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DOE OSTI2021

Developing Multi-Gene CRISPRa/I Programs to Accelerate DBTL Cycles in ABF Hosts Engineered for Chemical Production (CRADA 468)

Bacterial metabolism is comprised of large and complex gene networks that can produce valuable chemical products. Sophisticated organism engineering efforts are required to optimize production of high-value compounds from these networks. In principle, synthetic multi-gene transcriptional programs could be constructed to reengineer these networks for efficient industrial chemical production. In practice, however, our incomplete ability to understand and model the underlying networks, combined with our limited ability to predictably control the expression of multiple genes makes achieving this goal difficult. To overcome these challenges, we will combine new CRISPR-Cas multi-gene expression programs with computational modeling, machine learning, and multi-omics data to enhance the efficacy of design-build-test-learn (DBTL) cycles. For industrially promising microorganisms in early stages of development, creating technologies for rapidly engineering complex multi-gene programs could be transformative for accelerating data- and model-driven strain design. New CRISPR-Cas tools allow programmable gene activation (CRISPRa) or repression (CRISPRi) at multiple genes simultaneously, using the catalytically inactive Cas9 protein (dCas9) with guide RNAs that recognize DNA targets through predictable Watson-Crick base pairing. To enable accelerated DBTL cycles, we will combine these technologies with advanced Agile BioFoundry (ABF) capabilities for multi-omics data collection and machine learning. We will demonstrate the immediate applicability of these tools by rapidly improving the production of an industrial aromatic in multiple ABF organisms. We recently identified and optimized new transcriptional activators that can be linked to programmable CRISPR-Cas DNA binding domains to activate gene expression in E. coli. We can now use these CRISPRa tools as generalizable trans-acting regulators for combinatorial multi-gene expression tuning that can be easily transferred to new pathways and networks without additional genome engineering. We anticipate these tools will also transfer to new hosts. We have recently found that CRISPRa systems developed in E. coli can be readily ported to Pseudomonas putida, suggesting that multi-gene CRISPRa/i programs for diverse ABF organisms may be within reach.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Analysis of the cyanobacterial amino acid metabolism with a precise genome-scale metabolic reconstruction of Anabaena sp. UTEX 2576

Filamentous cyanobacteria such as Anabaena sp. UTEX 2576 (a.k.a., Nostoc sp. PCC 7120) are a sustainable platform for commodity and specialty chemical production. Anabaena is a model cyanobacterium adopted to study production of nitrogen-containing metabolites useful for chemical, cosmetic, and pharmaceutical industries. Therefore, a precise description of the Anabaena metabolic network is desired to analyze their amino-acid metabolism and elucidate potential applications of these cyanobacteria as host organisms for biotechnological production. Secondary metabolite production depends on the metabolic availability of amino acids. We provide new insight on the biotechnological utilization of Anabaena after predicting phycocyanobilin and amino-acid production rates, using a genome-scale metabolic model (iDN1004). This metabolic reconstruction is a highly comprehensive representation of the global metabolism of Anabaena, which also contains experimental biomass equations and constraints under photoautotrophic and photodiazotrophic growth. Modelling results ranked proteinogenic amino acids based on predicted metabolic fluxes through amino-acid producing reactions. From these, l-aspartate, glycine, l-serine, l-valine, l-alanine, l-threonine, and l-leucine were selected as the best branching points to conduct metabolic engineering, where l-aspartate, l-serine, l-valine and glycine serve as potential precursors of the secondary metabolites Schizokinen (a siderophore), Sphingosine (a ceramide), Lyngbyatoxin A (an alkaloid), and Shinorine (a sunscreen).

