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NASA NTRS2026

Hydrology Copilot: A Cloud-Native Ai System for Hydrological Data Analysis

The emergence of AI-driven Earth observation systems promises to broaden access to petabyte-scale geospatial data beyond domain specialists. However, translating this vision into operational scientific infrastructure requires addressing fundamental challenges in data virtualization, code transparency, and domain-specific reasoning. We present Hydrology Copilot, a cloud-native AI framework for natural-language-driven analysis of Earth observation data. To demonstrate operational capabilities at scale, we implement the system using NASA's North American Land Data Assimilation System version 3 (NLDAS-3), which provides surface meteorological forcing and land-surface model output across North and Central America at 1-km resolution, from which drought diagnostics are derived. The system integrates five core contributions: (1) scalable data virtualization using Kerchunk-based cloud optimized access, achieving a 1.5 to 4.6 times improvement in I/O latency across benchmark queries spanning regional single-day extractions (4.6 times speedup) to continental monthly aggregations (1.5 times speedup); (2) transparent code generation through Microsoft Azure AI Foundry agents that expose executable Python workflows for scientific verification; (3) persistent conversational memory enabling multi-turn analytical discourse across sessions; (4) intelligent query validation that enforces dataset boundaries and resolves ambiguous requests before execution; and (5) a multi-agent architecture coordinating query parsing, code generation, and visualization. We evaluate the system through drought-monitoring workflows, demonstrating reliable code generation, accurate results validated against reference computations and the operational U.S. Drought Monitor, and efficient operation across increasingly complex tasks. By bridging natural-language interfaces with rigorous hydrological analysis, Hydrology Copilot advances beyond proof-of-concept demonstrations to provide a deployable framework for operational Earth science applications.

Data virtualization↗

DOE OSTI2026

A Sensitivity-driven Wide Area Protection (SWAP) Coordination Tool for High Penetration of Inverter-based Resources (IBR)

Traditionally, power system generation sources have been composed of synchronous generators, of which the fault current behavior is understood with minimal differences between generation size and types due to the physics of their construction. Present protection schemes and modeling methods are based upon these understood characteristics. Most renewable generation is composed of inverter-based resources (IBR), in which fault current is determined by switching control software and hardware limitations, each of which can vary between manufacturers and even between models of the same manufacturer. The resulting fault current is low in magnitude, low in negative-sequence current, unpredictable phase angles, and is a challenge to model. These characteristics also result in a challenge to traditional protection schemes and fault simulation software. To address several of these concerns, the project has the following goals: 1. Improve IBR models: Improve IBR models used in short circuit (SC) programs to accurately capture the response of IBRs at the bulk power system (BPS) level for fault and protection studies. 2. Develop automation tool: Develop an automation tool that allows engineers to identify protection coordination and sensitivity issues by performing SC and protection coordination studies in a high IBR-penetrated grid by applying variations to the IBR models, faults, contingencies, etc. 3. Develop schemes: Develop new protection mitigation solution schemes that complement the existing protection systems to ensure safe operation of the BPS with higher IBR penetration levels. The project team did not achieve this final goal, as the Department of Energy (DOE) stopped the project early due to changes in DOE funding priorities. The termination notice came at the beginning of the final project phase, while the team was identifying and beginning to investigate protection issues. It should be noted that the team discussed a 100% penetration scenario. However, this scenario would require the use of grid-forming IBR models that are not presently available. Since developing these models requires additional effort, the 100% penetration scenario was not pursued during this project. In the future, developing the methodology and models for the 100% scenario could benefit the industry.

14 SOLAR ENERGY↗

DOE OSTI2026

Modeling Flow in a Microreactor Core: From High-Fidelity CFD to Subchannel Analysis

