engines predates modern refinery optimization and today represents a small but significant fraction of liquid transportation fuels burnt each year. Similarly, white biotechnology has been used to produce many natural products through fermentation. The evolution of recombinant DNA technology into modern synthetic biology has expanded the scope of biofuels and bioproducts that can be made by biocatalysts. This opinion examines the current trends in this research space, highlighting the substantial growth in computational tools and the growing influence of renewable electricity in the design of metabolic engineering strategies. In short, advanced biofuel and bioproduct synthesis remains a vibrant and critically important field of study whose focus is shifting away from the conversion of lignocellulosic biomass towards a broader consideration of how to reduce carbon dioxide to fuels and chemical products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Operando S/TEM Reactions of Pt/TiO 2 Catalysts for Catalytic Fast Pyrolysis

Conversion of lignocellulosic biomass into the transportation fuel is an attractive concept. Using ex situ Catalytic Fast Pyrolysis (CFP) improves to upgrade bio-oils via catalytic deoxygenation of the biomass pyrolysis vapors. Zeolites are often used in this technique as the catalyst; however, in order to reduce minimum fuel selling price and improve carbon yields, ongoing research emphasizes catalyst optimization. By using bifunctional metal-acid catalysts and utilizing hydrogen, it is possible to improve carbon yields by minimizing the carbon-containing light gasses (since oxygen is removed aswater) and by reducing coke formation. Current research and development activities focuses on Pt/TiO 2 catalysts as the most promising catalyst candidates for the hydrodeoxygenation process. Here, our aim is to determine the structure-function relationships of the Pt/TiO 2 catalyst and the role of Pt dispersions within the catalyst on performance, using operando scanning transmission electron microscopy (S/TEM).

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Evaluating the Transport Mechanism for Lignocellulosic Inhibitors in Zymomonas mobilis

The conversion of lignocellulosic biomass to biofuels and bioproducts is limited by small inhibitory molecules generated during the pretreatment process. The diverse compounds within the cell wall hydrolysate including aldehydes, carboxylic acids, phenolics, and alcohols inhibit microbial growth by disrupting microbial membranes. Although Zymomonas mobilis combines high ethanol productivity with hopanoid-rich membranes that confer solvent tolerance, hydrolysate compounds still inhibit microbial growth. One proposed mitigation strategy is to pump these molecules out through active transport processes, reducing direct toxicity. However, if passive permeation rates are high, active transport would create a futile cycle where the exported molecules diffuse back in, creating a net drag on fitness. To investigate this hypothesis, we measure the passive permeation for thirty-three lignocellulose derived compounds using atomistic molecular dynamics simulations with enhanced sampling. Permeability coefficients determined spanned more than eight orders of magnitude driven largely by the balance between hydrophobicity and polarity. Hydrophobic inhibitors such as aromatic acids, aldehydes, and short chain alcohols readily partitioned into the membrane and showed high permeability consistent with rapid passive influx and strong toxicity. Most polar molecules have lower but still appreciable permeation rates, with only the most polar sugars having low permeabilities. By integrating these predictions together with hydrolysate composition information, we find that most of these molecules are so permeable that futile cycles where passive permeation overwhelms efflux is highly likely. These findings provide a predictive framework for strain engineering, highlighting that, for highly permeable inhibitors, reducing extracellular inhibitor concentrations through hydrolysate detoxification, pretreatment optimization, or dilution may be necessary to mitigate futile cycling and improve microbial productivity in industrial biorefineries.

Zymomonas mobilis↗
DOE OSTI2020

Isobutanol production freed from biological limits using synthetic biochemistry

Cost competitive conversion of biomass-derived sugars into biofuel will require high yields, high volumetric productivities and high titers. Suitable production parameters are hard to achieve in cell-based systems because of the need to maintain life processes. As a result, next-generation biofuel production in engineered microbes has yet to match the stringent cost targets set by petroleum fuels. Removing the constraints imposed by having to maintain cell viability might facilitate improved production metrics. Here, we report a cell-free system in a bioreactor with continuous product removal that produces isobutanol from glucose at a maximum productivity of 4 g L –1 h –1 , a titer of 275 g L –1 and 95% yield over the course of nearly 5 days. These production metrics exceed even the highly developed ethanol fermentation process. Our results suggest that moving beyond cells has the potential to expand what is possible for bio-based chemical production.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Energy utilization strategies in lignocellulosic biorefineries

