Development of a metagenomic approach for ultra-low biomass samples from spacecraft surfaces
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Frequently there is an inability to process and analyze samples of low biomass due to limiting amounts of relevant biomaterial in the sample. Furthermore, molecular biological protocols geared towards increasing the density of recovered cells and biomolecules of interest, by their very nature, also concentrate unwanted inhibitory humic acids and other particulates that have an adversarial effect on downstream analysis. A novel and robust fluorescence-activated cell-sorting (FACS)-based technology has been developed for purifying (removing cells from sampling matrices), separating (based on size, density, morphology), and concentrating cells (spores, prokaryotic, eukaryotic) from a sample low in biomass. The technology capitalizes on fluorescent cell-sorting technologies to purify and concentrate bacterial cells from a low-biomass, high-volume sample. Over the past decade, cell-sorting detection systems have undergone enhancements and increased sensitivity, making bacterial cell sorting a feasible concept. Although there are many unknown limitations with regard to the applicability of this technology to environmental samples (smaller cells, few cells, mixed populations), dogmatic principles support the theoretical effectiveness of this technique upon thorough testing and proper optimization. Furthermore, the pilot study from which this report is based proved effective and demonstrated this technology capable of sorting and concentrating bacterial endospore and bacterial cells of varying size and morphology. Two commercial off-the-shelf bacterial counting kits were used to optimize a bacterial stain/dye FACS protocol. A LIVE/DEAD BacLight Viability and Counting Kit was used to distinguish between the live and dead cells. A Bacterial Counting Kit comprising SYTO BC (mixture of SYTO dyes) was employed as a broad-spectrum bacterial counting agent. Optimization using epifluorescence microscopy was performed with these two dye/stains. This refined protocol was further validated using varying ratios and mixtures of cells to ensure homogenous staining compared to that of individual cells, and were utilized for flow analyzer and FACS labeling. This technology focuses on the purification and concentration of cells from low-biomass spacecraft assembly facility samples. Currently, purification and concentration of low-biomass samples plague planetary protection downstream analyses. Having a capability to use flow cytometry to concentrate cells out of low-biomass, high-volume spacecraft/ facility sample extracts will be of extreme benefit to the fields of planetary protection and astrobiology. Successful research and development of this novel methodology will significantly increase the knowledge base for designing more effective cleaning protocols, and ultimately lead to a more empirical and true account of the microbial diversity present on spacecraft surfaces. Refined cleaning and an enhanced ability to resolve microbial diversity may decrease the overall cost of spacecraft assembly and/or provide a means to begin to assess challenging planetary protection missions.
Nine predominantly cloud-free NOAA-7 advanced very high resolution radiometer images were obtained during a three-month period during the 1981 rainy season in the Sahel of Senegal. The 0.55-0.68 and 0.725-1.10-micron channels were used to form the normalized difference green leaf density vegetation index and the 11.5-12.5-micron channel was used as a cloud mask for each of the nine images. Changes in the normalized difference values among the various dates were closely associated with precipitation events. Six of the images spanning an eight-week period were used to generate a cumulative integrated index. Ground biomass samplings in the 30,000 sq km study area were used to assign total dry biomass classes to the cumulative index.
Identification of microorganisms in the spaceflight environment is critical for crew health risk assessment on the International Space Station (ISS). Since 2017, nanopore sequencing technology has been used to support thein situ identification of microbial species during spaceflight. Beginning in 2018, a culture-independent, swab-to-sequencer method was implemented onboard the ISS to provide a more thorough insight of the ISS microbiome. Eliminating microbial culture enables identification of difficult-to-culture organisms, reduces risks associated with potentially pathogenic cultures, and could significantly reduce the time from sample-to-answer. However, this molecular-based approach generates large metagenomic datasets that require substantial computational resources for analysis. To process nanopore-generated sequencing data, the JSC Microbiology Laboratory established a bioinformatics workflow on Amazon EC2 under the security guidance of the NASA Science Managed Cloud Environment (SMCE).This resource allows for the development, testing, and accessing of computational tools for processing large and complex datasets. The work described here will address the downlinking of data from the ISS, the automated pipeline developed to identify targeted bacterial and fungal organisms, and the time from sampling onboard to microbial identification. The pipelines have been enhanced to address high and low biomass samples using optimization based on sample source (air, water, or surface) and type of collection (filter, colony, or swab).The resulting microbiome data can be assessed beyond microbial identifications to gain understanding toward population changes over time, potential selective environmental pressures, and evaluating correlations with a wide range of additional data sets. Metagenome analysis pipelines in development could allow for simultaneous identification of microbial species, gene function, and gene pathways present in the environment. Beyond the ground processing, the developed analysis pipeline is currently deployed onboard the ISS to allow for near real-time assessments of the ISS microbiome. This study serves as a critical foundation for exploration missions, where rapid microbiome analyses will be required.