The accurate prediction of pressure drop in tightly packed, low–Reynolds number (Re) bare rod bundles is essential for the thermal-hydraulic design of the Microreactor Applications Research Validation and EvaLuation (MARVEL) reactor and other microreactor concepts. However, existing friction factor correlations, particularly the upgraded Cheng-Todreas (UCTD) correlation, have limited validation for the small pitch-to-diameter ratios (P/Ds) and transitional flow conditions characteristic of these systems. In this work, we perform high-fidelity large-eddy simulations (LESs) of both an infinite bare rod bundle and a finite bare 37-pin scalloped rod bundle across the range 1000≤ 𝑅𝑒 ≤5500. The simulations reveal strong gap vortex–driven transitional behavior and indicate that the UCTD may underpredict the friction factor by up to 28% at 𝑃/𝐷 = 1.05. Using the LES-calculated pressure drops, we formulate a new friction factor correlation that follows the Cheng-Todreas functional form but is calibrated for low-Re and tightly packed geometries representative of a MARVEL-like reactor. The correlation is implemented in the MOOSE (Multiphysics Object-Oriented Simulation Environment) subchannel module and compared against both the LES and UCTD predictions. Across all subchannel types, the proposed model reduces the streamwise velocity differences from as high as 44% (UCTD) to below 9%, and decreases the pressure gradient differences from 13% to 25% (UCTD) to 0.7% to 7% relative to the LES results. These results suggest that the new correlation has the potential to improve the pressure drop and flow field predictions for such geometries, highlighting the importance of high-fidelity simulations in supporting microreactor thermal-hydraulic model development and motivating future pressure drop experiments for compact rod bundles to further validate these findings.

21 - SPECIFIC NUCLEAR REACTORS AND ASSOCIATED PLAN↗

DOE OSTI2026

Innovations Driven by Advanced Characterization to Strategize Critical Mineral Production and Beneficial Reuse from Fossil Energy Waste

Critical minerals (CM), such as rare earth elements (REE), cobalt, nickel, and lithium, have important uses in modern electronics and advanced manufacturing, yet are vulnerable to potential supply chain disruptions. Relatively abundant and readily available fossil energy (FE) wastes, such as coal combustion ash, acid mine drainage (AMD) and treatment solids (AMD solids), and Oil and Gas (O&G) drilling wastes (drill cuttings and produced waters) are under consideration as CM feedstocks. The National Energy Technology Laboratory (NETL) has studied CM resources for various FE wastes as part of the U.S. Department of Energy’s mission of bolstering the domestic CM supply, and makes the data available to the public on EDX at sites such as the NEWTS group. Advanced characterization utilizing synchrotron x-ray techniques coupled with laboratory extractions has been performed to identify CM hosting phases in these FE wastes to inform CM recoverability mechanisms. Novel methods to selectively recover CMs while co-producing other valuable byproducts have been developed. Successful examples discussed here include: (1) The identification of REE/Co/Ni/Sc binding and hosting phases in select FE waste (coal combustion ash and AMD solids), resulting in the development of a patented CM step-extraction process, (2) coupled production of functional sorbents from these extraction wastes and for CM recovery. A pilot-scale testing to evaluate the patent’s technical feasibility for extracting REE from coal ash on a barrel scale has been successfully performed. Additionally, (3) evaluation and measurements of brine geochemistry from U.S. O&G produced waters has informed a high Li recovery potential from Marcellus Shale produced water. NETL researchers have been developing tailored pre-treatment processes, an innovative and highly durable lithium sorbent, and geochemical model guided precipitation to accelerate Li production from the Marcellus Shale produced waters. These innovations driven by characterization are integral for maximizing and advancing the potential for CM recovery while offsetting the cost and environmental footprint for FE waste management.

critical mineral processing↗

DOE OSTI2026

In situ electric field X-ray total scattering reveals composition-dependent electromechanical strain mechanisms in (1 − x )BiFe 2/8 Ti 3/8 Mg 3/8 O 3 – x PbTiO 3 ceramics

Ferroelectric materials find many applications in energy and aerospace industries. A major challenge for ferroelectric materials is maintaining their properties at elevated temperatures. The (1 − x )BiFe 2/8 Ti 3/8 Mg 3/8 O 3 – x PbTiO 3 (BFTM–xPT) ferroelectric solid solution is a promising candidate for high-temperature applications as it has a high piezoelectric response while also maintaining its ferroelectric phase until a high Curie temperature (T C ) of 650 °C. In this work, we investigate the piezoelectric mechanism in BFTM–xPT, a novel high-T C ferroelectric ceramic. To elucidate the origin of its enhanced piezoelectric performance, relative to other piezoceramics of similar T C , in situ electric field X-ray total scattering experiments were performed. Total scattering combines Bragg scattering (diffraction) and diffuse scattering (pair distribution function), providing insight on various length scales. With information spanning different length scales, extrinsic contributions (i.e., domain wall motion and interphase boundary motion) can be separated from intrinsic contributions (i.e., piezoelectric lattice strain). We show that, for compositions within the morphotropic phase boundary (MPB, x = 0.325), the piezoelectric response is dominated by intrinsic piezoelectric lattice strain, whereas outside the MPB (x = 0.375) the major component of the piezoelectric response is from extrinsic mechanisms including an electric-field-induced phase transition and tetragonal domain wall motion. This article reveals that the origin of piezoelectric response in BFTM–xPT is composition-dependent and shows that high-performance materials may also be located outside the MPB. These findings can help guide material design to optimize properties for specific applications.

