Engineering topics

Beckham, Gregg T

Publications and source records attributed to Beckham, Gregg T.

14 records

DOE OSTI2020

Exoproteomics Reveals Outer Membrane Vesicle Mediated Catabolism of Lignin-Derived Aromatics by P. putida KT2440

Lignin is an abundant component of plant cell walls which is heterogeneous and highly resistant to degradation. Biological funneling of lignin via microbes represents an efficient approach for reducing chemical heterogeneity and generating molecules which can be valorized into performance-advantaged materials. Pseudomonas putida KT2440 (P. putida) catabolizes lignin-derived aromatic monomers rapidly and efficiently via the b-ketoadipate (bKA) pathway and our data suggest P. putida may depolymerize HMW lignin as well [1]. The mechanism by which HMW lignin is depolymerized by bacteria remains unknown. Intriguingly, we found that P. putida secretes outer membrane vesicles (OMVs) of two distinct sizes in the presence of lignin. Here, we describe the temporal proteome of OMVs and the vesicle-free secretome (VFS) in the presence or absence of lignin. Enzymes involved in aromatic catabolism via the bKA pathway are temporally enriched into OMVs in the presence of lignin. Isolated OMVs enhance growth of P. putida mutants unable to grow on model lignin-derived aromatics, and turnover protocatechuic acid in vitro. Together, this work leads us to hypothesize mechanisms by which OMVs contribute to extracellular aromatic catabolism by P. putida. Ongoing and future work seeks to further understand this phenomena in the context of both fundamental an applied lignin conversion research areas.

09 BIOMASS FUELS↗

DOE OSTI2019

Production of a Biofuel Intermediate from Biomass Sugars by Clostridium tyrobutyricum

Butyric acid is a valuable chemical that can be fermentatively produced in Clostridium tyrobutyricum and catalytically upgraded to drop in biofuels. Despite considerable study of glucose fermentation in C. tyrobutyricum, information about glucose and xylose co-fermentation to produce butyric acid is limited. In this work, fed-batch fermentations of corn stover hydrolysate at pH 5.0 demonstrated the co-fermentation of glucose and xylose. However, the fermentation stalled at approximately 48 hours after reaching inhibitory levels of butyric acid (18 g/L). When the fermentation pH was increased to 6.0, higher butyric acid titers (50 g/L) were achieved but the fermentation again stalled after 98 hours due to product inhibition. To demonstrate the potential of a continuous co-fermentation process, in situ product recovery via membrane-based liquid-liquid extraction (pertraction) was applied to remove butyric acid. The extracted acids were then recovered from the organic solvent via back extraction into an alkaline solution, producing a high purity product. Results from pertractive fermentations of real corn stover hydrolysate are shown below.

09 BIOMASS FUELS↗

DOE OSTI2019

Molecular Interactions at the Cellulose-Lignin Interface Explored via Molecular Simulation

Lignin and cellulose are found ubiquitously in plant cell walls, making them two of the most abundant biopolymers on Earth. In spite of their ubiquity, comparatively little is known quantitatively about their interactions within native biomass, and by extension how their interaction may play a role in biomaterials. The possible binding faces for lignin on crystalline cellulose I-ss and the heterogeneous chemistries found in native lignin typically preclude the isolation of specific interactions for quantitative comparison in the laboratory. In this study, we use molecular simulation to examine individual combinations of binding face and lignin chemistry to quantify lignin-cellulose interactions, including contributions from both specific hydrogen bonds and nonspecific van der Waals interactions. For all lignin chemistries simulated, the hydrophobic 200 face is the preferred for cellulose interactions. For hydrophilic crystalline faces, lignin is found to bind most effectively to the 110 face. The affinity between lignin and cellulose increases as additional lignin methoxy groups are added. These groups increase the lignin-cellulose contact area, increasing the binding affinity. These trends are extended to larger lignin polymers, where the relationships between lignin polymer size and binding affinity are quantified through biased simulation. In this case, the binding surface area between the lignin polymer and the cellulose surface dictates the interaction strength.

09 BIOMASS FUELS↗

DOE OSTI2019

The Dissociation Mechanism of Processive Cellulases

Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.

