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At least 55 records · Page 3
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DOE OSTI2022

Characterization of the Biomass Degrading Enzyme GuxA from Acidothermus cellulolyticus

Microbial conversion of biomass relies on a complex combination of enzyme systems promoting synergy to overcome biomass recalcitrance. Some thermophilic bacteria have been shown to exhibit particularly high levels of cellulolytic activity, making them of particular interest for biomass conversion. These bacteria use varying combinations of CAZymes that vary in complexity from a single catalytic domain to large multi-modular and multi-functional architectures to deconstruct biomass. Since the discovery of CelA from Caldicellulosiruptor bescii which was identified as one of the most active cellulase so far identified, the search for efficient multi-modular and multi-functional CAZymes has intensified. One of these candidates, GuxA (previously Acel_0615), was recently shown to exhibit synergy with other CAZymes in C. bescii, leading to a dramatic increase in growth on biomass when expressed in this host. GuxA is a multi-modular and multi-functional enzyme from Acidothermus cellulolyticus whose catalytic domains include a xylanase/endoglucanase GH12 and an exoglucanase GH6, representing a unique combination of these two glycoside hydrolase families in a single CAZyme. These attributes make GuxA of particular interest as a potential candidate for thermophilic industrial enzyme preparations. Here, we present a more complete characterization of GuxA to understand the mechanism of its activity and substrate specificity. In addition, we demonstrate that GuxA exhibits high levels of synergism with E1, a companion endoglucanase from A. cellulolyticus. We also present a crystal structure of one of the GuxA domains and dissect the structural features that might contribute to its thermotolerance.

09 BIOMASS FUELS↗
DOE OSTI2022

Biogas Biocatalysis

Biogas derived from anaerobic digestion of waste streams such as biorefinery wastewater, animal, agricultural, and municipal solid waste, offers a versatile renewable energy source. Total domestic methane potential from landfill material, animal manure, wastewater, and organic waste, combined with biogas generated from AD of lignocellulosic biomass, is estimated to offer >4 quadrillion Btu potential energy. This energy could displace nearly half of current domestic natural gas consumption in the electric power sector and all consumption in the transportation sector. However, despite this promise of this feedstock, its gaseous state prevents facile integration with extant transportation and industrial infrastructure. Microbial conversion of biogas to liquid fuel and chemical intermediates offers valorization potential. However, biogas biocatalysis is currently limited by poor substrate gas-to-liquid mass transfer, low conversion efficiencies, and incomplete biogas utilization. To this end, the Biogas Biocatalysis AOP aims to develop a carbon- and energy-efficient biogas bioconversion process via techno-economic-informed strain and fermentation engineering strategies. Efforts here will improve both process economics and sustainability via process-intensified, carbon-efficient biogas bioconversion to value-added platform molecules, enabling bolt-on deployment for valorization of biogas derived from standalone AD infrastructure.

BASIC BIOLOGICAL SCIENCES,BIOMASS FUELS↗
DOE OSTI2023

Enhanced microbial production of protocatechuate from engineered sorghum using an integrated feedstock-to-product conversion technology

Building a stronger bioeconomy requires production capabilities that are largely generated through microbial genetic engineering. Plant feedstocks can additionally be genetically engineered to generate desirable feedstock traits and provide precursors for direct microbial conversion into desired products. The oleaginous yeast Rhodosporidium toruloides is a promising organism for this type of conversion as it can grow on a wide range of deconstructed biomass and consume a variety of carbon sources. Here, we leveraged R. toruloides native p-coumaric acid consumption pathway to accumulate protocatechuate (PCA) from 4-hydroxybenzoate (4HBA) released from a sorghum feedstock line genetically engineered to overproduce 4HBA. We did so by generating and evaluating an R. toruloides strain that accumulates PCA, RSΔ12623. We then show that at two scales a cholinium lysinate pretreatment with enzymatic saccharification successfully extracts 95% of the 4HBA from the engineered sorghum biomass while producing deconstructed lignin that can be more efficiently depolymerized in a subsequent thermochemical reaction. We also demonstrate that strain RSΔ12623 can convert more than 95% of 4HBA to PCA while consuming >95% of the glucose and >80% of the xylose present in sorghum hydrolysates. Finally, to evaluate the scalability of such fermentations, we conducted the conversion of 4HBA to PCA in a 2 L bioreactor under controlled conditions. Importantly, this work demonstrates the potential of purposefully producing aromatic precursors in planta that can be liberated during biomass deconstruction for direct microbial conversion to desirable bioproducts.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Microbial assimilation of lignin-derived aromatic compounds and conversion to value-added products

