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149 records · Page 9

A Transportable Gravity Gradiometer Based on Atom Interferometry

A transportable atom interferometer-based gravity gradiometer has been developed at JPL to carry out measurements of Earth's gravity field at ever finer spatial resolutions, and to facilitate high-resolution monitoring of temporal variations in the gravity field from ground- and flight-based platforms. Existing satellite-based gravity missions such as CHAMP and GRACE measure the gravity field via precise monitoring of the motion of the satellites; i.e. the satellites themselves function as test masses. JPL's quantum gravity gradiometer employs a quantum phase measurement technique, similar to that employed in atomic clocks, made possible by recent advances in laser cooling and manipulation of atoms. This measurement technique is based on atomwave interferometry, and individual laser-cooled atoms are used as drag-free test masses. The quantum gravity gradiometer employs two identical atom interferometers as precision accelerometers to measure the difference in gravitational acceleration between two points (Figure 1). By using the same lasers for the manipulation of atoms in both interferometers, the accelerometers have a common reference frame and non-inertial accelerations are effectively rejected as common mode noise in the differential measurement of the gravity gradient. As a result, the dual atom interferometer-based gravity gradiometer allows gravity measurements on a moving platform, while achieving the same long-term stability of the best atomic clocks. In the laboratory-based prototype (Figure 2), the cesium atoms used in each atom interferometer are initially collected and cooled in two separate magneto-optic traps (MOTs). Each MOT, consisting of three orthogonal pairs of counter-propagating laser beams centered on a quadrupole magnetic field, collects up to 10(exp 9) atoms. These atoms are then launched vertically as in an atom fountain by switching off the magnetic field and introducing a slight frequency shift between pairs of lasers to create a moving rest frame for the trapped atoms. While still in this moving-frame molasses, the laser frequencies are further detuned from the atomic resonance (while maintaining this relative frequency shift) to cool the atom cloud's temperature to 2 K or below, corresponding to an rms velocity of less than 2 cm/s. After launch, the cold atoms undergo further state and velocity selection to prepare for atom interferometry. The atom interferometers are then realized using laser-induced stimulated Raman transitions to perform the necessary manipulations of each atom, and the resulting interferometer phase is measured using laser-induced fluorescence for state-normalized detection. More than 20 laser beams with independent controls of frequency, phase, and intensity are required for this measurement sequence. This instrument can facilitate the study of Earth's gravitational field from surface and air vehicles, as well as from space by allowing gravity mapping from a low-cost, single spacecraft mission. In addition, the operation of atom interferometer-based instruments in space offers greater sensitivity than is possible in terrestrial instruments due to the much longer interrogation times available in the microgravity environment. A space-based quantum gravity gradiometer has the potential to achieve sensitivities similar to the GRACE mission at long spatial wavelengths, and will also have resolution similar to GOCE for measurement at shorter length scales.

