Search NASA⌕ Search

SEARCH · Search NASA

Results for “advanced construction”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 307 records · Page 17

Measurements of the Flow in the Vicinity of an Additively Manufactured Turbine Leading-Edge Using X-Ray Particle Tracking Velocimetry

X-ray particle tracking velocimetry (PTV) is performed, for the first time, to measure the velocity field inside a leading-edge of a turbine blade made by laser-additive-manufacturing (LAM) process. The traditional showerhead holes were replaced by a porous matrix in the leading-edge. The flow through such a leading-edge piece cannot be faithfully recreated by traditional prototype testing methods due to the surface roughness and imperfections caused by LAM process. Hence, direct measurement is the only option. However, it is difficult to measure flow inside such pieces with traditional velocimetry measurements due to the existence of metallic walls. Moreover, small internal size and high flow speeds call for a measurement technique with high spatial and temporal resolutions. To address these issues, we performed time-resolved X-ray PTV using the Advanced Photon Source (APS) synchrotron facility at the Argonne National Laboratory (ANL). A hydraulic system was constructed to run water, mixed with seeding particles, through the leading-edge piece. A high-speed camera captured the images of the seeding particles, which were later processed to create particle tracks. The time-averaged velocity field showed distinct pairs of vortices located in front of the porous outlet inside the leading-edge piece. The inlet channel showed reversed flow due to partial obstruction by the porous inlet of the test piece. Such knowledge of the flow field inside a leading-edge of a turbine blade will help us to design better cooling paths leading to higher cooling efficiency and increased life-span of a turbine blade.

42 ENGINEERING↗

Creation Synthetic Data to Train a Digital Twin to Predict Reactor Operations

Understanding techniques to strengthen the nuclear safeguards regime is crucial in preventing nuclear proliferation due to recent advancements in the nuclear energy industry such as Generation IV reactors and microreactors. Prior to the construction of a nuclear power plant, it is necessary to understand the proliferation potential of the plant's reactor. Digital twins serve as a unique solution to recognizing reactor behavior indicative of nuclear proliferation. A digital twin is defined as a virtual model that works in unison to represent a physical asset, with a transference of data between the virtual and physical assets [1]. This work serves as validation for training a digital twin on synthetic data fabricated via means of Serpent reactor physics and point kinetics equations simulations. In this case this work is based on parameters of Idaho State University's AGN-201 reactor. The synthetic data can then be utilized to train machine learning models in the future to further investigate the utility of these methods. The accuracy of the predicted data is measured against real operational data to verify the reliability of the synthetic data creation methods and decide whether these methods should be used in the future to inform inspectors of a reactor's proliferation potential.

98 NUCLEAR DISARMAMENT, SAFEGUARDS, AND PHYSICAL P↗

The Analysis Description Language Ecosystem: Latest developments and physics applications

We present latest developments in Analysis Description Language (ADL), a declarative domain-specific language describing the physics algorithm of a HEP data analysis decoupled from software frameworks. Analyses written in ADL can be integrated into any framework for various tasks. ADL is a multipurpose construct with uses ranging from analysis design to preservation, reinterpretation, queries, visualisation, combination, etc. The most advanced infrastructure to execute ADL on events is the CutLang runtime interpreter. Recent technical developments include an automated interface with different data types, generation of the abstract syntax tree, a visualization tool that that auto-converts analysis flows to graphs, incorporation of trained machine learning models and a Jupyter-based plotting tool. We also report physics implications including a large scale LHC analysis implementation and validation effort for beyond the standard model reinterpretation purposes and studies with ATLAS and CMS open data.

Sekmen, Sezen [Kyungpook National Univ., Daegu (Ko↗

Design of a High-Pressure Fuel System for Use with Dimethyl Ether

The paper documents the modeling and experimental work on a common rail fuel injection system for Dimethyl Ether, a potential diesel substitute with a low carbon intensity signature. The DME fuel system is deployed on a light duty 2.2L compression ignition engine. The paper describes the injector optimization to shift to higher flows to account for the lower heating value and density of the DME when compared to diesel. The type of the injection system used for the DME application is an advanced rendering of the Common rail noted for a one-piece piston-needle injector construction and a solenoid driven spill valve featuring a pressure balanced poppet. A dedicated high-pressure fuel pump designed to pressurize DME is used. The design results in a fast acting open and close injection event, reduced leakage, with reduced cavitation in the fuel injector volume. Design parameters for system optimization included fill and spill orifices, needle lift, bias spring, and injector hole size. The design model provides good correlation of the instantaneous rates of injection with experiments across a wide range of pressure and injection timings. Proposed performance milestones for the design included similar DME injection duration to the diesel counterpart for same fuel energy injected into the cylinder to retain high engine cycle efficiency. Here, the dedicated DME design provided reduced hydraulic delays of 50%. Tests demonstrated sustained operation at pressures of 1000 bar, with capability to reach 1500bar. Durability tests showed no cavitation-deterioration over a 200-hour test cycle by means of spray imaging and hardware inspection.