09 BIOMASS FUELS↗

DOE OSTI2019

Computational Insights into Fuels and Chemicals Extraction from Microbial Biorefineries

Over the past two decades, substantial investments have been made in engineering microorganisms to produce specific fuels and chemicals as part of the global bioeconomy. Many target molecules accumulate intracellularly, and a challenge is how to effectively extract the product from the cells without needing to destroy them due to the barrier imposed by the cell membrane. For some hydrophobic compounds, an organic overlay is an effective strategy for nondestructive product extraction, although the relationship between functional groups on the product and the rate of extraction are not well understood. Through both biased and unbiased molecular dynamics simulations for a range of fatty acyl compounds and terpenoids, we directly compute permeability coefficients for different steps of the extraction process. Via comparative analysis between the calculated permeability coefficients and observed interactions between the compounds and the membrane, we determine how the rate limiting steps vary depending on product chemistry. For instance, fatty aldehydes are found to transfer very rapidly across the membrane bilayer relative to alcohols, although their comparable rate of extraction into the organic phase makes them equally effective at extraction from the cell. In assessing the terpenoids, it is found that in general a modestly hydrophilic product improves desorption rates into an organic phase sufficiently to make up for their lower bilayer crossing rate. With this new insight, we can more effectively engineer microorganisms towards the production of these modestly hydrophilic fuel precursors or chemicals.

09 BIOMASS FUELS↗

DOE OSTI2019

Methyl Ketone Condensation over Tailored Metal Oxides

Methyl ketones represent promising oxygenate intermediates that can be derived from lignocellulosic biomass via numerous conversion routes and subsequently coupled to produce precursors for renewable diesel and aviation fuels. Basic metal oxides are known to promote ketone coupling via condensation reactions, with magnesium oxide (MgO) providing an ideal catalytic system for evaluating the reaction mechanism and role of water on observed activity. Recent studies demonstrate that the MgO(111) surface facet can be promoted to enhance activity for base catalyzed reactions; however, to our knowledge, there is a dearth of information regarding the impact of MgO(111) faceting and surface termination under reaction conditions for ketone coupling. Therefore, this talk will discuss our recent investigation into the catalytic coupling of methyl ketones over MgO(100) and MgO(111) catalysts. Results will be presented that address the synthesis and detailed characterization of MgO(111) nanosheets, as well as their activity and selectivity for ketone coupling when compared to MgO(100). The impact of water on MgO surface termination will be discussed, as well as a proposed reaction mechanism for ketone condensation over clean and hydroxyl terminated surfaces for both facets. Findings will include outcomes from experimental studies, computational modeling, and advanced surface characterization by neutron scattering. Collectively, this work provides further insight into the role of tailored metal oxide catalysts for this important class of biomass conversion chemistry.

09 BIOMASS FUELS↗

DOE OSTI2019

Dissociation Mechanism of Processive Cellulases Explored Through Molecular Simulation

Cellulose is the most abundant biopolymer on the planet, with significant potential as a feedstock for bioderived products. In nature, fungi and bacteria secrete cellulases to hydrolyze cellulose into smaller carbohydrates for incorporation into their own metabolic processes. For effective biomass utilization at industrial scale, these cellulases are coupled together to produce a clean sugar stream. One such cellulase is Cel7A from Trichoderma reesei, which is used extensively in biotechnological applications. However, dissociation of Cel7A from a bound cellulose strand is rate-limiting, reducing catalytic efficiency and yields within industrial applications. To explore this unknown dissociation mechanism and devise potential enzyme engineering strategies to mitigate slow dissociation, we conduct Hamiltonian replica exchange molecular dynamics simulations. Two postulated dissociation mechanisms were tested ('dethreading' of the bound cellulose strand from the binding tunnel and 'clamshell'-like opening of the binding tunnel and recrystallization of the bound cellulose). For each mechanism, free energy profiles were determined together with estimated diffusion coefficients. The kinetics for both mechanisms were then estimated and compared against experimentally measured dissociation rates, which suggest that the dethreading mechanism ought to be preferred. These findings are further supported by dissociation rates measured on engineered mutants incapable of undergoing the clamshell mechanism.

09 BIOMASS FUELS↗

DOE OSTI2019

Binding Affinity Dependence of Lignin-Cellulose Complexes on Cellulose Faces and Lignin Composition

The interaction between lignin and cellulose is essential to the structural integrity of plant cell walls, and these strong interactions are key to biomass recalcitrance. Previous studies have indicated that the hydrophobic face of cellulose is the primary face of interaction between cellulose and lignin. However, due to the structural inhomogeneity of lignin and the small lengthscales that would need to be probed, the relative binding affinities for lignin to different cellulose faces has not been previously determined. Through the creation of idealized infinite cellulose faces for the (200), (110), (1-10) and, (010) planes, we determine through computation the relative binding affinities for selected lignin compounds to these different faces of cellulose. The preference for individual lignin monomers to bind to hydrophobic faces of cellulose is found to be remarkably weak. We develop a mathematical model to determine the structural features of lignin have the strongest relationship with the computed binding affinities for large lignin polymers.

