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DOE OSTI2025

Microreactor Optimization Using Simulation And Economics (mouse)

Microreactor Optimization Using Simulation and Economics (MOUSE) is a tool that integrates both nuclear microreactor design and reactor economics to provide comprehensive evaluations and optimizations. This tool enables stakeholders to explore the interplay between technical and economic variables, guiding them towards effective and competitive microreactor solutions. For the reactor core simulations, MOUSE leverages the OpenMC Monte Carlo Particle Transport Code to perform detailed core simulations for various microreactor designs. The included OpenMC models are 2D core designs of a Liquid Metal Thermal Microreactor (LMTR), a Gas-Cooled TRISO-Fueled Microreactor (GCMR), and a Heat Pipe Microreactor. Beyond core design, MOUSE includes simplified calculations for: - Calculating the masses of heat exchangers within the system. - Mechanical power of pumps. - Estimating the area occupied by various buildings within the nuclear plant. For the economic analysis, MOUSE provides detailed bottom-up cost estimates, encompassing a wide range of costs including preconstruction costs, direct costs, indirect costs, training costs, financial costs, operation & maintenance (O&M) costs, and fuel costs. These cost estimations are developed using data from the MARVEL project and additional literature sources, enabling the calculation of total capital costs and levelized cost of energy for both first-of-a-kind and nth-of-a-kind microreactors. MOUSE also enables analysis of the cost drivers and competitiveness in the electricity market. MOUSE allows users to modify a wide array of technical and economic parameters to evaluate different scenarios and their impacts. Examples of these parameters include: Fuels, coolants, or reflector materials Enrichment levels Control drum materials and geometry Fuel pin geometry and materials Moderator pin geometry and materials Reactor core and reflector dimensions Packing factor for the TRISO particles Nuclear reactor power and reactor burnup Number of sensors Shielding thickness Reactor vessel and guard vessel dimensions Operational staff requirements Number of emergency shutdowns Levelization period Interest rate Construction duration Since MOUSE is powered by the WATTS toolkit, it supports optimization studies, parametric analyses, and uncertainty calculations/propagation. The optimization techniques enable users to identify optimal design and economic configurations. The parametric analysis tools allow users to explore the sensitivity of various parameters, while uncertainty propagation helps quantify the impact of uncertainties on overall performance and cost. User Interface and Workflow: Currently, MOUSE is a command-line-based tool. Users can input various reactor design or economic parameters, modify the designs, run simulations, and visualize results through comprehensive data visualization and reporting capabilities. The typical workflow involves setting up the reactor model, defining economic parameters, running simulations, and analyzing the results to make informed decisions. By combining advanced design calculations with detailed economic modeling, MOUSE provides a robust framework for optimizing nuclear microreactor technologies, enhancing their competitiveness, and guiding stakeholders towards innovative and cost-effective solutions.

Hanna, Botros [Idaho National Laboratory (INL), Id↗

DOE OSTI2021

TracKlinic: Diagnosis of Challenge Factors in Visual Tracking

Generic visual object tracking is difficult due to many challenge factors (e.g., occlusion, blur, etc.). Each of these factors may cause serious problems for a tracker, and when they work together can make things even more complicated. Despite a great amount of efforts devoted to understanding the behavior of trackers, reliable and quantifiable ways for studying the per factor tracking behavior remain barely available. Addressing this issue, in this paper we contribute to the community a tracking diagnosis toolkit, TracKlinic, for diagnosis of challenge factors of tracking algorithms. TracKlinic consists of two novel components focusing on the data and analysis aspects, respectively. For the data component, we carefully prepare a set of 2,390 annotated videos, each involving one and only one major challenge factor. When analyzing an algorithm for a specific challenge factor, such one-factor-per-sequence rule greatly inhibits the disturbance from other factors and consequently leads to more faithful analysis. For the analysis component, given the tracking results on all sequences, it investigates the behavior of the tracker under each individual factor and generates the report automatically. With TracKlinic, a thorough study is conducted on ten state-of-the-art trackers on nine challenge factors (including two compound ones). The results suggest that, heavy shape variation and occlusion are the two most challenging factors faced by most trackers. Besides, out-of-view, though does not happen frequently, is often fatal. By sharing TracKlinic1, we expect to make it much easier for diagnosing tracking algorithms, and to thus facilitate developing better ones.