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Off-Road Vehicle Decarbonization and Energy Systems Integration: R&D Gaps and Opportunities

This report summarizes findings from the Off-Road Decarbonization and Energy Systems Integration workshop, hosted by the National Renewable Energy Laboratory (NREL) from March 22-24, 2022. The workshop focused on the importance of collaboration among the off-road vehicle industry and government to address barriers and opportunities for decarbonization. The workshop aligns with priorities of the U.S. Department of Energy (DOE) Vehicle Technologies Office and Hydrogen and Fuel Cell Technologies Office to decarbonize transportation in the agriculture, mining, construction, and military industries. This decarbonization effort is also intended to support original equipment manufacturers, industry associations, technology developers, utilities, and consultants. The sections within this report correspond to the three topic areas covered in the 3-day workshop: a high-level perspective of needs and challenges, vehicle and equipment decarbonization strategies, and energy systems integration opportunities.

33 ADVANCED PROPULSION SYSTEMS↗

DOE OSTI2024

Viruses of Nitrogen-Fixing Mesorhizobium Bacteria in Globally Distributed Chickpea Root Nodules

Legume nodules are specialized environments on plant roots that are induced and dominated by nitrogen-fixing bacteria. Bacteriophages (phages) in these nodules could potentially provide top-down controls on the population size and, therefore, the function of nitrogen-fixing symbionts. Here we sought to characterize the diversity and biogeographical patterns of phages that infect nitrogen-fixing Mesorhizobium symbionts isolated from root nodules, leveraging 266 genomes of Mesorhizobium isolated from nodules and 648 nodule metagenomes collected from three species of chickpea plants ( Cicer spp.) under different agricultural management practices, spanning eight countries on five continents. We identified 106 phage populations (viral operational taxonomic units [vOTUs]) in Mesorhizobium draft genomes, 37% of which were confirmed as likely prophages. These vOTUs were detected in 64% of the Mesorhizobium-dominated nodule metagenomes and 58% of the Mesorhizobium isolates. Per metagenome, 1 to 16 putative Mesorhizobium vOTUs were detected, with more than half of the nodules containing only one such vOTU. The majority of vOTUs were detected exclusively in Ethiopia, followed by India and Morocco, with the lowest richness of putative Mesorhizobium phages in countries that applied industrial Mesorhizobium inoculants to crops. Two vOTUs were identified in five or more countries and in nodules dominated by different strains of Mesorhizobium, suggesting infection of diverse Mesorhizobium hosts and long-term interactions. Beta-diversity of these Mesorhizobium phage assemblages was significantly correlated with the dominant Mesorhizobium strain, but not with measured environmental parameters. Our findings indicate that nitrogen-fixing nodules in chickpea plants can contain distinct viral assemblages, with potential impacts on the nodule microbiome that bear further exploration.

Microbiology↗

DOE OSTI2020

From Long-distance Entanglement to Building a Nationwide Quantum Internet: Report of the DOE Quantum Internet Blueprint Workshop

A Quantum Path Forward. Today, many scientific experts recognize that building and scaling quantum-protected and enhanced communication networks are among the most important technological frontiers of the 21st century. The international research community perceives the construction of a first prototype global quantum network-the Quantum Internet-to be within reach over the next decade. In February 2020, the U.S Department of Energy (DOE)'s Office of Advanced Scientific Computing Research hosted the Quantum Internet Blueprint workshop to define a potential roadmap toward building the first nationwide quantum Internet. The workshop participants included representatives from DOE national laboratories, universities, industry, and other U.S. agencies with serious interests in quantum networking. The goal was to provide an outline of the essential research needed, detail any engineering and design barriers, and suggest a path forward to move from today's limited local network experiments to a viable, secure quantum Internet.

97 MATHEMATICS AND COMPUTING↗

DOE OSTI2020

Estimation of Large-Scale Wind Field Characteristics Using Supervisory Control and Data Acquisition Measurements

As the wind energy industry continues to push for increased power production and lower cost of energy, the focus of research has expanded from individual turbines to entire wind farms. Among a host of interesting problems to be solved when considering the wind farm as a whole, we consider the challenge of scalar field estimation, based on information already collected at the individual turbine level. We aim to estimate the large-scale, low-frequency characteristics of the wind field, such as the mean wind direction and the overall decrease in wind speed across the farm, and employ a Kalman filter that models the wind field using a polynomial function. We compare the proposed method’s performance to both a simple averaging technique and filtering of individual turbine measurements. The method presented is not limited to wind turbines and is applicable in other situations where multiple remote agents are used to estimate a scalar field.

