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At least 37 records · Page 2

Phylogenomics and genetic analysis of solvent-producing Clostridium species

Abstract The genus Clostridium is a large and diverse group within the Bacillota (formerly Firmicutes), whose members can encode useful complex traits such as solvent production, gas-fermentation, and lignocellulose breakdown. We describe 270 genome sequences of solventogenic clostridia from a comprehensive industrial strain collection assembled by Professor David Jones that includes 194 C. beijerinckii , 57 C. saccharobutylicum , 4 C. saccharoperbutylacetonicum , 5 C. butyricum , 7 C. acetobutylicum , and 3 C. tetanomorphum genomes. We report methods, analyses and characterization for phylogeny, key attributes, core biosynthetic genes, secondary metabolites, plasmids, prophage/CRISPR diversity, cellulosomes and quorum sensing for the 6 species. The expanded genomic data described here will facilitate engineering of solvent-producing clostridia as well as non-model microorganisms with innately desirable traits. Sequences could be applied in conventional platform biocatalysts such as yeast or Escherichia coli for enhanced chemical production. Recently, gene sequences from this collection were used to engineer Clostridium autoethanogenum , a gas-fermenting autotrophic acetogen, for continuous acetone or isopropanol production, as well as butanol, butanoic acid, hexanol and hexanoic acid production.

59 BASIC BIOLOGICAL SCIENCES↗

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↗

Phenotypic and genomic characterization of Methanothermobacter wolfeii strain BSEL, a CO 2 -capturing archaeon with minimal nutrient requirements

A new variant of Methanothermobacter wolfeii was isolated from an anaerobic digester using enrichment cultivation in anaerobic conditions. Here, the new isolate was taxonomically identified via 16S rRNA gene sequencing and tagged as M. wolfeii BSEL. The whole genome of the new variant was sequenced and de novo assembled. Genomic variations between the BSEL strain and the type strain were discovered, suggesting evolutionary adaptations of the BSEL strain that conferred advantages while growing under a low concentration of nutrients. M. wolfeii BSEL displayed the highest specific growth rate ever reported for the wolfeii species (0.27 ± 0.03 h –1 ) using carbon dioxide (CO 2 ) as unique carbon source and hydrogen (H 2 ) as electron donor. M. wolfeii BSEL grew at this rate in an environment with ammonium (NH 4 + ) as sole nitrogen source. The minerals content required to cultivate the BSEL strain was relatively low and resembled the ionic background of tap water without mineral supplements. Optimum growth rate for the new isolate was observed at 64°C and pH 8.3. In this work, it was shown that wastewater from a wastewater treatment facility can be used as a low-cost alternative medium to cultivate M. wolfeii BSEL. Continuous gas fermentation fed with a synthetic biogas mimic along with H 2 in a bubble column bioreactor using M. wolfeii BSEL as biocatalyst resulted in a CO 2 conversion efficiency of 97% and a final methane (CH 4 ) titer of 98.5%v, demonstrating the ability of the new strain for upgrading biogas to renewable natural gas.

09 BIOMASS FUELS↗

Multiphysics Computational-Fluid-Dynamics (CFD) for Design and Scale-Up of Gas Bioreactors that Utilize CO2

Gas fermentation technology offers sustainable and high carbon-efficiency routes to fuels and chemicals. However, efficiently delivering low-solubility substrate gases to production-scale bioreactors is a significant technical challenge that could limit productivity. We perform fundamental and applied multiphysics computational fluid dynamics (CFD) research targeting specific challenges of gas delivery in fermentation systems that utilize varied gas-compositions, for e.g. CO2/CO/CH4/H2 substrates: bubble dynamics, coupled mass-transfer and reaction, that can lead to optimal reactor designs at scale. This work will support BETO goals towards greenhouse gas reduction, derisking technologies toward commercialization/deployment and sustainable-aviation-fuel synthesis targets.

BIOMASS FUELS↗

Production of Bioproducts from Electrochemically-Generated C1 Intermediates

The overarching objective of the project was to develop an integrated process that converts carbon dioxide (CO 2 ) into the platform chemical isopropanol (IPA) using a combination of two key technologies: CO 2 electrolysis and gas fermentation. The final goal was to demonstrate the full process at bench scale with a minimum carbon conversion efficiency of 37% and to evaluate the economics and environmental impact of scaling up the electrochemical-biochemical process.