Lignocellulosic biorefineries can generate various energy-rich side streams, including biogas from anaerobic digestion, a lignin stream from biomass pretreatment, and conversion residues. This study evaluates different chemical energy utilization strategies in lignocellulosic biorefineries based on energy and carbon efficiencies, cost, and greenhouse gas mitigation potential. Specifically, using modeling and optimization, we examine strategies that include electricity generation, biogas upgrade to biomethane, lignin valorization, and carbon capture and storage. We find that upgrading biogas to biomethane demonstrates the highest energy efficiency and leads to the lowest minimum fuel selling price for the main product, ethanol. The biorefinery with carbon capture and storage achieves the lowest net carbon footprint. Lignin valorization has the highest carbon footprint due to the additional materials required for lignin depolymerization, despite its potential to produce high value bioproducts.

Aboagye, Emmanuel A. [Princeton Univ., NJ (United ↗
DOE OSTI2022

Mass spectral imaging showing the plant growth-promoting rhizobacteria's effect on the Brachypodium awn

The plant growth-promoting rhizobacteria (PGPR) on the host plant surface play a key role in biological control and pathogenic response in plant functions and growth. However, it is difficult to elucidate the PGPR effect on plants. Such information is important in biomass production and conversion. Brachypodium distachyon (Brachypodium), a genomics model for bioenergy and native grasses, was selected as a C3 plant model; and the Gram-negative Pseudomonas fluorescens SBW25 ( P.) and Gram-positive Arthrobacter chlorophenolicus A6 ( A.) were chosen as representative PGPR strains. The PGPRs were introduced to the Brachypodium seed's awn prior to germination, and their possible effects on the seeding and growth were studied using different modes of time-of-flight secondary ion mass spectrometry (ToF-SIMS) measurements, including a high mass-resolution spectral collection and delayed image extraction. We observed key plant metabolic products and biomarkers, such as flavonoids, phenolic compounds, fatty acids, and auxin indole-3-acetic acid in the Brachypodium awns. Furthermore, principal component analysis and two-dimensional imaging analysis reveal that the Brachypodium awns are sensitive to the PGPR, leading to chemical composition and morphology changes on the awn surface. Our results show that ToF-SIMS can be an effective tool to probe cell-to-cell interactions at the biointerface. This work provides a new approach to studying the PGPR effects on awn and shows its potential for the research of plant growth in the future.

metabolite↗
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 OSTI2019

Engineered Pseudomonas putida KT2440 co-utilizes galactose and glucose

Abstract Background Efficient conversion of plant biomass to commodity chemicals is an important challenge that needs to be solved to enable a sustainable bioeconomy. Deconstruction of biomass to sugars and lignin yields a wide variety of low molecular weight carbon substrates that need to be funneled to product. Pseudomonas putida KT2440 has emerged as a potential platform for bioconversion of lignin and the other components of plant biomass. However, P. putida is unable to natively utilize several of the common sugars in hydrolysate streams, including galactose. Results In this work, we integrated a De Ley–Doudoroff catabolic pathway for galactose catabolism into the chromosome of P. putida KT2440, using genes from several different organisms. We found that the galactonate catabolic pathway alone (DgoKAD) supported slow growth of P. putida on galactose. Further integration of genes to convert galactose to galactonate and to optimize the transporter expression level resulted in a growth rate of 0.371 h −1 . Additionally, the best-performing strain was demonstrated to co-utilize galactose with glucose. Conclusions We have engineered P. putida to catabolize galactose, which will allow future engineered strains to convert more plant biomass carbon to products of interest. Further, by demonstrating co-utilization of glucose and galactose, continuous bioconversion processes for mixed sugar streams are now possible.