Lignin is an abundant and sustainable source of aromatic compounds that can be converted to value-added products. However, lignin is underutilized, since depolymerization produces a complex mixture of aromatic compounds that is difficult to convert to a single product. Microbial conversion of mixed aromatic substrates provides a potential solution to this conversion challenge. Recent advances have expanded the range of lignin-derived aromatic substrates that can be assimilated and demonstrated efficient conversion via central metabolism to new potential products. Here, the development of additional non-model microbial hosts and genetic tools for these hosts have accelerated engineering efforts. However, yields with real depolymerized lignin are still low, and additional work will be required to achieve viable conversion processes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Evaluation of bacterial hosts for conversion of lignin-derived p-coumaric acid to 4-vinylphenol

Abstract Hydroxycinnamic acids such as p -coumaric acid (CA) are chemically linked to lignin in grassy biomass with fairly labile ester bonds and therefore represent a straightforward opportunity to extract and valorize lignin components. In this work, we investigated the enzymatic conversion of CA extracted from lignocellulose to 4-vinylphenol (4VP) by expressing a microbial phenolic acid decarboxylase in Corynebacterium glutamicum , Escherichia coli, and Bacillus subtilis . The performance of the recombinant strains was evaluated in response to the substrate concentration in rich medium or a lignin liquor and the addition of an organic overlay to perform a continuous product extraction in batch cultures. We found that using undecanol as an overlay enhanced the 4VP titers under high substrate concentrations, while extracting > 97% of the product from the aqueous phase. C. glutamicum showed the highest tolerance to CA and resulted in the accumulation of up to 187 g/L of 4VP from pure CA in the overlay with a 90% yield when using rich media, or 17 g/L of 4VP with a 73% yield from CA extracted from lignin. These results indicate that C. glutamicum is a suitable host for the high-level production of 4VP and that further bioprocess engineering strategies should be explored to optimize the production, extraction, and purification of 4VP from lignin with this organism.

4-hydroxystyrene↗
DOE OSTI2020

Emerging Strategies for Modifying Lignin Chemistry to Enhance Biological Lignin Valorization

Biological lignin valorization represents a promising approach contributing to sustainable and economic biorefineries. Here, the low level of valuable lignin–derived products remains a major challenge hindering the implementation of microbial lignin conversion. Lignin's properties play a significant role in determining the efficiency of lignin bioconversion. To date, despite significant progress in the development of biomass pretreatment, lignin fractionation, and fermentation over the last few decades, little efforts have gone into identifying the ideal lignin substrates for an efficient microbial metabolism. In this Minireview, emerging and state–of–the–art strategies for biomass pretreatment and lignin fractionation are summarized to elaborate their roles in modifying lignin structure for bioconversion. Fermentation strategies aimed at enhancing lignin depolymerization for microbial utilization are systematically reviewed as well. With an improved understanding of the ideal lignin structure elucidated by comprehensive metabolic pathways and/or big data analysis, modifying lignin chemistry could be more directional and effective. Ultimately, together with the progress of fermentation process optimization, biological lignin valorization will become more competitive in biorefineries.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Multiphase Reacting Flow Simulations and Optimization of Commercial-Scale Aerobic Bioreactors