Yu, Nan↗

Iterative-Transform Phase Retrieval Using Adaptive Diversity

A phase-diverse iterative-transform phase-retrieval algorithm enables high spatial-frequency, high-dynamic-range, image-based wavefront sensing. [The terms phase-diverse, phase retrieval, image-based, and wavefront sensing are defined in the first of the two immediately preceding articles, Broadband Phase Retrieval for Image-Based Wavefront Sensing (GSC-14899-1).] As described below, no prior phase-retrieval algorithm has offered both high dynamic range and the capability to recover high spatial-frequency components. Each of the previously developed image-based phase-retrieval techniques can be classified into one of two categories: iterative transform or parametric. Among the modifications of the original iterative-transform approach has been the introduction of a defocus diversity function (also defined in the cited companion article). Modifications of the original parametric approach have included minimizing alternative objective functions as well as implementing a variety of nonlinear optimization methods. The iterative-transform approach offers the advantage of ability to recover low, middle, and high spatial frequencies, but has disadvantage of having a limited dynamic range to one wavelength or less. In contrast, parametric phase retrieval offers the advantage of high dynamic range, but is poorly suited for recovering higher spatial frequency aberrations. The present phase-diverse iterative transform phase-retrieval algorithm offers both the high-spatial-frequency capability of the iterative-transform approach and the high dynamic range of parametric phase-recovery techniques. In implementation, this is a focus-diverse iterative-transform phaseretrieval algorithm that incorporates an adaptive diversity function, which makes it possible to avoid phase unwrapping while preserving high-spatial-frequency recovery. The algorithm includes an inner and an outer loop (see figure). An initial estimate of phase is used to start the algorithm on the inner loop, wherein multiple intensity images are processed, each using a different defocus value. The processing is done by an iterative-transform method, yielding individual phase estimates corresponding to each image of the defocus-diversity data set. These individual phase estimates are combined in a weighted average to form a new phase estimate, which serves as the initial phase estimate for either the next iteration of the iterative-transform method or, if the maximum number of iterations has been reached, for the next several steps, which constitute the outerloop portion of the algorithm. The details of the next several steps must be omitted here for the sake of brevity. The overall effect of these steps is to adaptively update the diversity defocus values according to recovery of global defocus in the phase estimate. Aberration recovery varies with differing amounts as the amount of diversity defocus is updated in each image; thus, feedback is incorporated into the recovery process. This process is iterated until the global defocus error is driven to zero during the recovery process. The amplitude of aberration may far exceed one wavelength after completion of the inner-loop portion of the algorithm, and the classical iterative transform method does not, by itself, enable recovery of multi-wavelength aberrations. Hence, in the absence of a means of off-loading the multi-wavelength portion of the aberration, the algorithm would produce a wrapped phase map. However, a special aberration-fitting procedure can be applied to the wrapped phase data to transfer at least some portion of the multi-wavelength aberration to the diversity function, wherein the data are treated as known phase values. In this way, a multiwavelength aberration can be recovered incrementally by successively applying the aberration-fitting procedure to intermediate wrapped phase maps. During recovery, as more of the aberration is transferred to the diversity function following successive iterations around the ter loop, the estimated phase ceases to wrap in places where the aberration values become incorporated as part of the diversity function. As a result, as the aberration content is transferred to the diversity function, the phase estimate resembles that of a reference flat.

Dean, Bruce H.↗

Investigating Low-Altitude Constellations of Ad-Hoc Lunar PNT System for Distributed Spacecraft Autonomy