97 MATHEMATICS AND COMPUTING↗

NASA NTRS2014

Applications of Satellite Remote Sensing for Response to and Recovery from Meteorological Disasters

Numerous on‐orbit satellites provide a wide range of spatial, spectral, and temporal resolutions supporting the use of their resulting imagery in assessments of disasters that are meteorological in nature. This presentation will provide an overview of recent use of Earth remote sensing by NASA's Short‐term Prediction Research and Transition (SPoRT) Center in response to disaster activities in 2012 and 2013, along with case studies supporting ongoing research and development. The SPoRT Center, with support from NASA's Applied Sciences Program, has explored a variety of new applications of Earth‐observing sensors to support disaster response. In May 2013, the SPoRT Center developed unique power outage composites representing the first clear sky view of damage inflicted upon Moore and Oklahoma City, Oklahoma following the devastating EF‐5 tornado that occurred on May 20. Subsequent ASTER, MODIS, Landsat‐7 and Landsat‐8 imagery help to identify the damaged areas. Higher resolution imagery of Moore, Oklahoma were provided by commercial satellites and the recently available International Space Station (ISS) SERVIR Environmental Research and Visualization System (ISERV) instrument. New techniques are being explored by the SPoRT team in order to better identify damage visible in high resolution imagery, and to monitor ongoing recovery for Moore, Oklahoma. This presentation will provide an overview of near real‐time data products developed for dissemination to SPoRT's partners in NOAA's National Weather Service, through collaboration with the USGS and other federal agencies. Specifically, it will focus on integration of various data sets within the NOAA National Weather Service Damage Assessment Toolkit, which allows meteorologists in the field to consult available satellite imagery while performing their damage assessment.

Molthan, Andrew I.↗

NASA NTRS2014

Applications of Satellite Remote Sensing for Response to and Recovery from Meteorological Disasters

Molthan, Andrew L.↗

DOE OSTI2021

Optimization and Augmentation for Data Parallel Contour Trees

Contour trees are used for topological data analysis in scientific visualization. While originally computed with serial algorithms, recent work has introduced a vector-parallel algorithm. Furthermore, this algorithm is relatively slow for fully augmented contour trees which are needed for many practical data analysis tasks. We therefore introduce a representation called the hyperstructure that enables efficient searches through the contour tree and use it to construct a fully augmented contour tree in data parallel, with performance on average 6 times faster than the state-of-the-art parallel algorithm in the TTK topological toolkit.

97 MATHEMATICS AND COMPUTING↗

NASA NTRS1989

Scientific visualization in computational aerodynamics at NASA Ames Research Center

The visualization methods used in computational fluid dynamics research at the NASA-Ames Numerical Aerodynamic Simulation facility are examined, including postprocessing, tracking, and steering methods. The visualization requirements of the facility's three-dimensional graphical workstation are outlined and the types hardware and software used to meet these requirements are discussed. The main features of the facility's current and next-generation workstations are listed. Emphasis is given to postprocessing techniques, such as dynamic interactive viewing on the workstation and recording and playback on videodisk, tape, and 16-mm film. Postprocessing software packages are described, including a three-dimensional plotter, a surface modeler, a graphical animation system, a flow analysis software toolkit, and a real-time interactive particle-tracer.