30 DIRECT ENERGY CONVERSION↗

Advances in systems metabolic engineering of autotrophic carbon oxide-fixing biocatalysts towards a circular economy

High levels of anthropogenic CO 2 emissions are driving the warming of global climate. If this pattern of increasing emissions does not change, it will cause further climate change with severe consequences for the human population. On top of this, the increasing accumulation of solid waste within the linear economy model is threatening global biosustainability. The magnitude of these challenges requires several approaches to capture and utilize waste carbon and establish a circular economy. Microbial gas fermentation presents an exciting opportunity to capture carbon oxides from gaseous and solid waste streams with high feedstock flexibility and selectivity. Here we discuss available microbial systems and review in detail the metabolism of both anaerobic acetogens and aerobic hydrogenotrophs and their ability to utilize C1 waste feedstocks. More specifically, we provide an overview of the systems-level understanding of metabolism, key metabolic pathways, scale-up opportunities and commercial successes, and the most recent technological advances in strain and process engineering. Finally, we also discuss in detail the gaps and opportunities to advance the understanding of these autotrophic biocatalysts for the efficient and economically viable production of bioproducts from recycled carbon.

59 BASIC BIOLOGICAL SCIENCES↗

Transcriptional control of Clostridium autoethanogenum using CRISPRi

Gas fermentation by Clostridium autoethanogenum is a commercial process for the sustainable biomanufacturing of fuels and valuable chemicals using abundant, low-cost C1 feedstocks (CO and CO 2 ) from sources such as inedible biomass, unsorted and nonrecyclable municipal solid waste, and industrial emissions. Efforts toward pathway engineering and elucidation of gene function in this microbe have been limited by a lack of genetic tools to control gene expression and arduous genome engineering methods. To increase the pace of progress, here we developed an inducible CRISPR interference (CRISPRi) system for C. autoethanogenum and applied that system toward transcriptional repression of genes with ostensibly crucial functions in metabolism.

59 BASIC BIOLOGICAL SCIENCES↗

Efficient Hydrogen Delivery for Microbial Electrosynthesis via 3D-Printed Cathodes

The efficient delivery of electrochemically in situ produced H 2 can be a key advantage of microbial electrosynthesis over traditional gas fermentation. However, the technical details of how to supply large amounts of electric current per volume in a biocompatible manner remain unresolved. Here, we explored for the first time the flexibility of complex 3D-printed custom electrodes to fine tune H 2 delivery during microbial electrosynthesis. Using a model system for H 2 -mediated electromethanogenesis comprised of 3D fabricated carbon aerogel cathodes plated with nickel-molybdenum and Methanococcus maripaludis, we showed that novel 3D-printed cathodes facilitated sustained and efficient electromethanogenesis from electricity and CO 2 at an unprecedented volumetric production rate of 2.2 L CH4 /L catholyte /day and at a coulombic efficiency of 99%. Importantly, our experiments revealed that the efficiency of this process strongly depends on the current density. At identical total current supplied, larger surface area cathodes enabled higher methane production and minimized escape of H 2 . Specifically, low current density (<1 mA/cm 2 ) enabled by high surface area cathodes was found to be critical for fast start-up times of the microbial culture, stable steady state performance, and high coulombic efficiencies. Our data demonstrate that 3D-printing of electrodes presents a promising design tool to mitigate effects of bubble formation and local pH gradients within the boundary layer and, thus, resolve key critical limitations for in situ electron delivery in microbial electrosynthesis.