Richtik, Brooke N. [University of Calgary, AB (Can↗

NASA NTRS1980

Intelsat-V: Nickel-hydrogen battery

A design program for the development of a nickel hydrogen battery is described. The design and design advantages of the battery are discussed. The general characteristics of the battery such as the strain gage cell pressure monitoring and the automatic low temperature heater control are reported along with the performance characteristics.

Vanommering, G.↗

NASA NTRS1981

Space Shuttle News Reference

A detailed description of the space shuttle vehicle and associated subsystems is given. Space transportation system propulsion, power generation, environmental control and life support system and avionics are among the topics. Also, orbiter crew accommodations and equipment, mission operations and support, and flight crew complement and crew training are addressed.

National Aeronautics and Space Administration↗

NASA NTRS1962

Manned Lunar Landing Via Rendezvous

In any mission description, the vehicles, the flight profiles, and the astrionics hardware to im- plement the mission are all tightly interwoven things. A final result evolves only after many iterations to the solution are made. This paper will describe one of these iterations in the Saturn C-5 Earth Orbit Rendezvous approach to the Manned Lunar Landing Program. Since the iter- ation to be described in an nthone, there exists some basis for the hope that the perturbation from the final solution is small. This paper is not concerned with the landing itself, but only with those operations leading to injection of the space craft into the lunar trans- fer trajectory. However, as is to be expected, it is the target conditions which set the pace for the overall operation. The entire operation must be sized to culminate at a time and place which places the lunar target in an attainable position. The procedure would call for a burst of activity lasting over a relatively short time as compared to the long and extensive preparations leading up to it. The activity must be aimed at the opening of the lunar "launch window". Figure 1 illustrates the variation in the velocity increment required to launch a vehicle into a lunar transfer trajectory from a 485 kilometer earth orbit. The minima are at irregularly spaced intervals and are a func- tion of the inclination of the lunar and the earth satellite planes and of the position of the moon in its orbit around the earth (i. e. , the day of the month). In an operations analysis these spacings will influence the number of vehicles on the launch pad (primary and back-up) , their state of readi- ness, the firing rate, and also the flight profile to be chosen. Whether it is decided to go by "con- necting" or by "tanking" mode, the objective must be to get the spacecraft in the launch ready state at the opening of one of these launch windows. It may be desired that the first vehicle be capable of remaining in a functionally capable state even after bridging one or more of the gaps between the windows. This consideration will influence the design of the vehicles as well as the operational modes to be designed into the flight control hard- ware. For example, a sleep switch may be de- sirable from the standpoint of savings in battery we ight.

LUNAR LANDING↗

DOE OSTI2024

WRF-Comfort: simulating microscale variability in outdoor heat stress at the city scale with a mesoscale model

Abstract. Urban overheating and its ongoing exacerbation due to global warming and urban development lead to increased exposure to urban heat and increased thermal discomfort and heat stress. To quantify thermal stress, specific indices have been proposed that depend on air temperature, mean radiant temperature (MRT), wind speed, and relative humidity. While temperature and humidity vary on scales of hundreds of meters, MRT and wind speed are strongly affected by individual buildings and trees and vary on the meter scale. Therefore, most numerical thermal comfort studies apply microscale models to limited spatial domains (commonly representing urban neighborhoods with building blocks) with resolutions on the order of 1 m and a few hours of simulation. This prevents the analysis of the impact of city-scale adaptation and/or mitigation strategies on thermal stress and comfort. To solve this problem, we develop a methodology to estimate thermal stress indicators and their subgrid variability in mesoscale models – here applied to the multilayer urban canopy parameterization BEP-BEM within the Weather Research and Forecasting (WRF) model. The new scheme (consisting of three main steps) can readily assess intra-neighborhood-scale heat stress distributions across whole cities and for timescales of minutes to years. The first key component of the approach is the estimation of MRT in several locations within streets for different street orientations. Second, mean wind speed and its subgrid variability are downscaled as a function of the local urban morphology based on relations derived from a set of microscale LES and RANS simulations across a wide range of realistic and idealized urban morphologies. Lastly, we compute the distributions of two thermal stress indices for each grid square, combining all the subgrid values of MRT, wind speed, air temperature, and absolute humidity. From these distributions, we quantify the high and low tails of the heat stress distribution in each grid square across the city, representing the thermal diversity experienced in street canyons. In this contribution, we present the core methodology as well as simulation results for Madrid (Spain), which illustrate strong differences between heat stress indices and common heat metrics like air or surface temperature both across the city and over the diurnal cycle.

