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DOE OSTI2022

Evidence for a genetic basis in functional trait tradeoffs with microbial growth rate but not growth yield

Tradeoffs in microbial functional traits have been a focus of recently described ecological frameworks and of mathematical models of microbial community functioning. Tradeoffs in key traits such as growth rate, growth yield, resource acquisition, and stress tolerance may have either a genetic basis or a physiological basis, and the type of tradeoff can inform how traits are modeled and measured. Here we provide evidence that growth rate/decomposition and growth rate/stress tolerance tradeoffs have a primarily genetic basis in a phylogenetically diverse suite of ten leaf litter-inhabiting fungi. In contrast, growth yield tradeoffs with functional traits are more likely to have a physiological basis. Consideration of the type of tradeoff, genetic or physiological, should help to inform efforts to model microbial contributions to ecosystem processes, especially when considering different scales. Consideration of physiological tradeoffs may be important for understanding short-term variability (e.g., pulse events) and fine spatial scales, whereas genetic tradeoffs are likely to be useful for understanding regional- to continental-scale and medium- to long-term contributions of microbes to ecosystem processes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

n-Butanol production by Rhodopseudomonas palustris TIE-1

Anthropogenic carbon dioxide (CO 2 ) release in the atmosphere from fossil fuel combustion has inspired scientists to study CO 2 to biofuel conversion. Oxygenic phototrophs such as cyanobacteria have been used to produce biofuels using CO 2 . However, oxygen generation during oxygenic photosynthesis adversely affects biofuel production efficiency. To produce n-butanol (biofuel) from CO 2 , here we introduce an n-butanol biosynthesis pathway into an anoxygenic (non-oxygen evolving) photoautotroph, Rhodopseudomonas palustris TIE-1 (TIE-1). Using different carbon, nitrogen, and electron sources, we achieve n-butanol production in wild-type TIE-1 and mutants lacking electron-consuming (nitrogen-fixing) or acetyl-CoA-consuming (polyhydroxybutyrate and glycogen synthesis) pathways. The mutant lacking the nitrogen-fixing pathway produce the highest n-butanol. Coupled with novel hybrid bioelectrochemical platforms, this mutant produces n-butanol using CO 2 , solar panel-generated electricity, and light with high electrical energy conversion efficiency. Overall, this approach showcases TIE-1 as an attractive microbial chassis for carbon-neutral n-butanol bioproduction using sustainable, renewable, and abundant resources.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

Model uncertainty obscures major driver of soil carbon

Understanding the formation and stabilization mechanisms of soil organic carbon (SOC) is important for managing land carbon (C) and mitigating climate change. Tao et al. reported that microbial C use efficiency (CUE) is the primary determinant of global SOC storage and that the relative impact of plant C inputs on SOC is minor. Although soil microbes undoubtedly play an important role in SOC cycling, we are concerned about the robustness of the approach taken by Tao et al. In conclusion, the potential biases in their analyses may lead to misleading, model-dependent results.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2020

An integrated process for butanol production from cellulosic biomass and CO 2 using engineered clostridia in a linear immobilized bioreactor

Engineered platforms using mixed microbial consortia to consolidate multiple reaction steps and perform disparate conversions simultaneously can substantially enhance the carbon efficiency of biomass conversion processes and lower the biofuel cost to an economically competitive level. In this project, an integrated process will be developed to produce n-butanol from cellulosic biomass and waste gas streams using two engineered clostridial strains, one butanol-tolerant solventogenic strain and one carboxydotrophic acetogen, co-immobilized in a “Linear Immobilized Bioreactor” (LIBR). The CO 2 and H 2 produced by the solventogenic strain will be used by the carboxydotrophic strain to produce acetate (and butyrate) that in turns can be used by the solventogenic strain to produce more butanol. Such a co-cultured fermentation has the potential to increase butanol production from biomass hydrolysate sugars by up to 50% with 100% reduction in CO 2 emission. Butanol produced in the fermentation will be stripped by the rising bubbles of fermentation produced gases (CO 2 and H 2 ) in the LIBR and separated via a condenser, alleviating butanol inhibition while achieving continuous butanol production at a high productivity and product titer, which can reduce both water and energy consumptions by over 50% compared to conventional ethanol and ABE fermentation processes. Phase I study proved the new bioprocess concept and verified our hypothesis that the proposed LIBR and co-cultured fermentation can enhance the conversion of lignocellulosic sugars to butanol with higher butanol yield, productivity and titer, leading to reduced biobutanol cost to compete more favorably with bioethanol and fossil fuels. A prototype lab-scale LIBR for multiphase fermentation was constructed to investigate and demonstrate the technical feasibility and advantages of the LIBR for multiphase fermentation involving sugars and gaseous substrates. The LIBR will be optimized and evaluated for its economic feasibility for biobutanol production from lignocellulosic biomass and industrial waste gases in Phase II.