03 NATURAL GAS↗

Computational Analysis of Different Sparging Systems and their Influence in the Fluid-Dynamic Behavior of Bubble Column Reactors

Bubble column bioreactors are being actively considered for gas fermentation applications, specifically for CO2 utilization, and sugars to fuels conversion. Their main advantages include good mass transfer without any moving parts and low-cost of operation and maintenance. However, the design and scale-up of such reactors is challenging specifically for carbon capture applications where a mixture of gases (e.g. CO2/CO/H2) with variable solubilities is used. The overall performance of scaled-up bioreactors (e.g., mass transfer rate) is largely affected by gas holdup, bubble size distribution (BSD), and multiphase hydrodynamics. We investigate the effect of gas sparger designs on the performance of these large-scale bioreactors using computational fluid dynamics simulations in this work, so as to improve CO2 conversion at scale. The gas distribution systems in bubble column reactors not only determines operational regime, but also affects the evolution of the BSD, which in turn influences interfacial mass transfer and ultimately the efficiency of the gas-liquid exchange process. In addition to the BSD, uniformity in gas sparging affects gas holdup and bubble residence time which constitute important metrics of performance in gas-liquid systems. In this work, we use computational models to simulate high fidelity representations of different sparger designs and their effect on the operation of a bubble column reactor. Four different types of spargers have been selected for the computational study (Fig. 1): ladder, multi-ring, single-ring and toroidal. Their effect on superficial velocity, gas holdup mixing efficiency, and BSD will be evaluated in this work. The model uses a multiphase Eulerian framework similar to [1] and include a composition of mixtures of H2/CO/CO2 gases, common in fermentation applications.

BIOMASS FUELS,MATHEMATICS AND COMPUTING↗

Collaboration to Enable Higher Penetrations of Solar Power Generation Using the Natural Gas Pipeline System for Energy Storage (CRADA CRD-14-00567)

This unique project explored one possible solution for photovoltaic (PV) generation to become a reliable and dispatchable energy resource similar to conventional baseload fossil fuel electricity generation. Solar power generation is a renewable resource that varies naturally from day-to-day as well as seasonally. To use solar power as a baseload generation asset requires a flexible storage system that can recover power on both a daily and seasonal basis. Converting solar power to natural gas (i.e., first to hydrogen and then to methane) and having access to utility-scale storage in the natural gas network has the potential to make solar power generation a baseload asset. This will: 1) Increase the net energy yield from solar resources by increasing the effective capacity factor and maximize the energy produced during the assets’ lifetimes. 2) Improve the economics of energy storage for solar project developers, thereby accelerating the deployment of new solar generation. 3) Reduce the impact of increasing solar penetration on congestion of the electrical transmission system by shifting power delivery in time using the gas pipeline network for long-duration storage. 4) Reduce the impact of increasing solar generation on electrical distribution system control. 5) Offer alternative revenue sources for solar power generation beyond electricity including: hydrogen (H 2 ) production for stationary fuel cells and fuel cell electric vehicles, conversion of carbon dioxide (CO 2 ) to methane (CH 4 ) as a direct drop-in replacement for fossil natural gas use, and transmission as high volumetrically-dense CH 4 for use in transportation, heating, power generation, chemical production or conversion back to H2 at the point of use. Additional work was added to this CRADA in modification 4 having a primary objective to characterize the performance of the electrolyzer, Southern California Gas (SoCalGas) bioreactor and balance of plant to demonstrate production of renewable natural gas (RNG) from renewable H 2 and CO 2 using single-cell, self-replicating organisms. The additional scope of work allowed researchers to ramp up gas flowrates and pressure of the system over the designed range to grow the cells and begin to show the load-following capabilities of this anaerobic gas fermentation process. These activities enabled the research team to predict system performance at much greater scales; namely 10’s of mega-watts (MWs) of electrolyzer nameplate capacity.

14 SOLAR ENERGY↗

An Arduino based automatic pressure evaluation system to quantify growth of non‐model anaerobes in culture

Abstract Measuring the growth rate of non‐model anaerobic microbes typically requires the use of time‐consuming and often destructive manual measurements. Here, an Arduino based automatic pressure evaluation system (A‐APES) was developed to automatically measure the rate of fermentation gas production as a proxy for microbial growth in anaerobic systems. The A‐APES system measures accumulated gas pressure in sealed cultures accurately at high‐resolution, while venting the system at programmed intervals to prevent over pressurization. The utility of A‐APES is demonstrated in this study by quantifying the growth rate and phases of a biomass‐degrading anaerobic gut fungus, which cannot be otherwise measured via conventional techniques due to its association with particulate substrates. Given the utility of the A‐APES approach, we provide a complete construction guide to fabricate the device, which is three times less expensive compared to existing commercial alternatives.