Bancroft, Gordon V.↗

DOE OSTI2018

Matminer: An open source toolkit for materials data mining

As materials data sets grow in size and scope, the role of data mining and statistical learning methods to analyze these materials data sets and build predictive models is becoming more important. This manuscript introduces matminer, an open-source, Python-based software platform to facilitate data-driven methods of analyzing and predicting materials properties. Matminer provides modules for retrieving large data sets from external databases such as the Materials Project, Citrination, Materials Data Facility, and Materials Platform for Data Science. It also provides implementations for an extensive library of feature extraction routines developed by the materials community, with 47 featurization classes that can generate thousands of individual descriptors and combine them into mathematical functions. Finally, matminer provides a visualization module for producing interactive, shareable plots. These functions are designed in a way that integrates closely with machine learning and data analysis packages already developed and in use by the Python data science community. We explain the structure and logic of matminer, provide a description of its various modules, and showcase several examples of how matminer can be used to collect data, reproduce data mining studies reported in the literature, and test new methodologies.

97 MATHEMATICS AND COMPUTING↗

DOE OSTI2021

Validation of the HyRAM+ physics models for use with propane

The Hydrogen Risk Assessment Model Plus (HyRAM+) toolkit combines quantitative risk assessment with simulations of unignited dispersion, ignited turbulent diffusion flames, and indoor accumulation with delayed ignition of fuels. HyRAM+ is differentiated from HyRAM in that it includes models and leak data for other alternate fuels. The models of the physical phenomena need to be validated for each of the fuels in the toolkit. This report shows the validation for propane which is being used as a surrogate for autogas, which is a mixture of propane and butane and used in internal combustion engines in vehicles. For flame length comparisons, five previously published experiments from peer reviewed journals were used to validate our models. The validation looked at flame lengths and flame widths with respect to different leak diameters, mass flow rates, and source pressures. Most of the sources included more than one set of experimental data, which were collected using different methods (CCD cameras, IR visualization etc.). In general, HyRAM+ overpredicts the flame lengths by around 65%. For heat and radiation models, we compared the heat flux and radiation data reported from two different sources to the values calculated by HyRAM+. For higher mass flow rates, the HyRAM+ calculated flame length results gave a better estimate of what is found in the experiments (65% error), but a higher error (85%) is observed between the HyRAM+ calculated lengths and the experimental flame lengthsfor lower mass flows. Some differences can be attributed to outdoor environmental effects (i.e. wind speed) and uncertainties in jet flame shapes. The propane flame trajectory is predicted for a high Reynolds number case with Re = 12,500 and a low Reynolds number case where Re = 2,000. The Re=12,500 case which is momentum dominated matches well with the experimental flame trajectory, but the agreement for the bouancy driven low Reynolds number case is not as good. Dispersion modeling for unignited propane was also analyzed. We compared the mole fraction, mixture fraction, mean velocity, concentration half width, and inverse mass concentration over an axial distance from different credible journals to the values calculated by HyRAM+. The results display good agreement but generally, HyRAM+ predicts a wider profile for mole fraction and mixture fraction experiments. Overall, HyRAM+’s results are reasonable for predicting the flame length, heat flux, flame trajectory, and dispersion for propane and can be used in risk analyses

08 HYDROGEN↗

DOE OSTI2017

URBAN-NET-TOOLKIT

Strengthening and maintaining secure, functioning, and resilient Critical Infrastructure Systems (CISs) is one of our nation's primary goals, and it requires proactive and coo0rdinated efforts among federal, state, local, public and private owners and operators of CISs. Recent worldwide extreme events have shown how interdependencies across CISs (electric grid, telecommunications, transportation, water networks, etc.) can drastically increase system vulnerability. To understand these complex interdependencies, ORNL has instantiated Urban-NET, a novel decision support system for CIS operators who need to identify vulnerabilities for minimizing the cascading impacts - saving utilities, the federal and state government, and private sector companies potentially billions by avoiding a complete CI failure and/or shutdown. The developed system includes a stack of software tools for preprocessing, modeling, interconnecting multiple CIS datasets as an integrated heterogeneous graph (i.e. network), identifying vulnerabilities through topological analysis, and predicting consequences based on simulations along with visualization.