estimation↗

DOE OSTI2020

From Long-distance Entanglement to Building a Nationwide Quantum Internet: Report of the DOE Quantum Internet Blueprint Workshop

A Quantum Path Forward. Today, many scientific experts recognize that building and scaling quantum-protected and enhanced communication networks are among the most important technological frontiers of the 21st century. The international research community perceives the construction of a first prototype global quantum network-the Quantum Internet-to be within reach over the next decade. In February 2020, the U.S. Department of Energy (DOE)'s Office of Advanced Scientific Computing Research hosted the Quantum Internet Blueprint workshop to define a potential roadmap toward building the first nationwide quantum Internet. The workshop participants included representatives from DOE national laboratories, universities, industry, and other U.S. agencies with serious interests in quantum networking. The goal was to provide an outline of the essential research needed, detail any engineering and design barriers, and suggest a path forward to move from today's limited local network experiments to a viable, secure quantum Internet.

Peters, Nicholas↗

DOE OSTI2023

Pathways for Negative-Emissions Hydrogen: Opportunities and R&D Needs

The Carbon-Negative Hydrogen Workshop, hosted by NREL in Golden, Colorado, focused on developing a shared understanding of the importance of and opportunities for generation and use of carbon-negative H2 in industrial decarbonization. Attendees helped define research needs, spot barriers to commercialization, and identify technical challenges, opportunities, and knowledge gaps for this emerging technology area. Workshop discussion themes included energy justice in early-stage research, R&D pathways for negative-emissions H2, biomass gasification to carbon-negative H2, and policy and carbon accounting considerations.