Since the Viking missions in the mid-1970s, traditional culture-based methods have been used for microbial enumeration by various NASA programs. Viable microbes are of particular concern for spacecraft cleanliness, for forward contamination of extraterrestrial bodies (proliferation of microbes), and for crew health/safety (viable pathogenic microbes). However, a "true" estimation of viable microbial population and differentiation from their dead cells using the most sensitive molecular methods is a challenge, because of the stability of DNA from dead cells. The goal of this research is to evaluate a rapid and sensitive microbial detection concept that will selectively estimate viable microbes. Nucleic acid amplification approaches such as the polymerase chain reaction (PCR) have shown promise for reducing time to detection for a wide range of applications. The proposed method is based on the use of a fluorescent DNA intercalating agent, propidium monoazide (PMA), which can only penetrate the membrane of dead cells. The PMA-quenched reaction mixtures can be screened, where only the DNA from live cells will be available for subsequent PCR reaction and microarray detection, and be identified as part of the viable microbial community. An additional advantage of the proposed rapid method is that it will detect viable microbes and differentiate from dead cells in only a few hours, as opposed to less comprehensive culture-based assays, which take days to complete. This novel combination approach is called the PMA-Microarray method. DNA intercalating agents such as PMA have previously been used to selectively distinguish between viable and dead bacterial cells. Once in the cell, the dye intercalates with the DNA and, upon photolysis under visible light, produces stable DNA adducts. DNA cross-linked in this way is unavailable for PCR. Environmental samples suspected of containing a mixture of live and dead microbial cells/spores will be treated with PMA, and then incubated in the dark. Thereafter, the sample is exposed to visible light for five minutes, so that the DNA from dead cells will be cross-linked. Following this PMA treatment step, the sample is concentrated by centrifugation and washed (to remove excessive PMA) before DNA is extracted. The 16S rRNA gene fragments will be amplified by PCR to screen the total microbial community using PhyloChip DNA microarray analysis. This approach will detect only the viable microbial community since the PMA intercalated DNA from dead cells would be unavailable for PCR amplification. The total detection time including PCR reaction for low biomass samples will be a few hours. Numerous markets may use this technology. The food industry uses spore detection to validate new alternative food processing technologies, sterility, and quality. Pharmaceutical and medical equipment companies also detect spores as a marker for sterility. This system can be used for validating sterilization processes, water treatment systems, and in various public health and homeland security applications.