In this study, we examine a low-altitude Lunar Position, Navigation, and Timing (LPNT) constellations and the localization performance of Centralized Extended Kalman Filter (CEKF) and Decentralized Extended Kalman Filter (DEKF) algorithms. The primary investigation involves a 100-node swarm operating at a 100 km altitude, in contrast to previous studies that examined a 21-node asset in a frozen-orbit at 5,500 km. The autonomous operation of large-scale swarm is based on two-way Inter-Satellite Link (ISL) measurements, which involve pseudoranges and relative velocities among swarm nodes. We perform a numerical assessment of the two filtering approaches, utilizing ‘fully sampled’ measurements from all available assets as well as ‘two ISL’ measurements where each spacecraft is restricted to only two antennas. This research includes an analysis of CEKF under 2-ISL constraints and evaluates the performance of DEKF in a 100-node swarm, which has not been explored in previous studies. In addition, we examine the impact of increasing the sampling frequency for DEKF, showing that the update cycle can be shortened from a 10-minute interval. A novel approach for ‘2-ISL limited’ DEKF will also be introduced, using a matching formulation that exhaustively enumerates all potential matches. This study provides valuable insights into large-scale distributed swarm operations, considering various filter configurations, sampling frequencies, matching strategies, and scalability of CEKF and DEKF for low-altitude LPNT applications. The Lunar PNT technology plays a key role in providing reliable and robust navigation services on the Moon's surface and the South pole, where the primary Lunar missions are planned. To support upcoming Lunar missions, including small satellites from NASA's Commercial Lunar Payload Services program, the Lunar PNT system must be adaptable to smaller platforms like CubeSats. Driven by the growing involvement of public and private exploration partnerships, the traditional low Earth orbit missions are shifting to beyond geosynchronous orbit [1]. These upcoming missions aim to foster a sustainable and innovative exploration program, in collaboration with commercial and international partners, to facilitate human expansion throughout the solar system and return new knowledge and opportunities to Earth [2]. As part of this trend, there are increasing efforts to utilize science missions in Lunar orbit to develop a non-dedicated and ad-hoc PNT network system. Two traditional approaches, the Deep Space Network (DSN) and the weak signal Global Positioning System (GPS), are established deep-space navigation technologies for missions beyond the geosynchronous orbit. Beginning in 1958, the DSN was developed to communicate with the Explorer 1 spacecraft based on the use of radiometric tracking in spacecraft navigation [3]. The DSN is capable of providing nearly unfettered coverage to spacecraft beyond low-Earth orbit (LEO), however, increased space mission volume has created concerns about future expectations of DSN usage for spacecraft navigation [4]. For cislunar mission applications, the position accuracy using DSN achieves 100 m (3σ) with at least three geometrically diverse ground stations when using radiometric tracking alone [5]. The DSN's dependence on Earth-based ground stations restricts its operational capabilities to periods of Earth visibility. This limitation, coupled with its poor localization performance, renders the DSN unsuitable for future lunar missions that demand continuous tracking and precise positioning. To satisfy the increasing requirements of DSN in Lunar applications, spacecrafts are also required to improve their onboard antenna power and efficiency of the transmission. However, there is an important aggregate cost trade between adding capabilities to every spacecraft and adding to a capacity on the ground that serves multiple spacecraft [6]. A weak GPS system can provide PNT service while the user spacecraft is bound to the Moon, leveraging a single, steerable high gain antenna with the relatively narrow beam which includes all the sources in its field of view [7]. However, the higher the altitude the receiver is above the GPS constellations, the poorer and the weaker are the relative geometry and the received signal powers, respectively, leading to a significant navigation accuracy reduction [8]. The transmitted power becomes weaker with increasing distance from the Earth as well as signals tracked from one of the side lobes of the GPS antenna pattern. As a results, the number of visible satellites and relative geometric condition of the GPS satellites at very high altitude drops dramatically and reduces the navigation solution accuracy. Therefore, the weak GPS system is also not an ideal way to provide PNT service to upcoming Lunar missions when considering its limited geometric condition and the recued navigation accuracy. Another navigation approach on the Moon is being developed, similar to the Global Navigation Satellite System (GNSS) on Earth, aiming to offer navigation service with continuous 24/7 coverage across the entire Lunar surface. For example, lunar communications relay and navigation systems (LCRNS) by NASA and Lunar navigation satellite systems (LNSS) by JAXA are designed to serve as dedicated Position, Navigation, and Timing (PNT) systems for the Moon. However, designing a dedicated LNSS and PNT service involves additional challenges, which are unique to the lunar environment, including limited payload capacity for the CubeSat platform, i.e., the size, weight, and power (SWaP) of the onboard clock, limited lunar ground monitoring stations, and limited financial investment as compared to the legacy Earth-GPS [9]. NASA’s focus on utilizing CubeSat platforms on the Moon leads to an alternative Lunar navigation platform that leverages the existing Lunar science and exploration assets. The small satellites used in Lunar missions can be used to create a low-cost, autonomous, ad-hoc, and on-demand mission-centric Lunar PNT swarm capable of providing PNT services to these low-cost lunar missions [10]. As upcoming Lunar missions will often operate at low-altitude about 30 km to 100 km for scientific observations and mapping purposes, the low-altitude orbital constellations