biomass↗

DOE OSTI2023

Energy Justice in Low-TRL Research

carbon-negative hydrogen↗

DOE OSTI2024

Carbon Management Projects (CONNECT) Database and Explorer

Overview The Carbon Management Projects (CONNECT) Toolkit is an online exploratory visualization tool developed by the U.S. Department of Energy's (DOE) Office of Fossil Energy and Carbon Management (FECM) with support from other federal agencies such as the U.S. Environmental Protection Agency (EPA) and the U.S. Department of Transportation (DOT). It provides a single point of access to authoritative information on federal agency investment in a portfolio of research, development, and demonstration (RD&D) projects that have been publicly announced to advance technologies for point source carbon capture, carbon dioxide removal, transport, storage, and conversion, collectively referred to as carbon management. The RD&D programs covered in this tool are authorized by annual congressional appropriations ("Base Program") and the 2021 Infrastructure Investment and Jobs Act (IIJA). The tool also incorporates public information on other federal initiatives, such as the Regional Clean Hydrogen Hubs, and public information released by other government agencies, such as the Environmental Protection Agency's (EPA) and Primacy States’ Underground Injection Control Class VI permits and EPA’s facility level greenhouse gas (GHG) emissions. Developed in a geographic information system, the tool organizes carbon management projects into five groups based on the primary technology that a project aims to advance, each visually represented as a digital layer ("carbon management project layer"). Only federally funded projects are included, which can be awarded projects that are completed or ongoing, or projects that have been selected but are currently under negotiation. Project information can be viewed in the map or in the attribute table below it when turned on. In the map view, each project is displayed at either its host site (for field work), where available, or its performer site (project lead's location, further explained in the table below). Host sites and performer sites are represented in distinct icons. Several reference layers offer additional public information on infrastructural and natural resource environment for carbon management. These reference layers, combined with multiple geographical basemaps, enable users to visualize the carbon management project layers in context. Carbon management project information will be updated monthly based on feedback and information availability. Carbon management project layers Point Source Carbon Capture (PSC) This layer contains DOE-funded projects focused on capturing carbon dioxide (CO2) from power plants or industrial facilities. Carbon Dioxide Removal (CDR) This layer contains DOE-funded projects focused on capturing CO2 from the atmosphere, including direct air capture (DAC) and DAC hubs, direct ocean capture, enhanced mineralization, and biomass carbon removal and storage. For projects with multiple host sites, each of the sites are displayed individually with the project cost and cost sharing information representing the total for the entire project. Carbon Transport This layer contains DOE- and DOT-funded projects focused on CO2 transport. The Transport Research and Development sublayer contains projects that do not involve physical infrastructure; the Proposed Transport Corridor sublayer contains projects for which either a route for the transport infrastructure has been proposed or a general area for the transport infrastructure has been identified. Carbon Storage This layer contains DOE-funded key projects focused on CO2 storage. For projects with multiple field-work sites, each of the sites are displayed individually on the map with the project cost and cost sharing information representing the overall total for the entire project. Carbon Conversion This layer contains DOE-funded projects focused on converting CO2 into economically valuable products. Reference layers The following layers provide additional information in the geographic proximity of carbon management projects. Users should reference the original sources for more details (weblinks provided below and in pop-up windows on the map). Regional Clean Hydrogen Hub and Facility These layers illustrate the approximate areas of the Regional Clean Hydrogen Hubs announced by DOE's Office of Clean Energy Demonstrations (OCED) and the approximate locations of individual facilities that constitute the hubs (see "Where are the H2Hubs located?" on the webpage linked above). EPA Facility Level GHG Emissions (direct emitter) This layer shows direct CO2 emissions from stationary sources in 2022, using data extracted from EPA's Facility Level Information on GreenHouse gases Tool (FLIGHT). Captured and injected CO2 are not deducted from direct emitters’ total emissions. Contact EPA for additional details. Underground Injection Control Class VI permit/permit application This layer shows the locations of CO2 injection wells that are granted or in the process of applying for an Underground Injection Control Class VI permit by EPA or a Primacy State (currently Louisiana, North Dakota, and Wyoming). The URLs for the permits or permit applications are provided in the pop-up windows associated with the well locations. Contact EPA for additional details. Carbon Storage Resource This layer contains information on prospective CO2 storage resources in saline formations and oil and gas reservoirs provided by the National Carbon Sequestration Database and Geographic Information System (NATCARB) spatial database. Contact NETL for additional details. Existing CO2 pipeline This layer shows active CO2 pipelines based on information digitized from the map issued by the Pipeline and Hazardous Materials Safety Administration (PHMSA). Contact PHMSA for additional details.

Carbon Conversion↗

DOE OSTI2023

Fluctuating pH for efficient photomixotrophic succinate production

Cyanobacteria are attracting increasing attention as a photosynthetic chassis organism for diverse biochemical production, however, photoautotrophic production remains inefficient. Photomixotrophy, a method where sugar is used to supplement baseline autotrophic metabolism in photosynthetic hosts, is becoming increasingly popular for enhancing sustainable bioproduction with multiple input energy streams. In this study, the commercially relevant diacid, succinate, was produced photomixotrophically. Succinate is an important industrial chemical that can be used for the production of a wide array of products, from pharmaceuticals to biopolymers. In this system, the substrate, glucose, is transported by a proton symporter and the product, succinate, is hypothesized to be transported by another proton symporter, but in the opposite direction. Thus, low pH is required for the import of glucose and high pH is required for the export of succinate. Succinate production was initiated in a pH 7 medium containing bicarbonate. Glucose was efficiently imported at around neutral pH. Utilization of bicarbonate by CO 2 fixation raised the pH of the medium. As succinate, a diacid, was produced, the pH of the medium dropped. By repeating this cycle with additional pH adjustment, those contradictory requirements for transport were overcome. pH affects a variety of biological factors and by cycling from high pH to neutral pH processes such as CO 2 fixation rates and CO 2 solubility can vary. In this study the engineered strains produced succinate during fluctuating pH conditions, achieving a titer of 5.0gL -1 after 10 days under shake flask conditions. Finally, these results demonstrate the potential for photomixotrophic production as a viable option for the large-scale production of succinate.