The synergistic use of active and passive remote sensing (i.e., data fusion) demonstrates the ability of spaceborne light detection and ranging (LiDAR), synthetic aperture radar (SAR) and multispectral imagery for achieving the accuracy requirements of a global forest biomass mapping mission (+/-20 Mg/ha or 20%, the greater of the two, for at least 80% of grid cells). A data fusion approach also provides a means to extend 3D information from discrete spaceborne LiDAR measurements of forest structure across scales much larger than that of the LiDAR footprint. For estimating biomass, these measurements mix a number of errors including those associated with LiDAR footprint sampling over regional-global extents. A general framework for mapping above ground live forest biomass density (AGB) with a data fusion approach is presented and verified using data from NASA field campaigns near Howland, ME, USA, to assess AGB and LiDAR sampling errors across a regionally representative landscape. We combined SAR and Landsat-derived optical (passive optical) image data to identify contiguous areas (>0.5 ha) that are relatively homogenous in remote sensing metrics (forest patches). We used this image-derived data with simulated spaceborne LiDAR derived from orbit and cloud cover simulations and airborne data from NASA's Laser Vegetation Imaging Sensor (LVIS) to compute AGB and estimate LiDAR sampling error for forest patches and 100 m, 250 m, 500 m, and 1 km grid cells. At both the patch and grid scales, we evaluated differences in AGB estimation and sampling error from the combined use of LiDAR with both SAR and passive optical and with either SAR or passive optical alone. First, this data fusion approach demonstrates that incorporating forest patches into the AGB mapping framework can provide sub-grid forest information for coarser grid-level AGB reporting. Second, a data fusion approach for estimating AGB using simulated spaceborne LiDAR with SAR and passive optical image combinations reduced forest AGB sampling errors 12%-38% from those where LiDAR is used with SAR or passive optical alone. In absolute terms, sampling errors were reduced from 14-40 Mg/ha to 11-28 Mg/ha across all grid scales and prediction methods, where minimum sampling errors were 11, 15, 18, and 22 Mg/ha for 1 km, 500 m, 250 m, and 100 m grid scales, respectively. Third, spaceborne global scale accuracy requirements were achieved whereby at least 80% of the grid cells at 100 m, 250 m, 500 m, and 1 km grid levels met AGB accuracy requirements using a combination of passive optical and SAR along with machine learning methods to predict vegetation structure metrics for forested areas without LiDAR samples. Finally, using either passive optical or SAR, accuracy requirements were met at the 500 m and 250 m grid level, respectively..
The synergistic use of active and passive remote sensing (i.e., data fusion) demonstrates the ability of spaceborne light detection and ranging (LiDAR), synthetic aperture radar (SAR) and multispectral imagery for achieving the accuracy requirements of a global forest biomass mapping mission. This data fusion approach also provides a means to extend 3D information from discrete spaceborne LiDAR measurements of forest structure across scales much larger than that of the LiDAR footprint. For estimating biomass, these measurements mix a number of errors including those associated with LiDAR footprint sampling over regional - global extents. A general framework for mapping above ground live forest biomass (AGB) with a data fusion approach is presented and verified using data from NASA field campaigns near Howland, ME, USA, to assess AGB and LiDAR sampling errors across a regionally representative landscape. We combined SAR and Landsat-derived optical (passive optical) image data to identify forest patches, and used image and simulated spaceborne LiDAR data to compute AGB and estimate LiDAR sampling error for forest patches and 100m, 250m, 500m, and 1km grid cells. Forest patches were delineated with Landsat-derived data and airborne SAR imagery, and simulated spaceborne LiDAR (SSL) data were derived from orbit and cloud cover simulations and airborne data from NASA's Laser Vegetation Imaging Sensor (L VIS). At both the patch and grid scales, we evaluated differences in AGB estimation and sampling error from the combined use of LiDAR with both SAR and passive optical and with either SAR or passive optical alone. This data fusion approach demonstrates that incorporating forest patches into the AGB mapping framework can provide sub-grid forest information for coarser grid-level AGB reporting, and that combining simulated spaceborne LiDAR with SAR and passive optical data are most useful for estimating AGB when measurements from LiDAR are limited because they minimized forest AGB sampling errors by 15 - 38%. Furthermore, spaceborne global scale accuracy requirements were achieved. At least 80% of the grid cells at 100m, 250m, 500m, and 1km grid levels met AGB density accuracy requirements using a combination of passive optical and SAR along with machine learning methods to predict vegetation structure metrics for forested areas without LiDAR samples. Finally, using either passive optical or SAR, accuracy requirements were met at the 500m and 250m grid level, respectively.