could be employed to create an ad-hoc Lunar PNT system. However, several issues must be addressed, such as the instability of these orbits, which often require maintenance or are only suitable for short-duration missions, operating for fewer than 90 days. Additionally, at an altitude of 100 km, the satellites have a limited period during which they are above the horizon and capable of providing PNT service to users. The implementation of a non-dedicated, ad-hoc Lunar navigation constellation facilitates on-demand PNT services. A preliminary study of ad-hoc Lunar PNT system was conducted using 21 spacecraft in 5,5000 km altitude frozen orbits to test its feasibility and a basic performance of orbital asset localization among ad-hoc Lunar constellations in small satellites format [10]. These swarm assets are designed for autonomous localization with minimal Earth interaction, reducing dependency on bandwidth and ground resources. The design in [10] demonstrated the feasibility of a decentralized PNT approach, specifically employing a DEKF approach for state estimation, which helps minimize onboard operating costs. The DEKF method distributes computation across individual satellites, which lightens the computational load while maintaining accuracy in orbit ephemeris and clock offsets, similar to centralized systems [11]. In a follow-on study [12], each spacecraft was limited to 2 communications antennae, forcing the selection of measurements and scheduling spacecraft activities to perform the measurements. A matching algorithm is implemented to select the best measurements and schedule position estimation updates. The decentralized localization performance is also investigated with increasing levels of network degradation for swarm assets considering the impact of intermittent and permanent communication failure, to demonstrate the robustness and fidelity of the decentralized Lunar PNT service [13]. This study confirmed that the ad-hoc PNT constellations in frozen orbit are highly robust and resilient to communication failures. However, unlike frozen orbit swarm assets, the low-altitude satellites have a limited ground view at an altitude of 100 km, where the ad-hoc Lunar constellation consists of 98 low-altitude satellites, evenly distributed across seven circular polar orbital planes, alongside two satellites in a frozen orbit at an altitude of 5,500 km (Figure 1). Therefore, the number of satellites visible to ground users is significantly limited in low-altitude orbit constellations. As each visibility of a spacecraft remains intact for only a few ticks before it moves out of the field of view, the ground user encounters challenges in maintaining continuous navigation service, resulting in sparse availability and provision of Lunar PNT system. Consequently, service availability is primarily restricted to the Lunar South Pole region (Figure 2). Given these limitations and concerns, the localization performance of low-altitude swarm assets will be assessed in this study. We focus on the investigation of the localization performance of low-altitude swarm assets and ground users near the Lunar South Pole. The overall flow of the Lunar PNT simulation incorporates the DEKF approach of asset localization and the weighted least-squares approach in user localization (Figure 3). The autonomous Lunar PNT simulation is primarily implemented in MATLAB, where the DEKF based on the matching scheduler is implemented with Google’s OR-tools as a model builder and Gurobi optimization tool as a backend solver. The General Mission Analysis Tool (GMAT) is utilized to generate ephemeris data for swarm assets, and accounts for satellite orbital details, mass, and perturbations like solar radiation pressure and drag coefficients. Each ephemeris dataset is produced in the Moon International Celestial Reference Frame (ICRF) inertial coordinate system. For state estimation, the distributed swarm assets rely on two-way Inter-Satellite Link (ISL) measurements, which involve tracking pseudoranges and relative velocities between visible satellites and anchor nodes during each observation. Numerical evaluations of the decentralized localization process are conducted to demonstrate the feasibility of the low-altitude PNT system in providing reliable navigation services. The main approach involves using DEKF and CEKF to localize 100 satellites in low-altitude constellations, where the CEKF is implemented to serve as a baseline for comparing the performance of distributed algorithms. In both cases, we evaluate ‘fully sampled’ measurements from all available assets, and ‘two ISL’ measurements when spacecraft are constrained to have only two antennas. We test four estimation techniques: CEKF fully sampled, CEKF two ISL, DEKF fully sampled, and DEKF two ISL filters. As the DEKF update cycle is comprised of network setup, communication, and computations, a global broadcast network and 2-way ISL network setup will take from 4 to 6 minutes as maximum [12]. In this simulation, the DEKF update cycle is set to 10 minutes, including a 4-minute latency for obtaining and computing the actual measurement updates. We experiment an increased update cycle to demonstrate the feasibility and evaluate the impact on localization performance using various tuning values for measurement noise covariances (Figures 4 and 5). By comparing centralized and decentralized approaches using a matching algorithm, we analyze the influence of cross-correlation factors in the covariance matrix, assuming 100% reliability of all assets and measurements. The increased frequency and the adjustments of tuning parameters reveal distinct error patterns between the two scenarios. The localization accuracy of the swarm assets and ground users is assessed by taking the median error across 100 assets and one ground user (84.9°S, 137.5°E) over 7-day simulation period (Table 1). Since the user localization accuracy is significantly affected by the performance of the swarm assets, it is crucial to maintain high localization accuracy within the swarm. This study will continue to explore decentralized filtering for autonomous LPNT operations, with further investigation of an 'iterative' matching approach which enumerates every valid matching pair, planned for the following month.