De Ojeda, William [WM International Engineering, L↗

First Experiments: New Science Opportunities at the Spallation Neutron Source Second Target Station (abridged)

This brochure is an abridged version of a more comprehensive companion report. DOE proposes to build a Second Target Station (STS) at SNS to produce beams of cold neutrons with world-leading peak brightness at a repetition rate of 15 Hz, providing broad energy/wavelength ranges that can be used simultaneously. Construction of the STS will provide transformative capabilities that allow thousands of users from national laboratories, universities, and industry to address grand scientific challenges [Hemminger 2015], advance energy research, and accelerate industrial innovations through the combination of cold (long-wavelength) neutrons of unprecedented peak brightness and short neutron pulses with broad ranges of usable wavelengths or energy. The STS Project, now in progress at ORNL, will design, construct, install, and commission the facilities and equipment needed to create a world-leading source of cold neutrons of unprecedented peak brightness at SNS. Key aspects of the STS design, including preliminary descriptions of an illustrative suite of eight instruments, are presented in the section “The STS at SNS.” The capabilities offered by the STS instruments (22 when fully built out) will complement those of the FTS and HFIR, providing the United States with unparalleled resources for neutron scattering at the world’s leading high–peak brightness cold neutron source.

43 PARTICLE ACCELERATORS↗

Identifying Limitations of ASME Section III Division 5 For Advanced SMR Designs

This report provides an overview of the ASME Boiler & Pressure Vessel Section III, Division 5 rules for the design and construction of high temperature nuclear reactor components. The overview focuses on the application of the rules to the design of Small Modular Reactors (SMRs). The discussion covers the general ASME Code rules for base metal design and construction, the rules for designing weldments, and provides an overview of environmental degradation mechanisms affecting reactor structural materials. The analysis includes historical context on the development of the ASME design approach and a description of what actions could be taken to mitigate the gaps identified in the report. The report concludes with a summary of the key gaps identified in the rules, as they apply to SMR, and a list of recommendations on how those gaps might be addressed.

21 SPECIFIC NUCLEAR REACTORS AND ASSOCIATED PLANTS↗

Us participation in the construction of the T2k superfgd detector as part of the T2k ND280 upgrade

The neutrino physics field as a whole has made astonishing advances during the last two decades. Before 1998, there was no neutrino oscillation, meaning no neutrino mass and mixing. Today, neutrino oscillation is firmly established and we have measured all three mixing parameters and two mass parameters. Furthermore, these findings pave the way to determine the mass ordering/hierarchy and to explore Charge-Parity (CP) violation in the lepton sector, which may hold a critical key to our understanding of the matter−antimatter asymmetry in the universe, one of the most profound mysteries in science. In fact, T2K, a long baseline neutrino oscillation experiment based in Japan, has recently released the results that show initial hint of CP violation making the prospects of DUNE, a next generation long baseline neutrino oscillation experiment based in the US, discovering CP violation in neutrinos very bright.

72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS↗

Nuclear Safety [Vol. 29, No. 3, July-September 1988]