Aerobic bioreactors are widely used in the synthesis of value-added products in pharmaceutical and biotechnology industries, wherein microbial bioreactions aid in the conversion of sugars to macromolecules. These reactors are also being actively investigated as a cost-effective pathway for the production of low-value commodities such as biofuels and animal feed. Reactor scale-up is one of the important challenges when designing these systems. Gas and liquid phase transport, mass-transfer, and mixing physics at large length scales can significantly affect microbial conversion rates, particularly when the microbial reaction requires a narrow set of conditions. These phenomena are difficult to study in small-scale bench-top reactors that are typically well-mixed. Predictive computational fluid dynamics (CFD) based simulations can therefore aid in the design and optimization of these reactors. This work presents multiphase Euler-Euler CFD simulations of commercial-scale (~ 500 m3) aerobic bioreactors. Our mathematical model treats the gas and liquid as interpenetrating phases. This approach reduces the computational complexity of tracking individual gas bubbles that are several orders of magnitude smaller than reactor dimensions. We solve the Reynolds averaged Navier-Stokes (RANS) multiphase equations that account for phase and chemical species transport, interphase mass and momentum transfer and uses a phenomenological model for oxygen uptake by microbes. We use a customized solver derived from open-source CFD toolbox, OpenFOAM, to perform these simulations, which has been validated against small-scale reactors in our previous work. This work examines the performance of three different reactor designs, viz. bubble column reactor, airlift reactor with an internal draft tube, and a stirred-tank reactor with Rushton impellers. Reactor oxygen mass-transfer coefficient, gas hold-up, and oxygen distribution are critically analyzed among reactors, and sensitivity studies pertaining to gas flow rates and reactor geometry will be presented.

09 BIOMASS FUELS↗
DOE OSTI2022

Fermentative conversion of unpretreated plant biomass: A thermophilic threshold for indigenous microbial growth

Here, naturally occurring, microbial contaminants were found in plant biomasses from common bioenergy crops and agricultural wastes. Unexpectedly, indigenous thermophilic microbes were abundant, raising the question of whether they impact thermophilic consolidated bioprocessing fermentations that convert biomass directly into useful bioproducts. Candidate microbial platforms for biomass conversion, Acetivibrio thermocellus (basionym Clostridium thermocellum; T opt 60 °C) and Caldicellulosiruptor bescii (T opt 78 °C), each degraded a wide variety of plant biomasses, but only A. thermocellus was significantly affected by the presence of indigenous microbial populations harbored by the biomass. Indigenous microbial growth was eliminated at ≥75 °C, conditions where C. bescii thrives, but where A. thermocellus cannot survive. Therefore, 75 °C is the thermophilic threshold to avoid sterilizing pre-treatments on the biomass that prevents native microbes from competing with engineered microbes and forming undesirable by-products. Thermophiles that naturally grow at and above 75 °C offer specific advantages as platform microorganisms for biomass conversion into fuels and chemicals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

NREL's Biochemical Conversion Pilot Plant Capabilities

NREL's biochemical conversion plant is a flexible pilot plant for testing a wide range of bio-based technologies for producing renewable fuels and byproducts. The biochemical conversion pilot plant is housed in the Integrated Biorefinery Research Facility located on the National Renewable Energy Laboratory's (NREL's) main campus. The facility provides industry partners the opportunity to test and develop their own biorefining technology using NREL equipment or rental equipment that can be brought into the plant. The pilot plant can process materials up to one dry ton per day in an integrated fashion using a variety of operating modes including batch, fed-batch, and continuous processing.

biochemical↗
DOE OSTI2023

Multiplexed fitness profiling by RB-TnSeq elucidates pathways for lignin-related aromatic catabolism in Sphingobium sp. SYK-6

Bioconversion of lignin-related aromatic compounds relies on robust catabolic pathways in microbes. Sphingobium sp. SYK-6 (SYK-6) is a well-characterized aromatic catabolic organism that has served as a model for microbial lignin conversion, and its utility as a biocatalyst could potentially be further improved by genome-wide metabolic analyses. To this end, we generate a randomly barcoded transposon insertion mutant (RB-TnSeq) library to study gene function in SYK-6. The library is enriched under dozens of enrichment conditions to quantify gene fitness. Several known aromatic catabolic pathways are confirmed, and RB-TnSeq affords additional detail on the genome-wide effects of each enrichment condition. Selected genes are further examined in SYK-6 or Pseudomonas putida KT2440, leading to the identification of new gene functions. The findings from this study further elucidate the metabolism of SYK-6, while also providing targets for future metabolic engineering in this organism or other hosts for the biological valorization of lignin.