Yeji Kim↗

A Transmission Electron Microscopy Study of a Refractory Metal Grain from a Calcium-Aluminum-Rich Inclusion in the Leoville CV3 Chondrite

Introduction: Calcium-aluminum-rich inclusions (CAIs) are an important component of chondritic meteorites. They can contain materials that are thermodynamically predicted and isotopically age dated to be among the first-formed solids in our solar system [1-5]. Observed in some CAIs are micron to sub-micron sized inclusions rich in Fe, Ni, and high-Z elements such as Pt, Os, Ir and W, in the form of refractory metal nuggets (RMNs), fremdlinges, and ‘nugget like objects’ (NLOs) [1,6]. Refractory siderophile elements such as Os, Ir and Ru are thermodynamically predicted to condense at temperatures well in excess of the major CAI phases such as melilite, perovskite, spinel and hibonite [2,7-9]. These refractory metal inclusions in CAIs can therefore serve as probes into the thermodynamic landscape of the early solar protoplanetary disk. Here we report on a refractory grain identified in a CAI of the Leoville CV3 chondrite. This work is part of an ongoing effort to gain insight into the thermochemistry of the early solar system through systematic analyses of the structure and chemistry of various components in CAIs [10-13]. Sample and Analytical Techniques: A fluffy type A CAI (Fig. 1A) was identified in a section of the Leoville, CV3 chondrite (Center for Meteorite Studies, Arizona State University collection, #821_C_3) using a JOEL-JXA 8530F electron microprobe at Arizona State University. Backscattered electron (BSE) imaging and energy-dispersive X-ray spectroscopy (EDS) were used to identify refractory metal grains in the CAI using a Thermo Fisher (formerly FEI) Helios NanoLab 660 G3 focused-ion-beam scanning-electron microscope (FIBSEM) located at the Kuiper Materials Imaging and Characterization Facility (KMICF) at the Lunar and Planetary Laboratory, University of Arizona. The FIB is equipped with an EDAX EDS system. We selected one of the larger (micron-sized) refractory metal grains, designated as ‘Spud’ (Fig. 1B) for further analysis. ‘Spud’ was extracted and thinned to electron transparency (<100 nm) using the FIB-SEM located in KMICF, following methods described by [14- 15]. The FIB section was analyzed using a 200 keV Hitachi HF5000 scanning transmission electron microscope (S/TEM) located at KMICF. The HF5000 is equipped with cold-field emission gun, 3rd-order spherical aberration corrector for STEM imaging, and an Oxford Instruments X-Max N 100 TLE energydispersive spectroscopy (EDS) system with dual 100 mm2 windowless silicon-drift detectors (Ω = 2.0 sr). Selected-area electron-diffraction (SAED) patterns were acquired to aid in determination of crystallinity and phase. Results: The mineralogy, texture, and morphology of the CAI are consistent with that of a fluffy type A (FTA) CAI [16]. BSE imaging at high magnifications revealed grains with high contrast, indicative of compositions rich in elements of higher atomic number relative to surrounding material. These high-Z grains have sizes that range from ∼250 nm to 4 µm. EDS analyses confirm that the bright grains are metal-rich inclusions. A minor fraction of the grains are composed of only Fe and Ni, but the majority (∼60%) of the identified inclusions also contained various refractory siderophiles including Os, Ru, Zr, Ir and Mo. EDS analysis on the FIB-SEM of Spud shows that it contains Fe, Ni, Mo and Ru. High-angle annular dark-field (HAAFD) imaging and EDS mapping in the TEM (Fig. 2) show that Spud occurs in