Nuclear Safety is a review journal that covers significant developments in the field of nuclear safety. Its scope includes the analysis and control of hazards associated with nuclear energy, operations involving fissionable materials, and the products of nuclear fission and their effects on the environment. Primary emphasis is on safety in reactor design, construction, and operation; however, the safety aspects of the entire fuel cycle, including fuel fabrication, spent-fuel processing, nuclear waste disposal, handling of radioisotopes, and environmental effects of these operations, are also treated. Table of Contents for this issue follows. GENERAL SAFETY CONSIDERATIONS: 259 Fifteenth Water Reactor Safety Information Meeting by E. G. Silver; CONTROL AND INSTRUMENTATION: 284 Reliability Technology to Improve and/or Maintain Emergency Diesel Generator Performance by S. Karimian and J. H. Taylor, 293 A Noise Diagnostics System for Operator Advice by G. Hessel, P. Liewers, P. Schumann, W. Schmitt, and F.-P. Weiss; PLANT SAFETY FEATURES: 307 A Passive Containment System for Advanced Light-Water Reactors by O. B. Falls, Jr., and F. W. Kleimola; ENVIRONMENTAL EFFECTS: 318 Data Base Construction for a Computerized Radiological Risk Investigation System by L. M. Hively, J. E. Nyquist, J. L. Bledsoe, and A. L. Sjoreen, 326 Erratum to "Radiation Hormesis and Nuclear Safety," Vol. 29, No. 1; OPERATING EXPERIENCES: 327 Operational Safety Experience and Passive Safety Testing at the Fast Flux Text Facility by Q. L. Baird, J. L. Rathbun, D. D. Stepnewski, R. L. Stover, and A. E. Waltar, 344 Backfilling of Independent Residual Heat Removal Systems in West Germany and Switzerland by G. Eckert and Y. Salomon, 353 Reactor Shutdown Experience Compiled by J. W. Cletcher, 356 Selected Safety-Related Events Compiled by G. A. Murphy, 363 Operating U.S. Power Reactors Compiled by E. G. Silver; RECENT DEVELOPMENTS: 384 General Administrative Activities Compiled by E. G. Silver, 390 Reports, Standards, and Safety Guides by D. S. Queener, 395 Status of Power-Reactor Projects Undergoing Licensing Review Compiled by E. G. Silver, 400 Proposed Rule Changes as of Mar. 31,1988; ANNOUNCEMENTS: 283 International ENS/ANS Conference on Thermal Reactor Safety "NUCSAFE 88", 317 Northwestern University Short Course on Radiation Safety, 407 Second International Seminar on Small- and Medium-Sized Nuclear Reactors, 407 International Workshop on New Developments in Occupational Dose Control and ALARA Implementation at Nuclear Power Plants and Similar Facilities, 408 Fourth International Topical Meeting on Nuclear Reactor Thermal Hydraulics (NURETH-4), 404 The Authors.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗

Nuclear Safety [Vol. 29, No. 3, July-September 1988]

Nuclear Safety is a review journal that covers significant developments in the field of nuclear safety. Its scope includes the analysis and control of hazards associated with nuclear energy, operations involving fissionable materials, and the products of nuclear fission and their effects on the environment. Primary emphasis is on safety in reactor design, construction, and operation; however, the safety aspects of the entire fuel cycle, including fuel fabrication, spent-fuel processing, nuclear waste disposal, handling of radioisotopes, and environmental effects of these operations, are also treated. Table of Contents for this issue follows. GENERAL SAFETY CONSIDERATIONS: 259 Fifteenth Water Reactor Safety Information Meeting by E. G. Silver; CONTROL AND INSTRUMENTATION: 284 Reliability Technology to Improve and/or Maintain Emergency Diesel Generator Performance by S. Karimian and J. H. Taylor, 293 A Noise Diagnostics System for Operator Advice by G. Hessel, P. Liewers, P. Schumann, W. Schmitt, and F.-P. Weiss; PLANT SAFETY FEATURES: 307 A Passive Containment System for Advanced Light-Water Reactors by O. B. Falls, Jr., and F. W. Kleimola; ENVIRONMENTAL EFFECTS: 318 Data Base Construction for a Computerized Radiological Risk Investigation System by L. M. Hively, J. E. Nyquist, J. L. Bledsoe, and A. L. Sjoreen, 326 Erratum to "Radiation Hormesis and Nuclear Safety," Vol. 29, No. 1; OPERATING EXPERIENCES: 327 Operational Safety Experience and Passive Safety Testing at the Fast Flux Text Facility by Q. L. Baird, J. L. Rathbun, D. D. Stepnewski, R. L. Stover, and A. E. Waltar, 344 Backfilling of Independent Residual Heat Removal Systems in West Germany and Switzerland by G. Eckert and Y. Salomon, 353 Reactor Shutdown Experience Compiled by J. W. Cletcher, 356 Selected Safety-Related Events Compiled by G. A. Murphy, 363 Operating U.S. Power Reactors Compiled by E. G. Silver; RECENT DEVELOPMENTS: 384 General Administrative Activities Compiled by E. G. Silver, 390 Reports, Standards, and Safety Guides by D. S. Queener, 395 Status of Power-Reactor Projects Undergoing Licensing Review Compiled by E. G. Silver, 400 Proposed Rule Changes as of Mar. 31,1988; ANNOUNCEMENTS: 283 International ENS/ANS Conference on Thermal Reactor Safety "NUCSAFE 88", 317 Northwestern University Short Course on Radiation Safety, 407 Second International Seminar on Small- and Medium-Sized Nuclear Reactors, 407 International Workshop on New Developments in Occupational Dose Control and ALARA Implementation at Nuclear Power Plants and Similar Facilities, 408 Fourth International Topical Meeting on Nuclear Reactor Thermal Hydraulics (NURETH-4), 404 The Authors.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗

Empowering Lineworkers: The Case for Active Exoskeletons in Utility Work

Exoskeletons have evolved from early medical prototypes to advanced systems capable of addressing physical demands in various industries. This report explores the potential of active exoskeleton technology within the utility sector, focusing on its application for linemen who face significant risks of work-related musculoskeletal disorders (WMSDs). By analyzing existing literature on exoskeletons across industries such as construction, manufacturing, and military, the study identifies a gap in utility-specific applications. Task-specific design features like gravity compensation, limb support, and advanced safety measures, improve exoskeletons’ potential to alleviate physical strain, reduce workplace injuries, and enhance productivity. This review emphasizes the need for targeted research and development to optimize exoskeleton designs for the utility sector to provide benefits for workers, companies, and the broader community.

60 APPLIED LIFE SCIENCES↗

The Profiled Feldman-Cousins Method for Confidence Interval Construction for the Nova 3-Flavor Oscillation Analysis

The small interaction cross-section of neutrinos makes experimental neutrino physics particularly responsive to technological advancements. A significant development leveraged by the NOvA experiment is large-scale parallel processing, enabling novel computational approaches to longstanding experimental challenges. Central to managing the resulting high-throughput data is NOvA’s implementation of the Freight Train model, designed for efficient data production and handling.This dissertation details the methodology and execution of the NOvA 2024 3-Flavor Oscillation Analysis, supported by a comprehensive dataset spanning ten years. It emphasizes frequentist results refined through the Feldman-Cousins (FC) technique, specifically addressing confidence interval corrections in parameter estimation. The computational intensity associated with Feldman-Cousins arises from extensive Monte Carlo simulations, which were substantially mitigated through parallel computing on the Perlmutter supercomputer at the National Energy Research Scientific Computing Center (NERSC), employing the MPI framework.To further enhance computational efficiency, an Importance Sampling method is introduced and evaluated, demonstrating significant potential to reduce complexity, particularly in exploring extreme parameter space regions. This thesis presents both the successful application of advanced computational resources and the development of sophisticated statistical techniques, aiming to enhance the precision and scope of neutrino oscillation analyses.

Dye ajdye11190@gmail.com, Andrew Joseph [Mississip↗

BETO 2021 Peer Review: Algae Biotechnology Partnership - Technology Area Session: Advanced Algae Systems

Development of advanced genetic and genomic tools for targeted algal metabolic engineering pursuits will be integral to achieving target biomass productivity and, ultimately, the BETO goal of cost-competitive biofuels derived from algal biomass by 2022. However, at present, broad-host-range tool development is currently hindered by strain-specific negative regulatory mechanisms. Indeed, the biological processes controlling algal transcription and translation are subject to complex host regulation, which often presents hurdles for targeted genetic engineering strategies. Advanced genetic approaches offer a means to rewire these regulatory systems and/or introduce novel functionality into algal biocatalysts. Synthetic systems biology approaches also present a means to construct novel genetic regulatory networks and rewire natural biological systems to establish an “orthogonal central dogma,” wherein non-native control elements are introduced into or evolved in host microbes for bypass of host control. To this end, the Algae Biotechnology Partnership aims to develop advanced genetic editing tools and synthetic and orthogonal genetic regulatory systems to enable universal metabolic engineering strategies in top-candidate deployment algal strains. Successful development will ultimately open the door for targeted strain-engineering strategies, aimed at maximizing algal outdoor biomass production, composition, and strain robustness.

biocatalysis↗

State-to-State Molecular Reactions in the Ultracold Regime (Final Scientific Report)