09 BIOMASS FUELS↗

DOE OSTI2023

Mechanism of furfural toxicity and metabolic strategies to engineer tolerance in microbial strains

Abstract Lignocellulosic biomass represents a carbon neutral cheap and versatile source of carbon which can be converted to biofuels. A pretreatment step is frequently used to make the lignocellulosic carbon bioavailable for microbial metabolism. Dilute acid pretreatment at high temperature and pressure is commonly utilized to efficiently solubilize the pentose fraction by hydrolyzing the hemicellulose fibers and the process results in formation of furans—furfural and 5-hydroxymethyl furfural—and other inhibitors which are detrimental to metabolism. The presence of inhibitors in the medium reduce productivity of microbial biocatalysts and result in increased production costs. Furfural is the key furan inhibitor which acts synergistically along with other inhibitors present in the hydrolysate. In this review, the mode of furfural toxicity on microbial metabolism and metabolic strategies to increase tolerance is discussed. Shared cellular targets between furfural and acetic acid are compared followed by discussing further strategies to engineer tolerance. Finally, the possibility to use furfural as a model inhibitor of dilute acid pretreated lignocellulosic hydrolysate is discussed. The furfural tolerant strains will harbor an efficient lignocellulosic carbon to pyruvate conversion mechanism in presence of stressors in the medium. The pyruvate can be channeled to any metabolite of interest by appropriate modulation of downstream pathway of interest. The aim of this review is to emphasize the use of hydrolysate as a carbon source for bioproduction of biofuels and other compounds of industrial importance.

5-hydroxymethyl furfural↗

DOE OSTI2024

Predictions of rhizosphere microbiome dynamics with a genome-informed and trait-based energy budget model

Abstract Soil microbiomes are highly diverse, and to improve their representation in biogeochemical models, microbial genome data can be leveraged to infer key functional traits. By integrating genome-inferred traits into a theory-based hierarchical framework, emergent behaviour arising from interactions of individual traits can be predicted. Here we combine theory-driven predictions of substrate uptake kinetics with a genome-informed trait-based dynamic energy budget model to predict emergent life-history traits and trade-offs in soil bacteria. When applied to a plant microbiome system, the model accurately predicted distinct substrate-acquisition strategies that aligned with observations, uncovering resource-dependent trade-offs between microbial growth rate and efficiency. For instance, inherently slower-growing microorganisms, favoured by organic acid exudation at later plant growth stages, exhibited enhanced carbon use efficiency (yield) without sacrificing growth rate (power). This insight has implications for retaining plant root-derived carbon in soils and highlights the power of data-driven, trait-based approaches for improving microbial representation in biogeochemical models.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Self-Assembling Cell-Free Systems for Scalable Bioconversion

This project focused on developing cell-free systems to directly express multi-enzyme catalysts and perform CO2 bioconversions for industrial chemical production. The use of cell-free expression (CFE) systems derived from bacterial lysates is emerging as a promising approach for biomanufacturing. CFEs are genetically programmable, permit the expression of toxic enzymes, and allow for rapid prototyping of metabolic pathways. Research Contributions: 1. Understanding the Area Investigated: This research advances the understanding of cell-free systems by demonstrating their capability to perform complex multi-enzyme reactions. By directly expressing multi-gene systems, CFEs avoid the high costs and inefficiencies associated with producing and purifying enzymes for multi-step pathways. 2. Technical Effectiveness and Economic Feasibility: The project successfully engineered a CFE-based multienzyme biocatalyst for the de novo synthesis of serine and glycine from CO2 equivalents (formate and bicarbonate) and ammonia. This method achieved a 30% conversion rate of formate into these industrially important amino acids. Additionally, an 8-gene CFE biocatalyst was developed to produce malate, conserving 43% of carbon that would otherwise be lost as CO2. This approach has the potential to reach higher carbon efficiency than microbial production. 3. Public Benefit: The cell-free production of chemicals like serine, glycine, and malate using electrochemically generated formate could significantly reduce CO2 emissions. For example, satisfying the global malate market with this method could avoid approximately 400,000 tons of CO2 emissions annually. This work demonstrates the potential of CFE systems to produce platform chemicals, contributing to environmental sustainability and reducing reliance on petrochemicals. Future Prospects: The CFE-based biocatalyst process could be extended to produce a variety of chemicals, including other industrial di-acids, aromatics, terpenes, alcohols, and polymers. This project showcases the capabilities of cell-free expression systems for prototyping carbon-conserving pathways and sustainably bioproducing platform chemicals, marking a significant step towards economically-viable industrial processes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2025