09 BIOMASS FUELS↗
DOE OSTI2020

Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440

Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic–catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could pote

biological funneling↗
DOE OSTI2023

Gas-Liquid Flow Modeling for Renewable Fuels Production

Aerobic/anaerobic and gas fermentation pathways have emerged as promising new technologies for the generation of renewable fuels/chemicals from biomass derived sugars, and mixtures of greenhouse/energy rich gas streams (CO2/CH4/H2/CO) via microbial action. Example pathways include sugars-to-ethanol conversion, biomethanation (CO2/H2 to CH4), biogas upgrading, CO fermentation and wet-waste conversion. Gas and liquid phase transport, mass-transfer, and mixing physics at large length scales can significantly affect microbial conversion rates, particularly when the microbial reaction requires a narrow set of conditions. These phenomena are difficult to study in small-scale bench-top reactors that are typically well-mixed. Predictive computational fluid dynamics (CFD) based simulations can therefore aid in the scale-up, design and optimization of these reactors. This work presents multiphase Euler-Euler CFD simulations of at-scale (~500 m3) bioreactors. Our mathematical model treats the gas and liquid as interpenetrating phases. This approach reduces the computational complexity of tracking individual gas bubbles that are several orders of magnitude smaller than reactor dimensions. We solve the Reynolds averaged Navier-Stokes (RANS) multiphase equations that account for phase and chemical species transport, interphase mass and momentum transfer and uses a phenomenological model for gas uptake by microbes. We use a customized solver derived from open-source CFD toolbox, OpenFOAM [1], to perform these simulations, which has been validated against small-scale reactors in our previous work [2]. There is currently a knowledge-gap regarding bubble-size distributions when using gas mixtures with vastly different properties, which can have a significant impact overall mass-transfer. For example, hydrogen bubbles are more buoyant compared to other relatively heavier gases (CO2/CH4/CO), resulting in a large distribution of residence times and bubble sizes. This work therefore develops a deeper understanding of bubble dynamics and interphase mass transfer in such heterogenous gas mixtures through well-resolved computational models. We use a population balance model (PBM) for bubble-size-distribution modeling that is validated against small-scale experiments in our solver with an uncertainty quantification study for bubble coalescence and break-up model parameters. Results pertaining to multiple simulations of gas-fermentation reactors are presented where gas mixtures with varying compositions of CO2/CH4/CO/H2 are imposed at the sparger boundaries. The spatio-temporal variations in bubble-size distribution and mass transfer coefficient are analyzed for varying superficial velocities and gas-compositions for varying sizes of bubble-column and airlift reactors. This work will also examine the performance of different reactor designs, viz. bubble column reactor, airlift reactor with an internal draft tube, and a stirred-tank reactor with Rushton impellers. Reactor mass-transfer coefficient, gas hold-up, and dissolved gas distribution are critically analyzed among reactors, and sensitivity studies pertaining to gas flow rates and reactor geometry will be presented. [1] Weller, H., Tabor, G., Jasak, H. and Fureby, C., A tensorial approach to computational continuum mechanics using object-oriented techniques, Computers in physics, 12, 6, 620--631, 1998. [2] Rahimi, M., Sitaraman, H., Humbird, D. and Stickel, J., Computational fluid dynamics study of full-scale aerobic bioreactors: Evaluation of gas-liquid mass transfer, oxygen uptake, and dynamic oxygen distribution, Chemical Engineering Research and Design, 139: 283-295.

BIOMASS FUELS↗
DOE OSTI2024

Necromass responses to warming: A faster microbial turnover in favor of soil carbon stabilisation

Microbial byproducts and residues (hereafter ‘necromass’) potentially play the most critical role in soil organic carbon (SOC) sequestration. However, little is known about the influence of climate warming on necromass accumulation in the agroecosystem and the underlying mechanisms associated with microbial life strategies. Here, in order to address these knowledge gaps, we used amino sugars as biomarkers of microbial necromass, and investigated their variation through an 8-year trial in an agroecosystem with two warming levels (+1.6 and + 3.2 °C) compared to ambient temperature. The results showed that the lower warming level had no impact on total microbial necromass carbon. Conversely, warming the soil 3.2 °C above ambient increased total microbial necromass by 17 % and its contribution to SOC by 21.3 %, mainly by increasing fungal necromass (+19.8 %), whereas +3.2 °C warming had no impact on bacterial necromass. At the phylum level, compared with the ambient control, +3.2 °C warming induced an increase in the abundance of Proteobacteria and a decrease in both Acidobacteria and Actinobacteria, whereas in the fungal community, Ascomycota increased and Mortierellomycota decreased. This indicates that r-strategists outcompete K-strategists in warmer climates, which led to increased microbial necromass production and accumulation, as supported by the positive correlation between r-strategists and microbial necromass. Stronger microbial competition for resources also resulted in a higher biomass turnover rate, greater cell death, and greater production of microbial necromass. This was supported by the lower bacterial and fungal network complexity and trophic links under warming conditions. In addition, the necromass generated from accelerated microbial turnover further offsets warming-induced deceases in microbial biomass. Consequently, bulk SOC did not change, despite microbial necromass having a much greater response to warming than the soil C pool. Therefore, future climate warming may influence the composition and persistence of SOC during microbial degradation.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