We present a first view of data collected during a recent field campaign aimed at measuring biomass burning aerosol above clouds from airborne platforms. The NASA ObseRvations of CLouds above Aerosols and their intEractionS (ORACLES) field campaign recently concluded its first deployment sampling clouds and overlying aerosol layer from the airborne platform NASA P3. We present results from the Spectrometer for Sky-Scanning, Sun-Tracking Atmospheric Research (4STAR), in conjunction with the Solar Spectral Flux Radiometers (SSFR). During this deployment, 4STAR sampled transmitted solar light either via direct solar beam measurements and scattered light measurements, enabling the measurement of aerosol optical thickness and the retrieval of information on aerosol particles in addition to overlying cloud properties. We focus on the zenith-viewing scattered light measurements, which are used to retrieve cloud optical thickness, effective radius, and thermodynamic phase of clouds under a biomass burning layer. The biomass burning aerosol layer present above the clouds is the cause of potential bias in retrieved cloud optical depth and effective radius from satellites. We contrast the typical reflection based approach used by satellites to the transmission based approach used by 4STAR during ORACLES for retrieving cloud properties. It is suspected that these differing approaches will yield a change in retrieved properties since light transmitted through clouds is sensitive to a different cloud volume than reflected light at cloud top. We offer a preliminary view of the implications of these differences in sampling volumes to the calculation of cloud radiative effects (CRE).
The following concepts were introduced: a) Bayesian adaptive sampling for solving biomass estimation; b) Characterization of MISR Rahman model parameters conditioned upon MODIS landcover. c) Rigorous non-parametric Bayesian approach to analytic mixture model determination. d) Unique U.S. asset for science product validation and verification.
A new method is able to circumvent the bias to which one commercial DNA extraction method falls prey with regard to the lysing of certain types of microbial cells, resulting in a truncated spectrum of microbial diversity. By prefacing the protocol with glass-bead-beating agitation (mechanically lysing a much more encompassing array of cell types and spores), the resulting microbial diversity detection is greatly enhanced. In preliminary studies, a commercially available automated DNA extraction method is effective at delivering total DNA yield, but only the non-hardy members of the bacterial bisque were represented in clone libraries, suggesting that this method was ineffective at lysing the hardier cell types. To circumvent such a bias in cells, yet another extraction method was devised. In this technique, samples are first subjected to a stringent bead-beating step, and then are processed via standard protocols. Prior to being loaded into extraction vials, samples are placed in micro-centrifuge bead tubes containing 50 micro-L of commercially produced lysis solution. After inverting several times, tubes are agitated at maximum speed for two minutes. Following agitation, tubes are centrifuged at 10,000 x g for one minute. At this time, the aqueous volumes are removed from the bead tubes and are loaded into extraction vials to be further processed via extraction regime. The new method couples two independent methodologies in such as way as to yield the highest concentration of PCR-amplifiable DNA with consistent and reproducible results and with the most accurate and encompassing report of species richness.
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Largely used in several independent estimates of fire emissions, fire products based on MODIS sensors aboard the Terra and Aqua polar-orbiting satellites have a number of inherent limitations, including (a) inability to detect fires below clouds, (b) significant decrease of detection sensitivity at the edge of scan where pixel sizes are much larger than at nadir, and (c) gaps between adjacent swaths in tropical regions. To remedy these limitations, an empirical method is developed here and applied to correct fire emission estimates based on MODIS pixel level fire radiative power measurements and emission coefficients from the Fire Energetics and Emissions Research (FEER) biomass burning emission inventory. The analysis was performed for January 2010 over the northern sub-Saharan African region. Simulations from WRF-Chem model using original and adjusted emissions are compared with the aerosol optical depth (AOD) products from MODIS and AERONET as well as aerosol vertical profile from CALIOP data. The comparison confirmed an 30-50% improvement in the model simulation performance (in terms of correlation, bias, and spatial pattern of AOD with respect to observations) by the adjusted emissions that not only increases the original emission amount by a factor of two but also results in the spatially continuous estimates of instantaneous fire emissions at daily time scales. Such improvement cannot be achieved by simply scaling the original emission across the study domain. Even with this improvement, a factor of two underestimations still exists in the modeled AOD, which is within the current global fire emissions uncertainty envelope. Plain Language Summary Polar-orbiting satellites sensors, such as MODIS, have limitations in detecting fires under clouds or when viewing angles are large or in the gaps among satellites' different ground swaths. Here we developed an empirical method to mitigate the effect of these limitations in fire emission estimate. The method is applied to a fire emission inventory (FEER) based on MODIS. We show that, with our method, the adjusted emission inventory improves WRF-Chem simulation of smoke transport and distribution.