Chinthavali, Supriya↗

DOE OSTI2022

GridDS: Data Science Toolkit for Energy Grid Data

According to the U.S. Energy Information Administration (EIA), the demand for energy is expected to increase 50% by the year 20501. While energy standards, such as the Institute of Electrical and Electronics Engineers (IEEE) Standard 1547, (Basso 2015) and monitoring with wide area management systems (WAMS) (Liu 2017, Zhou 2016) have enabled large scale data collection and storage, the application of this data in mitigating costs associated with increased consumer demand is an ongoing focus for energy research. This ubiquitous data collection presents a promising opportunity for machine learning and data science to improve efficiency of distributed energy resources (DERs). The GridDS software toolkit is designed to leverage advanced metering infrastructure (AMI), outage management systems data (OMS), Supervisory control Data Acquisition (SCADA), and geographic information systems (GIS) to forecast future energy demands and detect incipient grid failures. GridDS is a python software library designed to be modular and generalizable to data recorded by DERs. In adapting to disparate datasets recorded by various WAMS, GridDS provides a range of unique functionality not presently implemented in current WAMS which have highly specific software infrastructure by design. GridDS functionality ranges from data specification and preparation, to training and validation for state of the art machine learning, to interactive data visualization. For data intake, GridDS combines: Pandera: a library for creating data specifications. TimeScaleDB: a postgresSQL database infrastructure for efficient storage of timeseries data. Dataset class: A custom dataset class / interface that ensures modularity between a range of synthetic and live recorded datasets. Is

Ladd, Alexander↗

NASA NTRS2014

Using ISERV and Commercial Satellite Imagery to Assess and Monitor Recovery Efforts in Urban Damaged Areas

NASA's Short-term Prediction, Research, and Transition (SPoRT) Center uses a wide array of satellites to monitor and assess the impacts of natural disasters, with support from NASA's Applied Sciences Program. One of the newest sensors SPoRT is utilizing in these activities is the International Space Station (ISS) SERVIR Environmental Research and Visualization System (ISERV) instrument. ISERV provides a unique view of the areas impacted and will play a big role in monitoring the recovery these areas. High‐resolution commercial satellite data is also used to monitor urban areas that have been impacted by natural disasters. SPoRT is developing techniques to measure the extent of these disasters and to monitor recovery. Several of these techniques include semi‐automatic feature detection and change as well as developing an experimental damage assessment based upon the visible damage observed by the satellites. Furthermore, throughout these activities SPoRT hopes to provide additional data to the NOAA National Weather Service Damage Assessment Toolkit, which will help to supplement those activities being performed in the field.

Bell, Jordan R.↗

NASA NTRS2011

Link Analysis in the Mission Planning Lab

The legacy communications link analysis software currently used at Wallops Flight Facility involves processes that are different for command destruct, radar, and telemetry. There is a clear advantage to developing an easy-to-use tool that combines all the processes in one application. Link Analysis in the Mission Planning Lab (MPL) uses custom software and algorithms integrated with Analytical Graphics Inc. Satellite Toolkit (AGI STK). The MPL link analysis tool uses pre/post-mission data to conduct a dynamic link analysis between ground assets and the launch vehicle. Just as the legacy methods do, the MPL link analysis tool calculates signal strength and signal- to-noise according to the accepted processes for command destruct, radar, and telemetry assets. Graphs and other custom data are generated rapidly in formats for reports and presentations. STK is used for analysis as well as to depict plume angles and antenna gain patterns in 3D. The MPL has developed two interfaces with the STK software (see figure). The first interface is an HTML utility, which was developed in Visual Basic to enhance analysis for plume modeling and to offer a more user friendly, flexible tool. A graphical user interface (GUI) written in MATLAB (see figure upper right-hand corner) is also used to quickly depict link budget information for multiple ground assets. This new method yields a dramatic decrease in the time it takes to provide launch managers with the required link budgets to make critical pre-mission decisions. The software code used for these two custom utilities is a product of NASA's MPL.