Wilken, St. Elmo↗

Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways

Gas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste. Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic part function, gene expression patterns, and biosynthetic pathway performance in vitro before implementing designs in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here in this paper, we report the development and optimization of a high-yielding (236 ± 24 μg/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map aerobic cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes.

59 BASIC BIOLOGICAL SCIENCES↗

Multiphysics Computational Fluid Dynamics for Design and Scale-Up of CO 2 /Syngas Bioreactors [Poster]

What are we trying to do? Develop validated, high fidelity computational models for gas fermentation using CO 2 , CO and H 2 . How is it done today? Microbiological advances are made at the lab scale in well mixed reactors. Process scale-up remains an open question when considering novel microorganisms/reaction systems. Why is this important? Lab-to-production scale transition is required to achieve CO 2 conversion at Mton/Gton scale and computational models can de-risk/accelerate this transition.

09 BIOMASS FUELS↗

Acetate as a Platform for Carbon-Negative Production of Renewable Fuels and Chemicals (Final Technical Report)

This project was an industrial-academic collaboration between experts at the University of Wisconsin-Madison, the University of Kentucky, and LanzaTech, a world leader in the use of gas fermentation to sustainably produce fuels and chemicals. The project developed technologies to create an integrated process for converting carbon dioxide and renewable hydrogen into molecules that can be blended with liquid transportation fuels or used in an array of chemical applications. The project was motivated by the Program Objectives of eliminating carbon dioxide release in the production of chemicals by integrating the unique and efficient capabilities of two microorganisms into a single process. The first microbe, an acetogen, produces acetate from carbon dioxide and hydrogen while the second microbe upgrades acetate from acetogen fermentation permeates to higher-value chemical products. The carbon dioxide released in the upgrading process is recycled internally to produce more acetate. As such, the process can be designed to operate with zero carbon dioxide release and net positive carbon dioxide capture. The process has the potential to provide an alternative paradigm to the current bioeconomy – one in which acetate is the primary energy carrier instead of sugars. Our process by-passes photosynthesis and the barriers created by biomass as primary chemical feedstock. As such, the process can be scaled to meet existing sources of carbon dioxide emissions and located anywhere renewable hydrogen can be provided. Our work developed microorganisms with optimized metabolism for producing acetate and other microorganisms with improved conversion of acetate to dodecanol and dodecyl-acetate. We developed synthetic biology tools for a promising non-model bacterium that could enhance metabolic engineering efforts to convert acetate to chemical products. We conducted protein engineering studies to improve the activity of key enzymes involved in our metabolic pathways. We conducted a full technoeconomic analysis that set technical targets for each strain to meet economic goals. We identified key technical barriers in our process and proposed strategies to overcome them.

09 BIOMASS FUELS↗

BETO 2021 Peer Review - Waste Carbon Gas Upgrading via Acetogens 2.3.2.106

Waste carbon gas represents a large and diverse set of feedstocks that could be captured and turned into useful products. This includes waste gas emitted from industrial activity, syngas from burned plant biomass or processed municipal waste, and electrochemical reduction of CO2. Currently, carbon gas is being microbially converted to ethanol as a main product. However, ethanol is a lower value product with a limited market size. While these microbes can make other higher value products, there are no commercial processes for generating these other products, leaving a gap in understanding potential implementation for commercialization. Expanding the products microbially produced from waste carbon gas requires several steps before commercial implementation. We are studying the acetogen Clostridium ljungdahlii as a biocatalyst to convert waste carbon gas to the chemical 3-hydroxybutyrate (3HB), a plastic monomer and fuel precursor. For that, we are studying and engineering microbial characteristics for novel 3HB product formation from waste gas streams. This includes metabolic characterization, genetic engineering, gas fermentation scaling, as well as technoeconomic and life cycle analysis.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗

Valorization of CO 2 through lithoautotrophic production of sustainable chemicals in Cupriavidus necator