CRADA Number NFE-19-07851 with Electro-Active Technologies Inc. (CRADA Final Report)

Cooperative Research and Development Agreement (CRADA) NFE-19-07851 between Oak Ridge National Laboratory (ORNL) and Electro-Active Technologies Inc. focused on developing a modular stack system to produce renewable hydrogen from food waste and renewable electricity. Developing technologies that can produce renewable, affordable hydrogen is key to enabling wider adoption of fuel cell technologies. Electro-Active has developed a microbial electrolysis process that leverages microbes growing on an anode to convert waste into electrons and protons, which are recombined and reacted with the help of an additional applied voltage to generate pure hydrogen at a cathode. The work done under the CRADA focused on developing a deeper understanding of the microbial community biocatalyst and how feedstock and process conditions affect structure and function in a prototype system in order to increase and sustain performance. Analysis indicates that developing a robust microbial community for conversion of food waste feedstocks at high rates within a microbial electrolysis system is possible, as well as maintaining this optimal microbial biocatalyst in larger systems. However, further work is needed to maintain performance in the system as it is scaled up to enable commercial deployment of the technology.

08 HYDROGEN↗
DOE OSTI2021

Activity-based, genome-resolved metagenomics uncovers key populations and pathways involved in subsurface conversions of coal to methane

Microbial metabolisms and interactions that facilitate subsurface conversions of recalcitrant carbon to methane are poorly understood. We deployed an in situ enrichment device in a subsurface coal seam in the Powder River Basin (PRB), USA, and used BONCAT-FACS-Metagenomics to identify translationally active populations involved in methane generation from a variety of coal-derived aromatic hydrocarbons. From the active fraction, high-quality metagenome-assembled genomes (MAGs) were recovered for the acetoclastic methanogen, Methanothrix paradoxum, and a novel member of the Chlorobi with the potential to generate acetate via the Pta-Ack pathway. Members of the Bacteroides and Geobacter also encoded Pta-Ack and together, all four populations had the putative ability to degrade ethylbenzene, phenylphosphate, phenylethanol, toluene, xylene, and phenol. Metabolic reconstructions, gene analyses, and environmental parameters also indicated that redox fluctuations likely promote facultative energy metabolisms in the coal seam. The active "Chlorobi PRB" MAG encoded enzymes for fermentation, nitrate reduction, and multiple oxygenases with varying binding affinities for oxygen. "M. paradoxum PRB" encoded an extradiol dioxygenase for aerobic phenylacetate degradation, which was also present in previously published Methanothrix genomes. Finaly, these observations outline underlying processes for bio-methane from subbituminous coal by translationally active populations and demonstrate activity-based metagenomics as a powerful strategy in next generation physiology to understand ecologically relevant microbial populations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Synthetic Microbial Consortium for Biological Breakdown and Conversion of Lignin

The plant polymer lignin is the most abundant renewable source of aromatics on the planet and conversion of it to valuable fuels and chemicals is critical to the economic viability of a lignocellulosic biofuels industry and to meeting the DOE’s 2022 goal of $\$2.50$/gallon mean biofuel selling price. Presently, there is no efficient way of converting lignin into valuable commodities. Current biological approaches require mixtures of expensive ligninolytic enzymes and engineered microbes. This project was aimed at circumventing these problems by discovering commensal relationships among fungi and bacteria involved in biological lignin utilization and using this knowledge to engineer microbial communities capable of converting lignin into renewable fuels and chemicals. Essentially, we aimed to learn from, mimic and improve on nature. We discovered fungi that synergistically work together to degrade lignin, engineered fungal systems to increase expression of the required enzymes and engineered organisms to produce products such as biodegradable plastics precursors.

09 BIOMASS FUELS↗