Geology↗

NASA NTRS2008

NASA Battery Working Group - 2007-2008: Battery Task Summary Report

This presentation provides a summary of the 2007-2008 NASA Battery Working Group efforts completed in support of the NASA Engineering Safety Center (NESC). The effort covered a series of pro-active tasks that address the following: Binding Procurements -- guidelines related to requirements for the battery system that should be considered at the time of contract award Wet Life of Ni-H2 Batteries -- issues/strategies for effective storage and impact of long-term storage on performance and life Generic Guidelines for Lithium-ion Safety, Handling and Qualification -- Standardized approaches developed and risk assessments (1) Lithium-ion Performance Assessment -- survey of manufacturers and capabilities to meet mission needs. Guidelines document generated (2) Conditions Required for using Pouch Cells in Aerospace Missions -- focus on corrosion, thermal excursions and long-term performance issues. Document defining requirements to maintain performance and life (3) High Voltage Risk Assessment -- focus on safety and abuse tolerance of battery module assemblies. Recommendations of features required for safe implementation (4) Procedure for Determination of Safe Charge Rates -- evaluation of various cell chemistries and recommendation of safe operating regimes for specific cell designs

Manzo, Michelle↗

DOE OSTI2026

Dark Energy Survey year 6 results: Magnification modeling and its impact on galaxy clustering and galaxy-galaxy lensing cosmology

Gravitational lensing magnification alters the observed spatial distribution of galaxies and must be accounted for to prevent biases in cosmological probes of the large-scale structure. We investigate its effects on the Dark Energy Survey Year 6 galaxy clustering and galaxy-galaxy lensing analyses using the fiducial lens (position tracer) sample M ag L im++. Magnification bias is parameterized by a coefficient that describes the response of the number of selected objects per unlensed area element to a change in the lensing convergence. We quantify this coefficient using the BALROG synthetic source injection catalog to account for the complexity of the selection function, and compare these results with simplified estimates. The resulting values of the magnification coefficients for each redshift bin are [3.16 ± 0.08, 2.76 ± 0.21, 4.09 ± 0.15, 4.42 ± 0.16, 4.90 ± 0.29, 4.83 ± 0.25]. Relative to Year 3, this analysis provides more precise and accurate magnification bias estimates through a larger BALROG area and reweighting to better match the data properties. Here, the cosmological results are robust when tested against various magnification parameter prior choices and also when adding cross-clustering between lens redshift bins. Neglecting magnification, however, introduces significant systematic shifts: relative to the fiducial analysis with Gaussian priors centered on the BALROG -derived estimates, we observe shifts of 1.37σ in S 8 and -0.84σ in Ω m (with cosmic shear included: -0.61σ in S 8 and -0.71σ in Ω m ), in agreement with findings from simulated data, demonstrating that magnification must be modeled to avoid biases. Freeing the magnification bias in lens bin 2 leads to unphysical negative values, further justifying its exclusion from the fiducial Year 6 analysis.

Cosmological parameters↗

NASA NTRS2026

Timing the Flames: Geostationary Satellite Detection of Diurnally Shifting Stubble Burning in Northwestern India

Post-monsoon open-field stubble burning in northwestern (NW) India—a key agricultural region known as the “breadbasket”—is a longstanding practice used to clear fields. Satellite observations spanning over two decades have revealed significant upward trends in crop production, vegetative greenness, and the frequency of post-harvest fires, with this last contributing to hazardous air quality during the peak burning season (mid-October to mid-November). Since 2022, thermal anomaly data from Aqua-MODIS and SNPP-VIIRS sensors have shown a sharp decline in reported fire events—an observation that contrasts starkly with the concurrent rise in regional aerosol loading detected from space. This apparent discrepancy became particularly pronounced in 2024–2025, prompting a closer examination using high-temporal-resolution imagery from the Advanced Meteorological Imager (AMI) on the geostationary satellite GEO-KOMPSAT-2A. These observations revealed a clear spike in fire-related signals occurring around and after 4:00 p.m. local time, i.e., outside the typical noon to 2:00 p.m. detection window of the MODIS and VIIRS. A fire detection algorithm exploiting the fire-sensitive shortwave-infrared 3.8 μm signal and its contrast to 11.2 μm infrared observations is designed to adopt AMI observations and applied to its multi-year observations (2019–2025). The resulting fire dataset unambiguously shows a gradual shift in stubble burning activity toward the late afternoon hours beginning in 2022 which is underreported by polar-orbiting satellites. The orbital drift of NASA’s MODIS sensor on the Aqua platform allows detection of some of the gradually shifting fires during afternoon hours, but the MODIS still misses a large number of fires occurring around and after 4 p.m. The AMI’s relatively coarse spatial resolution (~4 km), a consequence of its slant viewing geometry over NW India, imposes inherent limitations on quantifying the full extent of fire occurrences. The operational air quality forecasting models currently assimilate satellite fire detections predominantly captured during early afternoon overpasses of the MODIS and VIIRS. The temporal shift in fire activity complicates such forecast, leading to a substantial underestimation of emissions. Intense stubble burning and the resulting air pollution highlight the need for effective crop residue management practices for mitigating the frequency of open biomass burning and thereby reducing episodic degradation of air quality and its associated public health and economic impacts.