09 BIOMASS FUELS↗
DOE OSTI2020

Development of genetic tools for the thermophilic filamentous fungus Thermoascus aurantiacus

Abstract Background Fungal enzymes are vital for industrial biotechnology, including the conversion of plant biomass to biofuels and bio-based chemicals. In recent years, there is increasing interest in using enzymes from thermophilic fungi, which often have higher reaction rates and thermal tolerance compared to currently used fungal enzymes. The thermophilic filamentous fungus Thermoascus aurantiacus produces large amounts of highly thermostable plant cell wall-degrading enzymes. However, no genetic tools have yet been developed for this fungus, which prevents strain engineering efforts. The goal of this study was to develop strain engineering tools such as a transformation system, a CRISPR/Cas9 gene editing system and a sexual crossing protocol to improve the enzyme production. Results Here, we report Agrobacterium tumefaciens -mediated transformation (ATMT) of T. aurantiacus using the hph marker gene, conferring resistance to hygromycin B. The newly developed transformation protocol was optimized and used to integrate an expression cassette of the transcriptional xylanase regulator xlnR , which led to up to 500% increased xylanase activity. Furthermore, a CRISPR/Cas9 gene editing system was established in this fungus, and two different gRNAs were tested to delete the pyrG orthologue with 10% and 35% deletion efficiency, respectively. Lastly, a sexual crossing protocol was established using a hygromycin B- and a 5-fluoroorotic acid-resistant parent strain. Crossing and isolation of progeny on selective media were completed in a week. Conclusion The genetic tools developed for T. aurantiacus can now be used individually or in combination to further improve thermostable enzyme production by this fungus.

09 BIOMASS FUELS↗
DOE OSTI2021

Chimeric cellobiohydrolase I expression, activity, and biochemical properties in three oleaginous yeast

Consolidated bioprocessing using oleaginous yeast is a promising modality for the economic conversion of plant biomass to fuels and chemicals. However, yeast are not known to produce effective biomass degrading enzymes naturally and this trait is essential for efficient consolidated bioprocessing. We expressed a chimeric cellobiohydrolase I gene in three different oleaginous, industrially relevant yeast: Yarrowia lipolytica, Lipomyces starkeyi, and Saccharomyces cerevisiae to study the biochemical and catalytic properties and biomass deconstruction potential of these recombinant enzymes. Our results showed differences in glycosylation, surface charge, thermal and proteolytic stability, and efficacy of biomass digestion. L. starkeyi was shown to be an inferior active cellulase producer compared to both the Y. lipolytica and S. cerevisiae enzymes, whereas the cellulase expressed in S. cerevisiae displayed the lowest activity against dilute-acid-pretreated corn stover. Comparatively, the chimeric cellobiohydrolase I enzyme expressed in Y. lipolytica was found to have a lower extent of glycosylation, better protease stability, and higher activity against dilute-acid-pretreated corn stover.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Data for Optimizing Chemical-Free Pretreatment for Maximizing Oil/Lipid Recovery from Transgenic Bioenergy Crops and its Rapid Analysis Using Time Domain-NMR

Transgenic bioenergy crops have shown the potential to produce vegetative oil by accumulating energy-rich triacylglyceride molecules that can be converted into biofuels (biodiesel and biojet). These transgenic crops cater to improved biofuel yield by providing lipids along with cellulosic sugars. Efficient bioprocessing technologies are needed to utilize these transgenic plants to their maximum potential. To this end, this study investigates a low- and high-severity chemical-free hydrothermal pretreatment of transgenic oilcane 1566 bagasse with in situ lipids to maximize the recovery of lipids for biodiesel and fermentable sugars for ethanol with minimal inhibitor generation. Hydrothermal pretreatment at 170°C recovered ∼25% of total lipids in the pretreatment liquor, leaving the remainder in bagasse residue for hexane recovery post fermentation. The recovery of lipids in pretreatment liquor remained constant beyond 170°C. Along with lipids, ∼35% w/w and ∼50% w/w fermentable sugars were recovered post saccharification from bagasse pretreated at 170°C and 210°C for 20 min, respectively. Hydrothermal pretreatment at 170°C for 20 min provided the optimum conditions for maximum recovery of lipids and cellulosic sugars that resulted in enhanced biofuel yield per unit biomass. High severity pretreatment increased the generation of inhibitors beyond the tolerance of fermentation microorganisms. In addition, the application of time-domain proton NMR spectroscopy was extended to bioprocessing. NMR technology facilitated the analysis of total lipids, the composition of fatty acids, and the characterization of free and bound lipids in untreated and pretreated oilcane 1566 bagasse subsequent to each step of biomass to biofuel conversion.

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