Airborne scanning LiDAR (Light Detection and Ranging) has emerged as a promising tool to provide auxiliary data for sample surveys aiming at estimation of above-ground tree biomass (AGB), with potential applications in REDD forest monitoring. For larger geographical regions such as counties, states or nations, it is not feasible to collect airborne LiDAR data continuously ("wall-to-wall") over the entire area of interest. Two-stage cluster survey designs have therefore been demonstrated by which LiDAR data are collected along selected individual flight-lines treated as clusters and with ground plots sampled along these LiDAR swaths. Recently, analytical AGB estimators and associated variance estimators that quantify the sampling variability have been proposed. Empirical studies employing these estimators have shown a seemingly equal or even larger uncertainty of the AGB estimates obtained with extensive use of LiDAR data to support the estimation as compared to pure field-based estimates employing estimators appropriate under simple random sampling (SRS). However, comparison of uncertainty estimates under SRS and sophisticated two-stage designs is complicated by large differences in the designs and assumptions. In this study, probability-based principles to estimation and inference were followed. We assumed designs of a field sample and a LiDAR-assisted survey of Hedmark County (HC) (27,390 km2), Norway, considered to be more comparable than those assumed in previous studies. The field sample consisted of 659 systematically distributed National Forest Inventory (NFI) plots and the airborne scanning LiDAR data were collected along 53 parallel flight-lines flown over the NFI plots. We compared AGB estimates based on the field survey only assuming SRS against corresponding estimates assuming two-phase (double) sampling with LiDAR and employing model-assisted estimators. We also compared AGB estimates based on the field survey only assuming two-stage sampling (the NFI plots being grouped in clusters) against corresponding estimates assuming two-stage sampling with the LiDAR and employing model-assisted estimators. For each of the two comparisons, the standard errors of the AGB estimates were consistently lower for the LiDAR-assisted designs. The overall reduction of the standard errors in the LiDAR-assisted estimation was around 40-60% compared to the pure field survey. We conclude that the previously proposed two-stage model-assisted estimators are inappropriate for surveys with unequal lengths of the LiDAR flight-lines and new estimators are needed. Some options for design of LiDAR-assisted sample surveys under REDD are also discussed, which capitalize on the flexibility offered when the field survey is designed as an integrated part of the overall survey design as opposed to previous LiDAR-assisted sample surveys in the boreal and temperate zones which have been restricted by the current design of an existing NFI.
A remote sensing instrument was mounted on a 50-ft tower overlooking North Inlet Estuary, South Carolina in order to conduct a remote sensing study of benthic microalgae. The instrument was programmed to take multispectral imagery data along a 90 deg horizontal frame in six spectral bands ranging from 400-1050 nm and had a ground resolution of about 3 cm. Imagery measurements were encoded in digital form on magnetic tape and were stored, decoded, and manipulated by computer. Correlation coefficients were calculated on imagery data and chlorophyll a concentrations derived from ground truth data. The most significant correlation occurred in the blue spectral band with numerical values ranging from -0.81 to -0.88 for three separate sampling periods. Mean values of chlorophyll a for a larger section of mudflat were estimated using regression equations. The scanner has provided encouraging results and promises to be a useful tool in sampling the biomass of intertidal benthic microalgae.