Environmental matrix and moisture influence soil microbial phenotypes in a simplified porous media incubation

Soil moisture and porosity regulate microbial metabolism by influencing factors, such as system chemistry, substrate availability, and soil connectivity. However, accurately representing the soil environment and establishing a tractable microbial community that limits confounding variables is difficult. Here, we use a reduced-complexity microbial consortium grown in a glass bead porous media amended with chitin to test the effects of moisture and a structural matrix on microbial phenotypes. Leveraging metagenomes, metatranscriptomes, metaproteomes, and metabolomes, we saw that our porous media system significantly altered microbial phenotypes compared with the liquid incubations, denoting the importance of incorporating pores and surfaces for understanding microbial phenotypes in soils. These phenotypic shifts were mainly driven by differences in expression of Streptomyces and Ensifer, which included a significant decrease in overall chitin degradation between porous media and liquid. Our findings suggest that the success of Ensifer in porous media is likely related to its ability to repurpose carbon via the glyoxylate shunt amidst a lack of chitin degradation byproducts while potentially using polyhydroxyalkanoate granules as a C source. We also identified traits expressed by Ensifer and others, including motility, stress resistance, and carbon conservation, that likely influence the metabolic profiles observed across treatments. Together, these results demonstrate that porous media incubations promote structure-induced microbial phenotypes and are likely a better proxy for soil conditions than liquid culture systems. Furthermore, they emphasize that microbial phenotypes encompass not only the multi-enzyme pathways involved in metabolism but also include the complex interactions with the environment and other community members.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2021

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↗

DOE OSTI2024

Harnessing photoautotroph-methanotroph interactions for biogas conversion to fuels and chemicals using binary consortia (Project Final Technical Report)

Industrial, municipal, and agricultural waste streams containing stranded organic carbon represent a significant and underutilized feedstock to produce fuels and chemicals. With anaerobic digestion deployed at large scales to capture organic waste streams, over 6 million tons of biogas are available today. However, the utilization of biogas represents a significant challenge due to its low pressure and presence of contaminants such as H 2 S, ammonia, and volatile organic carbon compounds. To tap into this immense potential, effective biotechnologies that co-utilize both CO 2 and CH 4 are needed. Recent studies demonstrated that, in nature, microbial communities have developed a highly efficient way to recover energy and capture carbon from both CH 4 and CO 2 through metabolic coupling of methane oxidation to oxygenic photosynthesis. Using two synthetic methanotroph – photoautotroph (M-P) co-cultures that exhibit stable growth under a broad range of cultivation conditions, in this project we proposed to harness the interspecies interactions within these cocultures for biogas conversion to fuels and chemicals. To facilitate this overarching objective, we aim to develop experimental and computational tools to gain qualitative and quantitative understandings on the interactions and dynamics of the coculture at both systems and molecular levels, and to validate our findings through experiments and mutant development. The fundamental understanding on the interactions and dynamics of the photoautotroph-methanotroph will lay the foundation for the design and optimization of synthetic binary consortia for production of fuels and chemicals from biogas. We expect the knowledge gained from this project may be generally applicable to other cross-feeding binary consortium, and the tools developed can be adapted to study the interactions and dynamics of other multi-organism platforms.