We have achieved the proposed goal to experimentally probe the AB + CD and AB + C types of reactions with state-to-state resolution, which we also compared to advanced theoretical calculations to help elucidate the role of quantum mechanics in the processes of bond breakage and formation. Our approach uses reactants that are prepared at ultracold temperatures (< 1µK) such that the quantum effects of translational motion are an important factor. Specific example reactions, including the potassium-rubidium metathesis reaction KRb + KRb → K 2 + Rb 2 as well as the atom exchange reaction Rb + KRb → Rb2 + K, are chosen because the technology of quantum internal and motional state control of these types of molecules is particularly advanced. The results for the entire funding period are fruitful. For the majority of this grant, we have constructed a one-of-the-kind quantum degenerate gas apparatus that integrates ion detection and velocity map imaging capabilities, allowing us to explore the KRb + KRb → K 2 + Rb 2 bimolecular reaction in detail. Specifically, we first verified such a reaction indeed proceed at ultracold temperatures by direct detection of reaction products. We then mapped out the complete product state distribution, which was compared to a state-counting model based on statistical theory. Our results show an overall agreement with the statistical state counting model, but also reveal several deviating state-pairs. An exact quantum calculation for molecule-molecule collisions, that is needed to understand these deviations, is however beyond the current state-of-the-art. Beside scrutinizing the reaction products, we also directly observe the reaction intermediate complex, which was quite a surprise to us. The intermediate complexes are long-lived and can interact with the inferred light that we use to trap the ultracold gas. After molecule-molecule collisions, we then explored the more theoretically tractable Rb + KRb reaction, which is endothermic. Surprisingly, we observed an exceedingly long-lived KRb$^*_2$ collisional complexes, with our experimentally measured complex lifetime deviating from conventional theoretical calculations by five orders of magnitude. This discrepancy has motivated many explorations of possible underlying causes, though no model yet captures this phenomenon completely. In the final year and the work that continues today, we extend upon these atom-molecule collision experiments to explore the origin of the long-lived KRb$^*_2$ complex lifetime and develop means to control the outcome of the reaction complex. The 5-year funded work advanced our understanding of chemical reactions at the lowest possible temperatures and at the same time opened up many new questions that are beyond our initial imaginations.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Multi-Modal Characterization of Interfacial Corrosion of Ni-based Alloys in Chloride-based Molten Salts

The United States Department of Energy (DOE) is committed to the advancement of nuclear reactor technology through initiatives such as the Advanced Reactor Development Program (ARDP), to diversify the United States energy portfolio towards more sustainable energy options. The ARDP includes demonstration by industry partners of molten salt fast reactors (MSRs). Construction of molten salt reactor technology requires qualified nuclear structural materials. Unfortunately, there are no current materials that meet current qualification requirements dictated by the Nuclear Regulatory Commission for construction of MSRs. Adapting current structural material qualifications requires expansion of our current knowledgebase on corrosion performance. In this investigation, we assess microstructural changes in a Ni-based superalloy after exposure to a chloride-containing salt system through a correlated multi-modal approach combining several advanced characterization techniques. Namely we will highlight the impact of grain boundary phenomena at the onset of corrosion attack, including the role of intergranular crack propagation and development of internal corrosion products. The findings from this investigation will further expand our assessment of the corrosion performance of structural materials being investigated for construction of MSR components.

36 - MATERIALS SCIENCE↗

Solution and sensitivity analysis of nonlinear equations using a hypercomplex-variable Newton-Raphson method

Here, the classical Newton-Raphson (NR) method for solving nonlinear equations is enhanced in two ways through the use of hypercomplex variables and algebra. In particular, i) the Jacobian is computed in a highly accurate and automated way, and ii) the derivative of the solution to the nonlinear equations is computed with respect to any parameter contained within the system of equations. These advances provide two significant enhancements in that it is straightforward to provide an accurate Jacobian and to construct a reduced order model (ROM) of arbitrary order with respect to any parameter of the system. The ROM can then be used to approximate the solution for other parameter values without requiring additional solutions of the nonlinear equations. Several case studies are presented including 1D and 2D academic examples with fully functioning Python code provided. Additionally, a case of study of the catenary of an elastic cable subject to its own weight and a vertical point load. Derivatives up to 10th order were computed with respect to material, loading, and geometrical parameters. The derivatives were used to generate reduced order models of the cable deformation and reaction forces at its ends with respect to multiple input parameters. Results show that from a single hypercomplex evaluation of the cable under a single vertical point load, it is possible to generate an accurate reduced order model capable of predicting the cable deformation with 1.5 times the load in the opposite direction and with 3.5 times the load in the same direction without resolving the system of equations.