09 BIOMASS FUELS↗

DOE OSTI2019

Membrane Binding of Soluble Enzymes, Explored Through Simulation of Bacterial P450s

Members of the P450 enzyme family catalyze the oxidation of many different, but primarily lipophillic, substrates. In eukaryotic P450s, a membrane-anchored N-terminal helix tethers the enzyme to the membrane. However, in bacterial P450s, no N-terminal helix is present in the sequence, and the proteins are found to be soluble and amenable to solution experiments. Through unbiased classical molecular dynamics simulations, we demonstrate that these soluble enzymes bind to lipid membranes in the same orientation as tethered P450s. We postulate that the observed membrane binding improves the turnover of the enzyme by co-locating the enzyme at regions of high substrate concentration. Through mathematical modeling, we determine that the small cellular volume in prokaryotes is the primary reason that no N-terminal helix is found in these enzymes, as fewer enzymes are mistargeted into the small cytoplasmic space.

09 BIOMASS FUELS↗

DOE OSTI2019

Reaction: Proteins from Chemocatalysis; It’s What’s for Dinner

Supporting the agricultural needs of a rising global population is becoming increasingly difficult. In particular, supplying the animal-based protein demand is causing significant environmental impacts, including contributing to 12% of global greenhouse gas emissions and 30% of human-induced loss of terrestrial biodiversity. And the problem is getting worse. Even conservative estimates suggest that the demand for animal-based protein will double by 2050. Aside from systemic changes to the diets of billions, alternative protein sources must be developed to mitigate these issues. From an engineering perspective, the use of livestock as a 'process' to convert grown crops to protein is wildly inefficient and results in widespread environmental damage. Technological innovation is thus required to produce food-grade protein with lower environmental impacts.

09 BIOMASS FUELS↗

DOE OSTI2018

Lignin Valorization: Emerging Approaches

Lignin, an aromatic biopolymer found in plant cell walls, is a key component of lignocellulosic biomass and generally utilized for heat and power. However, lignin's chemical composition makes it an attractive source for biological and catalytic conversion to fuels and chemicals. Bringing together experts from biology, catalysis, engineering, analytical chemistry, and techno-economic/life-cycle analysis, Lignin Valorization presents a comprehensive, interdisciplinary picture of how lignocellulosic biorefineries could potentially employ lignin valorization technologies. Chapters will specifically focus on the production of fuels and chemicals from lignin and topics covered include (i) methods for isolating lignin in the context of the lignocellulosic biorefinery, (ii) thermal, chemo-catalytic, and biological methods for lignin depolymerization, (iii) chemo-catalytic and biological methods for upgrading lignin, (iv) characterization of lignin, and (v) techno-economic and life-cycle analysis of integrated processes to utilize lignin in an integrated biorefinery. The book provides the latest breakthroughs and challenges in upgrading lignin to fuels and chemicals for graduate students and researchers in academia, governmental laboratories, and industry interested in biomass conversion.

09 BIOMASS FUELS↗

DOE OSTI2018

Chapter 1: A Brief Introduction to Lignin Structure

Lignin is an alkyl-aromatic polymer found in the cell walls of terrestrial plants. Lignin provides structure and rigidity to plants, is a natural, highly effective barrier against microbial attack, and enables water and nutrient transport through plant tissues. Depending on the plant species, the constituents of lignin can vary considerably, leading to substantial diversity in lignin chemistry and structure. Despite nearly a century of research and development attempting to convert lignin into valuable products, lignin in most current and planned biorefinery contexts remains underutilized, most often being burned to generate heat and power. However, the drive towards effective lignin valorization processes has witnessed a significant resurgence in the past decade, catalyzed by advances in improved understanding of lignin chemistry, structure, and plasticity in parallel with new catalytic and biological approaches to valorize this important, prevalent biopolymer. As a preface to the subsequent chapters in this book, this chapter briefly highlights the known aspects of lignin structure.

09 BIOMASS FUELS↗

DOE OSTI2017

Renewable Acrylonitrile Production

Acrylonitrile (ACN) is a petroleum-derived compound used in resins, polymers, acrylics, and carbon fiber. We present a process for renewable ACN production using 3-hydroxypropionic acid (3-HP), which can be produced microbially from sugars. The process achieves ACN molar yields exceeding 90% from ethyl 3-hydroxypropanoate (ethyl 3-HP) via dehydration and nitrilation with ammonia over an inexpensive titanium dioxide solid acid catalyst. We further describe an integrated process modeled at scale that is based on this chemistry and achieves near-quantitative ACN yields (98 +/- 2%) from ethyl acrylate. This endothermic approach eliminates runaway reaction hazards and achieves higher yields than the standard propylene ammoxidation process. Avoidance of hydrogen cyanide as a by-product also improves process safety and mitigates product handling requirements.

09 BIOMASS FUELS↗

DOE OSTI2017

Hydroxide catalysts for lignin depolymerization

Solid base catalysts and their use for the base-catalyzed depolymerization (BCD) of lignin to compounds such as aromatics are presented herein. Exemplary catalysts include layered double hydroxides (LDHs) as recyclable, heterogeneous catalysts for BCD of lignin.

09 BIOMASS FUELS↗