McCarthy, Jessica A.↗

DOE OSTI2024

Transportation and System Analysis Collaborations in Support of a Federal Consolidated Interim Storage Facility

The U.S. Department of Energy’s Integrated Waste Management (IWM) program under the Office of Nuclear Energy is planning for the future transportation, storage, and eventual disposal of spent nuclear fuel (SNF) and high-level radioactive waste (HLW) from nuclear power plant and waste custodian sites across the United States. To better enable informed decision-making regarding the back end of the nuclear fuel cycle, the IWM program has been sponsoring the development and application of system analysis tools capable of analyzing various options for managing SNF and HLW. With these tools, integrated waste management system (IWMS) architecture analyses are being conducted to support the future deployment of a comprehensive nuclear waste management system that considers all major back-end aspects of the nuclear fuel cycle (i.e., transportation, storage, and disposal). System analyses and assessments typically use these modeling and simulation tools to investigate implications of changes in various assumptions and parameters such as acceptance rates, receipt logic, facility capacities and capabilities, use of standardized canisters, start and stop dates of facilities, etc. The Next Generation System Analysis Model (NGSAM) is an agent-based simulation toolkit that is used for a system-level simulation and analysis focused on SNF management in the United States. NGSAM’s primary purpose is to provide a system analyst with capabilities to model the IWMS and gain insights into SNF and HLW management alternatives including the impact of system choices, associated cost estimates, and development of integrated yet flexible approaches. An analyst using NGSAM can define several factors like the number of storage facilities, capacity at each facility, transportation schedules, shipment rates, and other conditions. IWM is also developing the Stakeholder Tool for Assessing Radioactive Transportation (START). START is a web-based geospatial tool developed to provide the visualization and initial evaluation of transportation options associated with future SNF and HLW shipment planning and operations. This includes characterizing safety, economic, and environmental conditions at and in proximity to shipment origins as well as along prospective transportation routes. Information from the START tool can be presented/shared as maps, graphics, geospatial files, and tabular form to enable easy data representation and export functionality. The system analysis, NGSAM, and START teams have been working closely for several years before formalizing this collaboration. START provides the SNF transportation routing information for use in NGSAM. System analysts use the NGSAM tool to generate results (schedule, costs, infrastructure acquisitions, shipment rates, etc.). Subject-matter experts and system analysts work together to inform how NGSAM should model the waste management system. Specifically, this paper discusses the collaborations between the system analysis, NGSAM, and START teams and their accomplishments over the past year. Some examples of these efforts include calibrating and adding data to the START output files to meet NGSAM needs, gaining a better understanding of START data used in NGSAM as well as how updates in START data could result in an improved NGSAM analysis. Detailed examples of various tasks that have been performed by the team will be discussed in the full paper. Continued efforts in this direction are expected in the coming years.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗

NASA NTRS2013

Near Real-Time Applications of Earth Remote Sensing for Response to Meteorological Disasters

Numerous on‐orbit satellites provide a wide range of spatial, spectral, and temporal resolutions supporting the use of their resulting imagery in assessments of disasters that are meteorological in nature. This presentation will provide an overview of recent use of Earth remote sensing by NASA's Short‐term Prediction Research and Transition (SPoRT) Center in response to disaster activities in 2012 and 2013, along with case studies supporting ongoing research and development. The SPoRT Center, with support from NASA's Applied Sciences Program, has explored a variety of new applications of Earth‐observing sensors to support disaster response. In May 2013, the SPoRT Center developed unique power outage composites representing the first clear sky view of damage inflicted upon Moore and Oklahoma City, Oklahoma following the devastating EF‐5 tornado that occurred on May 20. Subsequent ASTER, MODIS, Landsat‐7 and Landsat‐8 imagery help to identify the damaged area. Higher resolution imagery of Moore, Oklahoma were provided by commercial satellites and the recently available International Space Station (ISS) SERVIR Environmental Research and Visualization System (ISERV) instrument. New techniques are being explored by the SPoRT team in order to better identify damage visible in high resolution imagery, and to monitor ongoing recovery for Moore, Oklahoma. Other applications are being developed to refine light source detections with the VIIRS day‐night band and to map hail during the growing season through combination of available satellite and radar imagery. The aforementioned products and support are not useful unless they are distributed in a timely manner and within an appropriate decision support system. This presentation will provide an update on ongoing activities to support inclusion of these data sets within the NOAA National Weather Service Damage Assessment Toolkit, which allows meteorologists in the field to consult available satellite imagery while performing their damage assessment.