post-monsoon stubble burning; northwestern India;↗

NASA NTRS1992

Tenth Workshop for Computational Fluid Dynamic Applications in Rocket Propulsion, Part 1

Experimental and computational fluid dynamic activities in rocket propulsion were discussed. The workshop was an open meeting of government, industry, and academia. A broad number of topics were discussed including computational fluid dynamic methodology, liquid and solid rocket propulsion, turbomachinery, combustion, heat transfer, and grid generation.

R.W. Williams↗

DOE OSTI2026

Accessing the oceanic uranium reserve

The ocean contains a vast reserve of uranium that could satisfy global demand for generations. A newly proposed covalent organic framework utilizes structural asymmetry to increase uptake rates, enabling more efficient oceanic uranium recovery.

Wiechert, Alexander [ORNL]↗

DOE OSTI2026

Design and fabrication of ion traps for low RF power dissipation

Large surface-electrode ion traps with multiple trapping regions and junctions are a natural approach to scaling trapped ion quantum computers, supporting the connectivity and ion counts necessary for complex quantum algorithms. However, a major hurdle in this scaling is on-chip power dissipation from the applied RF voltage, which increases at a rate between linear and cubic relative to trap size, depending on whether the losses are dielectric or Ohmic. Here, we present two versions of a trap with features designed to reduce both types of RF power dissipation. The first variant contains a raised RF electrode that increases the electrode–ground distance to reduce capacitance. Different DC voltage sources are demonstrated on this trap to show that technical noise before the filter remains the dominant source of voltage noise and therefore motional heating. The second variant additionally includes a method for removing dielectric from beneath the RF electrode to further reduce dielectric losses. These traps were demonstrated at room temperature with 40 Ca + ions. In conclusion, the similar heating rates and heating rate axial frequency dependencies between 2.4 and 3.0 MHz illustrate that this dielectric modification is not detrimental to trap performance.

Sterk, J. D. [Sandia National Laboratories (SNL-NM↗

NASA NTRSDate not supplied

From Exploration Flight Test-1 to Artemis II--A NASA Langley's Orion Aerosciences Overview

The Orion Aerosciences program at NASA Langley has played a central role in advancing the aerodynamic and aeroheating prediction capabilities required for the Orion crew vehicle’s return from deep space. This presentation provides a technical overview of aerosciences contributions spanning Exploration Flight Test-1 (EFT-1), Artemis I, and the ongoing post-flight analysis of Artemis II. EFT-1 provided the first high-energy entry dataset for Orion, enabling critical validation of aerodynamic force and moment predictions, static and dynamic stability characteristics, and aeroheating environments at relevant flight Mach and Reynolds numbers. Flight-derived pressure data were used to refine the Flush Air Data System (FADS) methodology for atmospheric density reconstruction and to improve Best Estimated Trajectory (BET) solutions. The EFT-1 data also offered key insights into heat shield performance, including char layer recession, in-depth thermal response, and material retention behavior under flight conditions, informing updates to both thermal response models and uncertainty quantification practices. Building on EFT-1, Artemis I extended the database to true lunar-return conditions. Observations of heat shield performance, including localized char loss, bondline response, and recession variability, provided an unprecedented opportunity to reassess Thermal Protection System (TPS) and aeroheating modeling assumptions. Aerodynamic reconstruction efforts incorporated improved FADS calibration, enhanced atmospheric modeling, and refined force and moment databases to reduce trajectory and load uncertainties. Aeroheating comparisons between pre-flight predictions and flight data enabled targeted model updates, particularly in transitional flow environments and wake heating regions. For Artemis II, these lessons were systematically incorporated into the pre-flight prediction process. Updates included refined aerodynamic databases anchored to flight-validated corrections, improved density estimation and BET methodologies using enhanced database interpolation algorithm and FADS modeling, and revised aeroheating design environments informed by Artemis I material response observations. By the time of the workshop, Artemis II post-flight analysis will be underway, and preliminary findings will be presented where available, including early comparisons of aerodynamic reconstruction, atmospheric density estimation, and thermal protection system performance relative to updated predictions. Collectively, this body of work is a testament to the dedicated and multidisciplinary team whose sustained efforts have contributed to the program’s success and to the progressive maturation of Orion aerosciences modeling through numerical modeling, ground and flight data assimilation. The integrated advancement of aerodynamics, trajectory reconstruction, FADS-based density estimation, and aeroheating analysis has reduced predictive uncertainty and strengthened confidence for future crewed lunar and deep-space missions.