Coupling recent advancements in genetic engineering of diverse microbes and gas-driven fermentation provides a path toward sustainable commodity chemical production. Cupriavidus necator H16 is a suitable species for this task because it effectively utilizes H 2 and CO 2 and is genetically tractable. Here, we demonstrate the versatility of C. necator for chemical production by engineering it to produce three products from CO 2 under lithotrophic conditions: sucrose, polyhydroxyalkanoates (PHAs), and lipochitooligosaccharides (LCOs). We engineered sucrose production in a co-culture system with heterotrophic growth 30 times that of WT C. necator. We engineered PHA production (20-60% DCW) and selectively altered product composition by combining different thioesterases and phaCs to produce copolymers directly from CO 2 . And, we engineered C. necator to convert CO 2 into the LCO, a plant growth enhancer, with titers of ~1.4 mg/L—equivalent to yields in its native source, Bradyrhizobium. Here, we applied the LCOs to germinating seeds as well as corn plants and observed increases in a variety of growth parameters. Taken together, these results expand our understanding of how a gas-utilizing bacteria can promote sustainable production.

09 BIOMASS FUELS↗

Clostridium autoethanogenum alters cofactor synthesis, redox metabolism, and lysine-acetylation in response to elevated H 2 :CO feedstock ratios for enhancing carbon capture efficiency

Clostridium autoethanogenum is an acetogenic bacterium that autotrophically converts carbon monoxide (CO) and carbon dioxide (CO 2 ) gases into bioproducts and fuels via the Wood–Ljungdahl pathway (WLP). To facilitate overall carbon capture efficiency, the reaction stoichiometry requires supplementation of hydrogen at an increased ratio of H 2 :CO to maximize CO 2 utilization; however, the molecular details and thus the ability to understand the mechanism of this supplementation are largely unknown. In order to elucidate the microbial physiology and fermentation where at least 75% of the carbon in ethanol comes from CO 2 , we established controlled chemostats that facilitated a novel and high (11:1) H 2 :CO uptake ratio. We compared and contrasted proteomic and metabolomics profiles to replicate continuous stirred tank reactors (CSTRs) at the same growth rate from a lower (5:1) H 2 :CO condition where ~ 50% of the carbon in ethanol is derived from CO 2 . Our hypothesis was that major changes would be observed in the hydrogenases and/or redox-related proteins and the WLP to compensate for the elevated hydrogen feed gas. Our analyses did reveal protein abundance differences between the two conditions largely related to reduction–oxidation (redox) pathways and cofactor biosynthesis, but the changes were more minor than we would have expected. While the Wood–Ljungdahl pathway proteins remained consistent across the conditions, other post-translational regulatory processes, such as lysine-acetylation, were observed and appeared to be more important for fine-tuning this carbon metabolism pathway. Metabolomic analyses showed that the increase in H 2 :CO ratio drives the organism to higher carbon dioxide utilization resulting in lower carbon storages and accumulated fatty acid metabolite levels. This research delves into the intricate dynamics of carbon fixation in C. autoethanogenum, examining the influence of highly elevated H 2 :CO ratios on metabolic processes and product outcomes. The study underscores the significance of optimizing gas feed composition for enhanced industrial efficiency, shedding light on potential mechanisms, such as post-translational modifications (PTMs), to fine-tune enzymatic activities and improve desired product yields.

09 BIOMASS FUELS↗

Functional plasticity of HCO 3 – uptake and CO 2 fixation in Cupriavidus necator H16

Despite its prominence, the ability to engineer Cupriavidus necator H16 for inorganic carbon uptake and fixation is underexplored. We tested the roles of endogenous and heterologous genes on C. necator inorganic carbon metabolism. Deletion of β-carbonic anhydrase can had the most deleterious effect on C. necator autotrophic growth. Replacement of this native uptake system with several classes of dissolved inorganic carbon (DIC) transporters from Cyanobacteria and chemolithoautotrophic bacteria recovered autotrophic growth and supported higher cell densities compared to wild-type (WT) C. necator in batch culture. Strains expressing Halothiobacillus neopolitanus DAB2 (hnDAB2) and diverse rubisco homologs grew in CO 2 similarly to the wild-type strain. Our experiments suggest that the primary role of carbonic anhydrase during autotrophic growth is to support anaplerotic metabolism, and an array of DIC transporters can complement this function. This work demonstrates flexibility in HCO 3 - uptake and CO 2 fixation in C. necator, providing new pathways for CO 2 -based biomanufacturing.

59 BASIC BIOLOGICAL SCIENCES↗