Molthan, Andrew L.↗

NASA NTRS2013

Near Real-Time Applications of Earth Remote Sensing for Response to Meteorological Disasters

Numerous on‐orbit satellites provide a wide range of spatial, spectral, and temporal resolutions supporting the use of their resulting imagery in assessments of disasters that are meteorological in nature. This presentation will provide an overview of recent use of Earth remote sensing by NASA's Short‐term Prediction Research and Transition (SPoRT) Center in response to disaster activities in 2012 and 2013, along with case studies supporting ongoing research and development. The SPoRT Center, with support from NASA's Applied Sciences Program, has explored a variety of new applications of Earth‐observing sensors to support disaster response. In May 2013, the SPoRT Center developed unique power outage composites representing the first clear sky view of damage inflicted upon Moore and Oklahoma City, Oklahoma following the devastating EF‐5 tornado that occurred on May 20. Subsequent ASTER, MODIS, Landsat‐7 and Landsat‐8 imagery help to identify the damaged area. Higher resolution imagery of Moore, Oklahoma were provided by commercial satellites and the recently available International Space Station (ISS) SERVIR Environmental Research and Visualization System (ISERV) instrument. New techniques are being explored by the SPoRT team in order to better identify damage visible in high resolution imagery, and to monitor ongoing recovery for Moore, Oklahoma. Other applications are being developed to refine light source detections with the VIIRS day‐night band and to map hail during the growing season through combination of available satellite and radar imagery. The aforementioned products and support are not useful unless they are distributed in a timely manner and within an appropriate decision support system. This presentation will provide an update on ongoing activities to support inclusion of these data sets within the NOAA National Weather Service Damage Assessment Toolkit, which allows meteorologists in the field to consult available satellite imagery while performing their damage assessment.

Molthan, Andrew L.↗

NASA NTRS2021

EnDEVR: An Environment for Data Engineering in VR

Organizations within the public and private sectors are looking to improve their data science operations for research, development, and operational purposes. As this interest in data science grows, so too does interest in tools and programs that facilitating such operations. This paper presents EnDEVR, the Environment for Data Engineering in Virtual Reality, a user-extensible system that allows for the execution of custom data science functions. We describe the user-centered design process for the EnDEVR system, based on the needs of subject matter experts within the aerospace and aviation industries. Fundamental to this design is a method for users to integrate their existing analysis code (that may leverage third-party vendors) as usable objects in the virtual environment; this allows organizations to operate without the price tag and lifetime obligations associated with commercial data science toolkits, a concept known as “vendor lock-in”. We then present our system design with iterative implementations, and conclude with lessons learned. The presented work may provide insight into future user-centered design processes for VR applications, particularly for digital transformation stakeholders to circumvent vendor lock-in.