36 MATERIALS SCIENCE↗

DOE OSTI2021

Development of Efficient Process for Manufacturing of Thermoplastic Composites with Tailored Properties (CRADA 511)

This is a collaborative effort between Battelle Memorial Institute as manager and operator of Pacific Northwest National Laboratory (PNNL) and ESI North America Inc. (“ESI” or “Participant”) to apply computation and data analytics to the challenge of light weighting with a focus on the battery enclosures of electric vehicles (EVs). EVs use heavy batteries to increase range and power. A complex-shaped battery enclosure is required to meet a host of challenging performance requirements. The ability to virtually develop composite parts such as battery enclosure with tailored properties to meet required performance will be highly valuable to the automotive industry. However, efficient simulation of composite-manufacturing processes remains a challenging issue since simulation involves multiscale models in space and time, highly non-linear and anisotropic behavior, strongly coupled multi-physics, and complex geometries. This work will advance the state of the art by reducing the computational burden of composite optimization by using simulation data from a limited number of configurations off-line and then developing a reduced order model (ROM) using data analytics and machine learning (ML). Develop a data driven approach to link features of the material and manufacturing processes to the mechanical properties of thermoplastic composite parts.

42 ENGINEERING↗

DOE OSTI2026

Engineering Anti-Microbial Peptide Production in Fungi and Bioprocess Development (CRADA 502)

Crop Protection, specifically controlling agricultural diseases and pests, is essential for crop production at every agricultural scale. Biologics hold enormous potential in this equation to decrease use of more toxic pesticides. Currently, the widespread use of biologics in crop protection is severely hampered by the lack of available tools for their mass production. To ensure biologics are regulated under EPA Biopesticide regulations, the following must be true: The biopesticide is a naturally occurring substance or structurally similar and functionally identical to a naturally occurring substance with a history of exposure to humans and the environment demonstrating minimal toxicity. Since this pertains to the marketed product it is also essential that fermentation hosts have a history of exposure and safe use by humans This research focuses on developing a novel platform for the production and delivery of the cghSAMP peptide using the filamentous fungus Aspergillus niger. A. niger is a well-established industrial microorganism, widely recognized for its strong protein secretion capabilities, high-yield production of enzymes, and classification as a generally recognized as safe (GRAS) organism. By genetically modifying A. niger to express the cghSAMP peptide transgene, we aim to leverage the fungus's robust fermentation system to produce a high volume of the antimicrobial agent. This engineered system represents a potential breakthrough in sustainable disease management, offering an environmentally friendly method for localized application or large-scale production of a biopesticide designed to mitigate the effects of citrus greening disease. The main objectives of this project were: 1. Developing a high-yield and cost-efficient bioprocess to produce an AMP in a current ABF host or readily onboarded hosts. 2. Scale up the process in stirred tank reactors and produce at least 100 grams of AMP at 1- 5g/L

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

Discovering the Molecular Determinants of Phaeobacter inhibens Susceptibility to Phaeobacter Phage MD18

Bacteriophages are useful nonantibiotic therapeutics for bacterial infections as well as threats to industries utilizing bacterial agents. This study identified Phaeobacter virus MD18 , a phage antagonist of Phaeobacter inhibens , a bacterium with promising use as a probiotic for aquatic farming industries. Genomic analysis suggested that Phaeobacter phage MD18 has evolved to enhance its replication in P. inhibens by adopting favorable tRNA genes as well as through genomic sequence adaptation to resemble host codon usage. Lastly, a high-throughput analysis of P. inhibens transposon insertion mutants identified genes that modulate host susceptibility to phage MD18 and implicated the type IV pilus as the likely receptor recognized for adsorption. This study marks the first characterization of the relationship between P. inhibens and an environmentally sampled phage, which informs our understanding of natural threats to the bacterium and may promote the development of novel phage technologies for genetic manipulation of this host.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Workshop Report: Rural Electric Cooperative Distributed Energy Resource Business Model Development Workshops