97 MATHEMATICS AND COMPUTING↗

Cement and concrete as carbon sinks: Transforming a climate challenge into a carbon storage opportunity

Cement and concrete, while traditionally recognized as the main contributors to anthropogenic CO 2 emissions, also have untapped capacity to serve as substantial and scalable carbon sinks. This perspective examines how engineered mineral carbonation can transform cement-based materials into functional carbon storage systems, generating both environmental and economic value. We review the fundamental mechanisms of CO 2 uptake in cementitious systems, highlighting current limitations in reaction kinetics, phase control, and durability under varying environmental conditions. Emphasis is placed on the utilization of alkaline industrial residues and emerging magnesium-based cements, which offer synergistic pathways for carbon sequestration and circular resource use. We further assess the performance trade-offs associated with CO 2 uptake and the feasibility of deploying these technologies on industrial scales. A strategic roadmap is proposed that integrates scientific innovation, regulatory alignment, and carbon accounting in the life cycle to accelerate the adoption of carbon-storing concrete. This perspective provides a comprehensive framework to advance cement and concrete as engineered carbon sinks and supports the transition to a climate-positive construction industry.

Carbon storage↗

Three-dimensional mapping of mineral in intact shark centra with energy dispersive x-ray diffraction

The centra of shark vertebrae consist of cartilage mineralized by a bioapatite similar to bone's carbonated hy-droxyapatite, and, without a repair mechanism analogous to remodeling in bone, these structures still survive millions of cycles of high-strain loading. The main structures of the centrum are an hourglass-shaped double cone and the intermedialia which supports the cones. Little is known about the nanostructure of shark centra, spe-cifically the relationship between bioapatite and cartilage fibers, and this study uses energy dispersive diffraction (EDD) with polychromatic synchrotron x-radiation to study the spatial organization of the mineral phase and its crystallographic texture. The unique energy-sensitive detector array at beamline 6-BM-B, the Advanced Photon Source, enables EDD to quantify the texture within each sampling volume with one exposure while constructing 3D maps via specimen translation across the sampling volume. Herein this study maps a centrum from two shark orders, a carcharhiniform and a lamniform, with different intermedialia structures. In the blue shark (Prionace glauca, Carcharhiniformes), the bioapatite's c-axes are oriented laterally within the centrum's cone walls but axially within the wide wedges of the intermedialia; the former is interpreted to resist lateral deformation, the latter to support axial loads. In the shortfin mako (Isurus oxyrinchus, Lamniformes), there is some tendency for c-axis variation with position, but the situation is unclear because one dimension of the sampling volume is consid-erably larger than the thickness and spacing of the intermedialia's radially-oriented lamellae. Because elastic modulus in collagen plus bioapatite mineralized tissues varies significantly with both volume fraction of bio-apatite and crystallographic texture, the present 3D EDD-derived maps should inform future 3D numerical models of shark centra under applied load.

59 BASIC BIOLOGICAL SCIENCES↗

Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p -coumarate

We report Pseudomonas putida KT2440 is a well-studied bacterium for the conversion of lignin-derived aromatic compounds to bioproducts. The development of advanced genetic tools in P. putida has reduced the turnaround time for hypothesis testing and enabled the construction of strains capable of producing various products of interest. Here, we evaluate an inducible CRISPR-interference (CRISPRi) toolset on fluorescent, essential, and metabolic targets. Nuclease-deficient Cas9 (dCas9) expressed with the arabinose (8K)-inducible promoter was shown to be tightly regulated across various media conditions and when targeting essential genes. In addition to bulk growth data, single cell time lapse microscopy was conducted, which revealed intrinsic heterogeneity in knockdown rate within an isoclonal population. The dynamics of knockdown were studied across genomic targets in exponentially-growing cells, revealing a universal 1.75 ± 0.38 hour quiescent phase after induction where 1.5 ± 0.35 doublings occur before a phenotypic response is observed. To demonstrate application of this CRISPRi toolset, β-ketoadipate, a monomer for performance-advantaged nylon, was produced at a 4.39 ± 0.5 g/L and yield of 0.76 ± 0.10 mol/mol from p-coumarate, a hydroxycinnamic acid that can be derived from grasses. These cultivation metrics were achieved by using the higher strength IPTG (1K)-inducible promoter to knockdown the pcaIJ operon in the βKA pathway during early exponential phase. This allowed the majority of the carbon to be shunted into the desired product while eliminating the need for a supplemental carbon and energy source to support growth and maintenance.

59 BASIC BIOLOGICAL SCIENCES↗