09 BIOMASS FUELS↗

DOE OSTI2021

Physical protection regulates microbial thermal responses to chronic soil warming

Climate warming can affect the temperature sensitivity of microbial activity and growth efficiency, possibly explained by changes to microbially unavailable carbon (C) protected within in soil aggregates. Here, we assessed physical protection by crushing macroaggregates (250–2000 μm) and microaggregates (<250 μm) isolated from mineral soils exposed to 27 years of experimental warming (+5 °C). We hypothesized that removal of physical protection would increase microbial activity and reduce C use efficiency (CUE). We found that crushing increased microbial respiration and biomass turnover rate, but did not affect CUE. We also hypothesized that long-term warming would reduce the effect of physical protection on microbial activity, and that physical protection would attenuate microbial temperature sensitivity in heated compared to control soils. We found that long-term warming was associated with a smaller effect of physical protection for microbial respiration but with a larger effect for biomass turnover rate in macroaggregates. Physical protection reduced the temperature sensitivity of respiration but enhanced the temperature sensitivity of microbial biomass turnover rate in heated compared to control soils. Our work shows that long-term warming has contrasting effects on how microbial respiration, biomass turnover rate, and their thermal responses are mediated by physical protection within soil aggregates.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Efficient chito–oligosaccharide utilization requires two TonB–dependent transporters and one hexosaminidase in Cellvibrio japonicus

Chitin utilization by microbes plays a significant role in biosphere carbon and nitrogen cycling, and studying the microbial approaches used to degrade chitin will facilitate our understanding of bacterial strategies to degrade a broad range of recalcitrant polysaccharides. The early stages of chitin depolymerization by the bacterium Cellvibrio japonicus have been characterized and are dependent on one chitin-specific lytic polysaccharide monooxygenase and non-redundant glycoside hydrolases from the family GH18 to generate chito-oligosaccharides for entry into metabolism. Here, we describe the mechanisms for the latter stages of chitin utilization by C. japonicus with an emphasis on the fate of chito-oligosaccharides. Here, our systems biology approach combined transcriptomics and bacterial genetics using ecologically relevant substrates to determine the essential mechanisms for chito-oligosaccharide transport and catabolism in Cellvibrio japonicus. Using RNAseq analysis we found a coordinated expression of genes that encode polysaccharide-degrading enzymes. Mutational analysis determined that the hex20B gene product, predicted to encode a hexosaminidase, was required for efficient utilization of chito-oligosaccharides. Furthermore, two gene loci (CJA_0353 and CJA_1157), which encode putative TonB-dependent transporters, were also essential for chito-oligosaccharides utilization. This study further develops our model of C. japonicus chitin metabolism and may be predictive for other environmentally or industrially important bacteria.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Development of a molecularly informed biogeochemical framework for reactive transport modeling of subsurface carbon inventories, transformations and fluxes (Final Report)

The overall objective of the project was to combine new molecular-level characterization strategies with soil carbon flux measurements to develop and evaluate model representations of subsurface carbon cycling. We expanded on existing studies in the East River watershed, Colorado, in collaboration with the Berkeley Lab Watershed Function Scientific Focus Area (SFA), SLAC Groundwater Quality SFA, and Rocky Mountain Biological Laboratory (RMBL) to develop an elevation and vegetation gradient that is now the subject of long-term monitoring by the USGS. To achieve the overall objective, we combined field studies of soil respiration with laboratory analyses, ranging from spectroscopy to incubation studies. This combination of techniques enabled us to develop a new understanding of the drivers of high-elevation soil respiration. In the process, we developed three new modeling approaches that improve our ability to conceptualize and ultimately to represent soil respiration in numerical models. The first approach is a plot-scale transient inverse model that can be used to determine in situ CO 2 production rates from measured concentration profiles and surface fluxes. Application of this method revealed the importance of plant phenology and deep CO 2 production in moderating CO 2 fluxes to the atmosphere. The second modeling approach is a molecular-scale tool that enables spectroscopic and elemental data for carbon speciation to be transformed into functional group abundances, or the ‘SOC-fga model’. This method uniquely enables carbon speciation to be tracked within a reactive transport framework to partition carbon among different pathways and storage zones within the soil. The third modeling tool builds strongly on the previous approaches and captures the microbial processes driving heterotrophic respiration. This ‘dormancy model’ allows the native soil microbial population to respond transiently to the presence or absence of water in order to catalyze carbon respiration. This approach was also compared to the simpler and more widely used first-order model using two experimental datasets with different temporal and spatial resolutions. Our results illustrate that the simpler first order model provides a robust and efficient representation of deep (>1 meter) soil respiration, but that shallow soils, where most respiration occurs, require explicit representation of moisture-dependent activation and dormancy rates. By assessing soil organic carbon turnover at multiple scales, we see a complex array of controls emerge. Atte scale of a hillslope, spatial heterogeneity in soil respiration rates dominates and is uncorrelated with instantaneous soil moisture and plant community. At the profile scale, the balance between plant inputs and water availability is the dominant control. At the microbial to molecular scale, physiological processes associated with carbon use and carbon speciation are important controls. These scale-dependent controls emphasize the need for new modeling approaches that examine their interactions and hierarchies.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2022