The United States' energy future is being shaped by decades of technology innovation, rapidly changing consumer requirements, and governmental focus on securing reliable, low-cost energy sources. With expanded electrification and increased emphasis on grid reliability coupled with the heightened complexity and risks associated with transmission infrastructure, the use of distributed energy resources (DERs) is more commonly being considered a safe, efficient, and cost-effective way to maintain grid resilience and reliability. Amidst improvements in performance, decreasing costs, and unprecedented federal policy support for DERs, electric cooperatives are considering new opportunities to use these assets to ensure secure and reliable operations for millions of customers. In light of these developments, a series of workshops were held to collaborate on the development of business models for DERs in rural electric cooperative ecosystems. The first workshop was held July 11, 2024, at the Tri-State Generation and Transmission Association Inc. headquarters in Westminster, Colorado (hereafter referred to as the "Tri-State Workshop"). The second workshop was hosted by PNGC Power on Oct. 9, 2024, in Portland, Oregon (hereafter referred to as the "PNGC Power Workshop"). These workshops convened representatives from G&Ts, distribution cooperatives,2 the energy industry, the financial community, the U.S. government, national and regional organizations, and national laboratories to share perspectives on the unique needs, challenges, and opportunities that they face as DERs are further integrated into the grid. The workshops were designed to address the following objectives: (1) develop the enabling attributes of electric cooperative DER business models, (2) explore pathways for generating community benefits from these assets, and (3) identify technical challenges and market risks for DERs in rural electric cooperative service territories. This report outlines key findings and major themes identified by workshop participants that federal agencies, G&Ts, distribution cooperatives, and other entities can consider to advance electric cooperative DER business models.

24 POWER TRANSMISSION AND DISTRIBUTION↗

DOE OSTI2023

Initiation of fatty acid biosynthesis in Pseudomonas putida $\mathrm{KT2440}$

Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional β-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.

09 BIOMASS FUELS↗

DOE OSTI2025

Distributed Energy Resource Integration

This webinar focuses on the subject of DER integration, outlining the current industry state of the art and status of DER adoption in the US, a holistic view and roadmap of DER integration, DER interconnection standards, interconnection screening and study processes, interconnection automation, DER hosting capacity, AMI analytics, and non-wires alternatives.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗

DOE OSTI2025

Digital Droplet PCR and Mesocosm-Based Methods to Evaluate Biocontainment Strategies in a Native Soil Ecosystem

Genetically modified industrial production microbes and their associated bioproducts have emerged as an integral component of a sustainable bioeconomy. However, the rapid development of these innovative technologies raises biosecurity concerns, namely, the risk of environmental escape. Thus, the realization of a bioeconomy hinges not only on the development and deployment of microbial production hosts, but also on the development of secure biosystems and biocontainment designs. Current laboratory-based biocontainment testing systems do not accurately reflect the complexities found in natural environments, necessitating an environmentally relevant analysis pipeline that allows for the detection of rare escapees within a complex soil microbiome and differentiation between closely related strains. To this end, we have developed an approach that utilizes soil mesocosms and integrated digital droplet PCR (ddPCR) system to evaluate the efficacy of novel biocontainment strategies. We demonstrate the utility of this approach by modeling contamination with industrial microbial chasses versus their biocontained counterparts. Here we demonstrate the broad utility of this system by highlighting findings from strains of Saccharomyces cerevisiae that are contained with an inducible toxin anti-toxin system, strains of Synechocystis sp. PCC 6803 contained via gene knockout or toxin anti-toxin system, and strains of Escherichia coli that are contained via genomic recoding. We also show that ddPCR can be used to detect gene copies from E. coli equal to those counted by traditional spot plating assays. The resultant data demonstrates that this system has broad utility across diverse microbial chassis and biocontainment strategies and enables researchers to track the fate of our contaminating microbe with high sensitivity in the soil. The findings presented here support the use of this mesocosm-based approach to assess the environmental impact of industrial microbes and to validate biocontainment strategies.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