In a microgravity setting, such as the environment aboard the International Space Station (ISS), an ideal plant water delivery system is one that can grow edible crops with minimal resource consumption and minimal risk to crew members. There are also concerns associated with the ability to control fluid escape and biofilm formation resulting in potential dangers to systems, crops, or crewmembers. To identify an appropriate system, candidate systems were assembled and operated under simulated ISS environmental conditions (T,CO2,and RH) with red romaine lettuce (Lactuca sativa cultivar 'Outredgeous') as a model crop. Fluid reservoirs and randomly selected planting sites were sampled every seven days until maturity at which point edible plant biomass and root samples were also taken. Heterotrophic bacteria and fungi growth patterns throughout each planting cycle were determined by plate counts on appropriate agar media. The candidate systems were compared to a classic hydroponics system as a control and harvested crops were compared to controls as well as Veggie-grown and market produce. Plants harvested from candidate systems yielded lower average heterotrophic bacteria and fungi per gram of plant mass levels when compared to market and Veggie samples as well as those from the control system. Additional studies to evaluate the system sanitation regimen as well as testing additional crops should be considered to aid in the selection of an ideal system.
- The OCO-2 Level 3 assimilation product fills in gaps where OCO-2 does not have observations. - Although the Level 3 clearly shows the increased CO2 over the Amazon associated with the September 2017 biomass burning event, the total amount of CO2 may be underestimated because of a sampling bias. - The TCCON observations match fairly well with the OCO-2 observations and can be used to estimate the accuracy of the OCO observations and the temporal sampling bias. However, since it is a remotely sensed product, it may suffer the same sampling biases associated with biomass burning events as OCO-2. - Although aircraft data show that there is a significant variation in xCO2 with altitude, they suggest the OCO-2 fused products created by kriging may be better than the assimilated product at detecting large deviations from the average state on daily and regional scales.
Topics covered include: Instrument Suite for Vertical Characterization of the Ionosphere-Thermosphere System; Terahertz Radiation Heterodyne Detector Using Two-Dimensional Electron Gas in a GaN Heterostructure; Pattern Recognition Algorithm for High-Sensitivity Odorant Detection in Unknown Environments; Determining Performance Acceptability of Electrochemical Oxygen Sensors; Versatile Controller for Infrared Lamp and Heater Arrays; High-Speed Scanning Interferometer Using CMOS Image Sensor and FPGA Based on Multifrequency Phase-Tracking Detection; Ultra-Low-Power MEMS Selective Gas Sensors; Compact Receiver Front Ends for Submillimeter-Wave Applications; Dynamically Reconfigurable Systolic Array Accelerator; Blocking Losses With a Photon Counter; Motion-Capture-Enabled Software for Gestural Control of 3D Mod; Orbit Software Suite; CoNNeCT Baseband Processor Module Boot Code SoftWare (BCSW); Trajectory Software With Upper Atmosphere Model; ALSSAT Version 6.0; Employing a Grinding Technology to Assess the Microbial Density for Encapsulated Organisms; Demonstration of Minimally Machined Honeycomb Silicon Carbide Mirrors; Polyimide Aerogel Thin Films; Nanoengineered Thermal Materials Based on Carbon Nanotube Array Composites; Composite Laminate With Coefficient of Thermal Expansion Matching D263 Glass; Robust Tensioned Kevlar Suspension Design; Focal Plane Alignment Utilizing Optical CMM; Purifying, Separating, and Concentrating Cells From a Sample Low in Biomass; Virtual Ultrasound Guidance for Inexperienced Operators; Beat-to-Beat Blood Pressure Monitor; Non-Contact Conductivity Measurement for Automated Sample Processing Systems; An MSK Radar Waveform; Telescope Alignment From Sparsely Sampled Wavefront Measurements Over Pupil Subapertures; Method to Remove Particulate Matter from Dusty Gases at Low Pressures; Terahertz Quantum Cascade Laser With Efficient Coupling and Beam Profile; Measurement Via Optical Near-Nulling and Subaperture Stitching; 885-nm Pumped Ceramic Nd:YAG Master Oscillator Power Amplifier Laser System; Airborne Hyperspectral Imaging System; Heat Shield Employing Cured Thermal Protection Material Blocks Bonded in a Large-Cell Honeycomb Matrix; and Asymmetric Supercapacitor for Long-Duration Power Storage.