melilite (Ca1.9Al1.99Si1.06O7). Spud contains a subhedral to anhedral morphology and is compositionally heterogenous (polyphasic, Fig. 2). Local spatial correlation occurs among Fe, Ni, and Pt, and also among Os, Ru, and Mo. SAED patterns show that the Fe-Ni-Pt, Fe-Os-Mo-Ru and Fe-Pt regions are crystalline. Discussion: CAIs can contain various types of inclusions rich in Fe, Ni and refractory siderophiles such as Os, Ru, W and Pt [1]. RMNs are micron-sized, single phase alloy grains and can contain Os, Ir, Ru and Rh [1,7,17]. NLOs are also micron-sized inclusions, but contain two phases, a refractory metal, and an oxide [6]. Fremdlinge are the largest of such inclusions (tens of microns in size) and are complex aggregates of Fe-Ni alloy, silicates, oxides, and sulfides [1,17]. While the size of Spud matches previous descriptions of RMNs and NLOs, Spud is neither a single-phase alloy like RMNs, nor does it contain one metal phase and one oxide like NLOs. Spud does not match the above described categories of refractory metal inclusions. The presence of refractory siderophiles such as Mo, Os, Ru, and Pt suggests a high-temperature origin. Thermodynamic modelling by [7] indicates condensation temperatures of 1917 K, 1693 K, 1613 K and 1415 K for Os, Mo, Ru and Pt respectively. These models also show that following the initial condensation of a refractory metal, alloying of solutes such as Fe, Ni and W, occurs in levels proportional to their partial pressures in the surrounding gas. Such alloying occurs at temperatures above the condensation temperatures of common CAI phases such as melilite (1529 K), perovskite (1441 K), spinel (197 K) and forsterite (1354 K) [2]. The polyphasic nature of Spud could be the result of such high-temperature alloying, possibly shortly after the condensation of Mo and Ru at 1693 K and 1613 K respectively. That Spud occurs as an inclusion is consistent with it having formed prior to and at temperature above that of its host melilite in this FTA CAI, which is qualitatively consistent with such prior thermodynamic modeling. Further, the polyphasic nature of Spud is similar to refractory grains from a FTA CAI in the Northwest Africa (NWA) 8323, CV3 chondrite [11-13]. These data suggest that such refractory metal grains could have been widespread in the inner and early solar protoplanetary disk and represent some of the earliest formed solids to have condensed. Acknowledgments: Research and instrumentation supported by NASA grants #NNX12AL47G, #NNX15AJ22G and #80NSSC19K0509, and NSF grants #1531243 and #0619599. Fig 2. STEM data on ‘Spud’. HAADF Image (Top) False-color EDS Maps (Bottom) References: [1] MacPherson G. J. (2014) T. of Geochem. Vol I: Met. And Cosmochem. Processes, 139-179. [2] Lodders K. (2003) ApJ, 591, 1220-1247. [3] Ebel D. S. (2006) Met. & the Early S. Sys. II., 253- 277. [4] Amelin Y. (2002) Science, 297, 1678-1683. [5] Connelly J.N. et.al. (2012) Science, 338, 651-655. [6] Schwander D. et al. (2015) GCA, 18, 70-87. [7] Palme H. and Wlotzka F. (1976) EPSL, 33, 45-60. [8] Berg T. et al. (2009) ApJ, 702, 172-176. [9] Liffman K. et al. (2021) Icarus, 221, 89-105. [10] Zega T.J. et al. (2021) PSJ, 2, 115. [11] Ramprasad T. et al. (2020) LPSC LI, Abstract #2472. [12] Ramprasad T. et al. (2021) Microscopy & Microanalysis, S1, 2792-2794. [13] Ramprasad T. et al. (2021) 84th MetSoc, Abstract #6123. [14] Zega T.J. et al. (2007) MAPS, 42, 1373-1386. [15] Ramprasad T. et al. (2022) MAPS, in revision. [16] Grossman L. (1975), GCA, 39, 433-454. [17] El Goresy A. et al. (1978) LPSC IX, Abstract#1100