Human-centered computing↗

DOE OSTI2024

Transportation and Systems Analysis Collaborations in Support of a Federal Consolidated Interim Storage Facility

The U.S. Department of Energy’s Integrated Waste Management (IWM) program under the Office of Nuclear Energy is planning for the future transportation, storage, and eventual disposal of spent nuclear fuel (SNF) and high-level radioactive waste (HLW) from nuclear power plant sites across the United States. To better enable informed decision-making regarding the back end of the nuclear fuel cycle, the IWM program has been sponsoring the development and application of system analysis tools capable of analyzing various options for managing SNF and HLW. With these tools, integrated waste management system (IWMS) architecture analyses are being conducted to support the future deployment of a comprehensive nuclear waste management system that considers all major back-end aspects of the nuclear fuel cycle (i.e., transportation, storage, and disposal). System analyses and assessments typically use these modeling and simulation tools to investigate implications of changes in various assumptions and parameters such as acceptance rates, receipt logic, facility capacities and capabilities, use of standardized canisters, start and stop dates of facilities, etc. The Next Generation System Analysis Model (NGSAM) is an agent-based simulation toolkit that is used for a system-level simulation and analysis focused on SNF management in the United States. An analyst using NGSAM has the ability to define several factors like the number of storage facilities, capacity at each facility, transportation schedules, shipment rates, and other conditions. NGSAM’s primary purpose is to provide a system analyst with capabilities to model the IWMS and gain insights into SNF and HLW management alternatives including the impact of system choices, associated cost estimates, and development of integrated yet flexible approaches. IWM is also developing the Stakeholder Tool for Assessing Radioactive Transportation (START). START is a web-based geospatial tool developed to provide visualization and initial evaluation of transportation options associated with future SNF and HLW shipment planning and operations. This includes characterizing safety, economic, and environmental conditions on and in proximity to shipment origins as well as along prospective transportation routes. Information from the START tool can be presented/shared as maps, graphics, geospatial files, and tabular form to enable easy data representation and export functionality. The system analysis, NGSAM, and START teams had been working closely for several years before formalizing this collaboration. START provides the SNF routing information for use in NGSAM. System analysts use the NGSAM tool to generate results (schedule, costs, infrastructure acquisitions, shipment rates, etc.). Subject-matter experts and system analysts work together to inform how NGSAM should model the waste management system. Specifically, this paper discusses the collaborations between the system analysis, NGSAM, and START teams and their accomplishments over the past year. Some examples of these efforts include calibrating and adding data to the START output files to meet NGSAM needs, gaining a better understanding of START data used in NGSAM, as well as how updates in START data could result in an improved NGSAM analysis. Detailed examples of various tasks that have been performed by the team will be discussed in the full paper. Continued efforts in this direction are expected in the coming years.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗

DOE OSTI2020

Development of Strong Anaerobic Fluorescent Reporters for Clostridium acetobutylicum and Clostridium ljungdahlii Using HaloTag and SNAP-tag Proteins

One of the biggest limitations in the study and engineering of anaerobic Clostridium organisms is the lack of strong fluorescent reporters capable of strong and real-time fluorescence. Recently, we developed a strong fluorescent reporter system for Clostridium organisms based on the FAST protein. Here, we report the development of two new strong fluorescent reporter systems for Clostridium organisms based on the HaloTag and SNAP-tag proteins, which produce strong fluorescent signals when covalently bound to fluorogenic ligands. These new fluorescent reporters are orthogonal to the FAST ligands and to each other, allowing for simultaneous labeling and visualization. We used HaloTag and SNAP-tag to label the strictly anaerobic organisms Clostridium acetobutylicum and Clostridium ljungdahlii. We have also identified a new strong promoter for protein expression in C. acetobutylicum, based on the phosphotransacetylase gene (pta) from C. ljungdahlii. Furthermore, the HaloTag and the SNAP-tag, in combination with the previously described FAST system, were successfully used to measure cell populations in bacterial mixed cultures and showed the simultaneous orthogonal labeling of HaloTag and SNAP-tag together with the FAST protein reporter. Finally, we show the expression of recombinant fusion protein of FAST and the ZapA division protein (from C. acetobutylicum) in C. ljungdahlii. The availability of multiple strong fluorescent reporters is a major addition to the genetic toolkit of Here, Clostridium and other anaerobes that will lead to better understanding of these unique organisms.

59 BASIC BIOLOGICAL SCIENCES↗