Orion↗

DOE OSTI2026

Refractory-based thermal energy storage for industrial process heat: one-dimensional modeling, control, and optimization

The variable and weather-dependent output of wind and solar power plants present a substantial challenge for planning and operating electricity-systems, particularly in the absence of cost-effective and dispatchable energy storage technologies. This study investigates a high-temperature, electrically heated, refractory-based thermal energy storage (RTES) system that stores electrical energy as sensible heat in dense ceramic bricks over the 950–1800 °C range. The stored heat can be discharged as a controlled hot-gas stream for industrial heating, fuel substitution in high-temperature processes, or electricity generation. The main novelty is a comprehensive modelling, control, mapping, and optimization framework that integrates one-dimensional transient gas–solid heat transfer, fan-assisted discharge, bypass-flow regulation, reheating logic, fan-power evaluation, insulation-loss assessment, and genetic-algorithm-based design optimization. The model uses feedback from outlet temperature and delivered power to regulate discharge, while a two-stage genetic algorithm optimizes brick-channel geometry, gas-flow operation, and multilayer insulation thicknesses. Storage capacities below 50 MWh and discharge powers of 5–30 MW are analyzed to evaluate hold time, thermal delivery, fan-power penalty, heat loss, state-of-charge evolution, and indicative capital cost. Results demonstrate that optimized and well-insulated refractory-based thermal energy storage units can provide stable, efficient, and repeatable heat delivery over multiple discharge cycles. The generated performance and cost maps support modular refractory thermal energy storage as a practical option for large-scale integration of wind and solar generation and for high-temperature industrial process heat.

25 ENERGY STORAGE↗

DOE OSTI2026

Advancing Protein Display on Bacterial Spores through an Extensive Survey of Coat Components

The profound stability of bacterial spores makes them a promising platform for biotechnological applications like biocatalysis, bioremediation, drug delivery, etc. However, though the Bacillus subtilis spore is composed of >40 types of proteins, only ∼12 have been explored as fusion carriers for protein display. Here, we assessed the suitability of 33 spore proteins (SPs) as enzyme display carriers by direct allele tagging at native genomic loci. Of the 33 SPs investigated, 26 formed functional fusions with β-glucuronidase (GUS)─a ∼272 kDa homotetramer. This almost triples the number of SPs assessed for enzyme display and doubles the number of functional fusions documented in the literature. We quantitatively assessed 1) SP promoter activation dynamics, 2) GUS activity on spores, 3) surface availability, and 4) protection from thermal and proteolytic degradation. Multicopy expression and pairwise coexpression of the most promising SP-GUS fusions highlighted the complexity of spore structure/assembly and the difficulty in predicting compatibility between different SP fusions. We also assessed the suitability of engineered spores to degrade PET (polyethylene terephthalate) films and found that surface-exposed SPs were most effective. Beyond the broad survey, a key outcome of our work was the identification of SscA (small spore coat assembly protein A) as an effective spore display carrier. SscA supported enzyme activity at least 4-fold higher than any other SP, including the well-established anchor, CotY. We attribute this to its promoter, which demonstrated early and sustained activation relative to other SPs and its small size (∼3 kDa), which likely minimally interferes with enzyme folding, oligomerization, and activity. Labeling and genetic studies, its hydrophobic nature, and low surface availability suggest that SscA assembles within the inner spore coat, which makes it stabilizing and suitable for many biocatalytic applications. Overall, this work serves as a knowledge base to advance the biotechnological utility of B. subtilis spores.

Bacillus subtilis↗