T. Ramprasad↗

Does Collection Time Bias the Ecology of Cleanroom Air Samples?

Microbial monitoring of astromaterials collections has taken on increased importance with the return of biologically sensitive samples from the asteroids Ryugu and Bennu and the initiation of the Mars Sample Return Program. Terrestrial bacteria and fungi can alter the mineralogy and organic composition of our collections causing irreversible contamination of pristine samples and increasing the risk of false positives for life detection measurements. NASA has conducted routine microbial monitoring of its existing collections since 20181. Initial monitoring focused on surface samples collected with foam swabs. Although, airborne microbiology is often decoupled from surface microbiology in the built environment2 culture-based air sampling techniques like impactors were not compliant with existing contamination control requirements. Bringing organic rich media, gelatin or liquids into curation cleanrooms presents an unacceptable risk to pristine samples. In 2022 NASA purchased a materials complaint air sampler and began collecting air samples from the cleanrooms in addition to surface samples3. The new instrument uses an electret filter to collect samples that are suitable for cultivating organisms or for direct DNA sequencing. Preliminary DNA sequencing results appeared to indicate that longer sampling times biased the microbial community in favor of hearty, spore-forming bacteria3. We present the results of a study comparing overnight sampling (17 hours) to short (1 hour) sampling of unoccupied curation cleanrooms. The results will help us optimize our monitoring protocols and develop a more detailed inventory of the ecology of astromaterials curation cleanrooms. Methods: We analyzed 72 paired air samples from six different cleanrooms including the meteorite processing lab (ISO 7 equivalent, 16 samples), the lunar lab (ISO 6 equivalent, 10 samples), the stardust lab (ISO 5 equivalent 14 samples), the OSIRIS-REx lab (ISO 5 equivalent, 12 samples), the Hayabusa2 lab (ISO 5 equivalent, 14 samples), and the Genesis lab (ISO 4 equivalent, 6 samples). All the samples were collected with an InnovaPrep Bobcat air sampler operating at a sampling rate of 200 L/min. The sampler operates for 5 minutes out of every 20 minute period. Half of the samples were collected by filtering 3,000L (15 min. of active sampling) of air across an electret filter for one hour. The rest of the samples were collected by filtering approximately 51,000 L air across the filter overnight (~17 hours, 255 min. of active sampling). Cells were eluted from the filter using 6-7 ml of pressurized 0.15% tween 20 in PBS (phosphate buffered saline). This liquid was used to cultivate bacteria according to previously published methods1,4,5 and for DNA extraction and next generation sequencing. DNA was extracted with a Qiagen MagAttract PowerMicrobiome kit6. To identify bacteria and archaea, the 16S rRNA gene was amplified using Earth Microbiome primers for the V4 region 7. The amplified DNA was sequenced on an Illumina MiSeq using a V3 reagent kit. The resulting sequences were processed using DADA2 and QIIME2 as implemented on the EDGE bioinformatics platform8–10. Results: Only two of the 72 samples had no amplifiable DNA. Amplified DNA concentrations ranged from 2.67 – 0.272 ng/µl. The median concentration of amplified DNA for the 1 hour samples was 0.770 ± 0.368 ng/µl. The median concentration of amplified DNA for the overnight samples was 0.877 ± 0.434 ng/µl. On average the overnight samples had slightly more sequences (58,960 vs. 59,456) and ASV’s (amplicon sequence variants) (60 vs 64.5) than the one hour samples, but these differences are not statistically significant. The most abundant ASV in every sample mapped to the genus Cupravidus. ASV’s mapping to the genuses Bacillus, Schlegelella, Thermus, and Staphylococcus were also common. Discussion and Future Work: Alpha diversity statistics like Shannon Entropy and Faith Phylogenetic Diversity are used to describe the diversity of organisms in a single sample. If a longer sampling time was biasing the data, we would expect to see a change in these diversity statistics vs. sample time. However, we did not observe this in our data. The median Shannon entropy was slightly higher for the overnight samples (3.773 vs 3.611) as was the Faith Phylogenetic Diversity (4.042 vs 3.596), but both values were within a standard deviation of each other for the two sampling times (Fig. 1). It is unlikely, that the longer sampling time is introducing bias into our data. We do observe a significant decrease in diversity when comparing the air samples by lab. The Genesis lab (ISO 4 equivalent) has a lower median number of ASV’s (45.5) than the other labs (62). Median values for Shannon Entropy (3.717 vs. 3.430) and Faith Phylogenetic Diversity (3.796 vs. 3.548) are also lower for Genesis, but those values are with one standard deviation of each other for the different sampling times. This is consistent with previous culture-based results suggesting that the environment in cleanrooms tends to select for a core group of organisms capable of surviving under dry, low nutrient, conditions. The presence of the ASV’s mapping to Cupravidus and Thermus in our sequencing blanks and controls suggests that several of the most common organisms in our samples represent contaminants from the reagents used to perform the DNA extractions and sequencing. Further work is needed to identify these contaminants, remove them from our data and recalculate the diversity statistics. This is a systematic error. Therefore, we do not expect removing the sequencing contaminants to change our conclusions. Longer air sample collection times appear to result in slightly higher diversity and do not bias the results towards “hardy” bacteria like spore-formers. Based on these preliminary results we conclude that sampling at least 3,000 liters of air is sufficient to capture the microbial diversity of cleanrooms, and that air samples can also be collected overnight without negatively impacting diversity. These results allow us to be flexible when designing microbial monitoring plans so that they do not interfere with routine lab activity. References: 1. Regberg, A. B. et al. 49th Lunar and Planetary Science Conference (2018). 2. The United States Pharmacopeial Convention. USP General Chapter <1116> (2013). 3. Regberg, A. B., et al. 54th Lunar and Planetary Science Conference (2023). 4. Regberg, A. B. et al. 53rd Lunar and Planetary Science Conference ( 2022). 5. Davis, R. E.,et al. 50th Lunar and Planetary Science Conference (2019). 6. Qiagen. MagAttract® PowerMicrobiome® DNA/RNA EP Kit Handbook. (2018). 7. Walters, W. et al. mSystems 1, (2015). 8. Callahan, B. J. et al. Nat. Methods 13, 581–583 (2016). 9. Hall, M. & Beiko, R. G. Microbiome Analysis: Methods and Protocols113–129 (Springer, 2018). 10. Philipson, C. et al. Bio-Protoc. 7, e2622 (2017).

A. B. Regberg↗