Susceptibility of new soil organic carbon to mineralization during dry-wet cycling in soils from contrasting ends of a precipitation gradient

The persistence of soil organic carbon (SOC) is influenced by soil physicochemical properties, organic matter quality, and climatic conditions that govern its vulnerability to microbial activity. We compared the susceptibility of newly formed SOC to mineralization in two soils (Andosols) that developed under contrasting precipitation regimes. Soil from the high rainfall region (‘high rain ’) had higher SOC and lower iron concentrations than soils exposed to less rainfall (‘low rain ’). We amended soils with 13 C-labeled carbohydrates and measured the amount of substrate-derived SO 13 C mineralized when exposed to dry-wet cycling following months-long incubations. We hypothesized that susceptibility would differ due to iron content and mineralogy, initial SOC, substrate solubility (cellulose versus glucose amendment), and microbial substrate use efficiency (SUE). We found that SO 13 C was less susceptible to dry-wet cycling when more 13 C was assimilated into microbial biomass and co-localized with mineral surfaces than when co-localized with existing organo-mineral surfaces, according to microscale NanoSIMS imaging. Considerably less SO 13 C was susceptible to mineralization in the ferrihydrite-rich (low SOC) low rain soil than the leached (high SOC) high rain soil when C was added as either glucose (7.3-fold less C mineralized) or cellulose (15.2-fold less). Additionally, the SUE of glucose was comparable to cellulose in low rain soil where SO 13 C was less water soluble and coprecipitated with ferrihydrite, and used half as efficiently as cellulose in high rain soil. Our results show that the susceptibility of newly formed SOC to mineralization is modified by the effects of bioavailability on microbial metabolism and the availability of mineral surfaces for forming new organo-mineral complexes.

21 NanoSIMS↗

DOE OSTI2023

Defining the Minimal Set of Microbial Genes Required for Valorization of Lignin Biomass (Final Report)

Project Goals: Lignin is the second most abundant biopolymer on earth and represents a critically underutilized biomass resource for hydrocarbon feedstocks. Despite substantial effort, there is still no efficient process to convert lignin to useable carbon-based platform chemicals and materials. The goal of this project is identify a minimal set of microbial enzymes necessary for lignin breakdown and sufficient for the synthesis of valuable chemical intermediates from lignin isolated as a byproduct of lignocellulosic ethanol production. These genes will be then used to engineer functional whole cell biocatalysts for tunable lignin metabolism. To date, although a number of enzymes have been associated with lignin degradation, most have been tested in isolation (as individual enzymes) and on drastically different substrates -- often dyes that are not related to lignin. In contrast, lignin utilization in nature likely occurs by microbial consortia with multiple enzymes acting synergistically. We propose to examine two separate stages of lignin breakdown carried out by the microbes that do it best: (1) early breakdown of native polymeric lignin into soluble fragments by a set of sequenced wood-rotting fungal species, and (2) downstream metabolism of these soluble lignin fragments to useful chemical intermediates by a panel of sequenced soil saprophytes. Our approach involves testing sets of genes that will be assayed combinatorially in the context of a heterologous expression host. The resulting engineered strains will be systematically assayed using soluble lignin fragments, synthetic defined polymeric lignin, and finally lignin directly sourced from lignocellulosic processing streams. In addition to resulting in a functional whole cell biocatalyst for lignin utilization, we anticipate that this approach will allow us to address key unanswered questions about lignin metabolism in nature, including: (1) Why does the Trametes versicolor genome contain 25 different class II peroxidases? (2) What is the role of laccases in lignin metabolism? Why do some aggressive lignin degraders have many laccases (e.g. >7 in T. versicolor) while others have none (e.g. P. chrysosporium)? (3) How is peroxide provided in a controlled manner to drive peroxidase activity without causing the enzyme inhibition that is so often observed in vitro? (4) What strategies do microbial lignin degraders use to avoid the problem of repolymerization during active lignin degradation? and (5) Can microbial lignin metabolism be diverted for high level production of defined aromatics? A final critical question is whether combining key minimal sets of enzymes from a wide range of organisms will result in engineered strains capable of highly efficient, streamlined pathways for lignin utilization that can be tuned for a specific carbon output. This effort will leverage DOE investments in microbial genome sequencing, and secure a critical channel for lignin biomass utilization that will also help to render lignocellulosic a viable feedstock for the production of renewable liquid biofuels.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Innovative Polyhydroxyalkanoates (PHA) Production with Microbial Electrochemical Technology (MET)

The project “Innovative Polyhydroxyalkanoates (PHA) Production with Microbial Electrochemical Technology (MET)” addressed food waste disposal challenges by successfully converting food waste to bioplastics (known as PHAs). The novel process created by our team of researchers from universities, national labs, and industry substantially enhanced overall carbon conversion efficiency of food waste processing (> 50%), while reducing disposal costs (> 25%). The project showed economic viability potential at community scale through pilot-scale demonstration at a relevant scale (50 L reactor volume) with more than 100 hours of PHA production using realistic conditions. The project goal was to valorize food waste by shunting traditional anaerobic digestion processing and creating a value-added PHA processing route that improves the economics and sustainability of local, community-scale, wet organic waste treatment. First, the food waste undergoes microbial-based, dark fermentation to break down the food to small carbon chains known as volatile fatty acids (VFAs). Instead of microorganisms converting the VFAs into methane using normal anaerobic digestion processing, our innovative process inhibits methane production. This preserves the produced VFAs for extraction and use by a novel Haloferax mediterranei (HM) archaea, which effectively converts the VFAs to bioplastics. The project added microbial electrochemical cells (MEC) to the dark fermentation process to enhance the VFAs produced and optimize the type of bioplastics formed.

36 MATERIALS SCIENCE↗

DOE OSTI2022

Developing reactors for electrifying bio-methanation: a perspective from bio-electrochemistry

The integration of microbial synthesis with renewable electricity is an emerging route for both CO 2 utilization and seasonal energy storage in the form of stored bio-electrofuels. The major benefits of electrifying bioreactors include: using highly selective bio-catalysts for CO 2 conversion under mild reaction conditions; decoupling the production of more facile electrochemical intermediates, such as hydrogen, at the electrode from the production of bio-catalyzed multi-electron and/or carbon products, such as methane or acetate; using microbes as robust and self-regenerating catalysts enabling higher efficiency and durability in CO 2 conversion systems compared to inorganic catalysis. In this Perspective, we propose research aimed at developing electro-bioreactor components that will increase the productivity of the reactor while maintaining high energy efficiency and biocompatible reaction conditions to fully realize the benefits of electrified bioreactors. Furthermore, these developments include: flow reactors with tailored 3D electrodes to optimally use the reactor volume, electrocatalysts designed for peak performance in neutral pH electrolytes, high conductivity microbial media, and new membrane separator materials with high ion conductivity and low gas permeability. Production of methane via a hybrid electrical-biological approach is taken as a case study to motivate these developments. Finally, an iterative design–manufacture–test cycle, enabled by additive manufacturing and 3D printing technologies, is proposed to rapidly prototype components prior to large-scale manufacturing.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2024

Rational Optimization of Microbial Processing for High Yield CO 2 -to-Isopropanol Conversion: Cooperative Research and Development Final Report, CRADA Number CRD-20-17114

This project focuses on the production of the fuel blendstock isopropanol using a CO 2 -fixing Clostridium by metabolic engineering and process optimizations. The project will initiate from a baseline isopropanol producer and pursue isopropanol production at high carbon-conversion efficiency. We will lead engineering work by in-depth pathway analyses including thermodynamics optimization, enzyme expense analysis, metabolic robustness analysis, and -omics analysis. The isopropanol production will be optimized via genome editing followed by fermentation optimizations. This project will deliver a novel microbial process that efficiently converts waste CO 2 to isopropanol at ~g/L titer level within 18-months. This project will layout a solid knowledge basis and technology platform for renewable CO 2 valorization to bio-blendstock that help achieve Co-Optima and Shell's goals.

09 BIOMASS FUELS↗