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DOE OSTI2024

Endogenous Production and Vibrational Analysis of Heavy-Isotope-Labeled Peptides from Cyanobacteria

Stable isotope labeling is an extremely useful tool for characterizing the structure, tracing the metabolism, and imaging the distribution of natural products in living organisms using mass-sensitive measurement techniques. In this study, a cyanobacterium was cultured in 15 N/ 13 C-enriched media to endogenously produce labeled, bioactive oligopeptides. The extent of heavy isotope incorporation in these peptides was determined with LC–MS, while the overall extent of heavy isotope incorporation in whole cells was studied with nanoSIMS and AFM-IR. Up to 98 % heavy isotope incorporation was observed in labeled cells. Three of the most abundant peptides, microcystin-LR (MCLR), cyanopeptolin-A (CYPA), and aerucyclamide-A (ACAA), were isolated and further studied with Raman and FTIR spectroscopies and DFT calculations. This revealed several IR and Raman active vibrations associated with functional groups not common in ribosomal peptides, like diene, ester, thiazole, thiazoline, and oxazoline groups, which could be suitable for future vibrational imaging studies. More broadly, this study outlines a simple and relatively inexpensive method for producing heavy-labeled natural products. Finally, manipulating the bacterial culture conditions by the addition of specific types and amounts of heavy-labeled nutrients provides an efficient means of producing heavy-labeled natural products for mass-sensitive imaging studies.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

A new hypothesis for the origin of Amazonian Dark Earths

Amazonian Dark Earths (ADEs) are unusually fertile soils characterised by elevated concentrations of microscopic charcoal particles, which confer their distinctive colouration. Frequent occurrences of pre-Columbian artefacts at ADE sites led to their ubiquitous classification as Anthrosols (soils of anthropic origin). However, it remains unclear how indigenous peoples created areas of high fertility in one of the most nutrient-impoverished environments on Earth. Here, we report new data from a well-studied ADE site in the Brazilian Amazon, which compel us to reconsider its anthropic origin. The amounts of phosphorus and calcium—two of the least abundant macronutrients in the region—are orders of magnitude higher in ADE profiles than in the surrounding soil. The elevated levels of phosphorus and calcium, which are often interpreted as evidence of human activity at other sites, correlate spatially with trace elements that indicate exogenous mineral sources rather than in situ deposition. Stable isotope ratios of neodymium, strontium, and radiocarbon activity of microcharcoal particles also indicate exogenous inputs from alluvial deposition of carbon and mineral elements to ADE profiles, beginning several thousands of years before the earliest evidence of soil management for plant cultivation in the region. Our data suggest that indigenous peoples harnessed natural processes of landscape formation, which led to the unique properties of ADEs, but were not responsible for their genesis. If corroborated elsewhere, this hypothesis would transform our understanding of human influence in Amazonia, opening new frontiers for the sustainable use of tropical landscapes going forward.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2021

Characterizing major and trace element compositions in fallout melt glass from a near-surface nuclear test

We report the chemical and isotopic compositions of fallout melt glasses from nuclear tests contain a range of information constraining the physical conditions within the fireball and the mechanisms of fallout formation but historic studies tended to exclude the behavior of stable major and trace elements. Here, we present a large study specifically focused on major and trace element relationships within a population of macroscale fallout samples from a single event. We interpret these data to better constrain how fallout melt glass formation in near surface environments is influenced by that environment and demonstrate how major and trace element abundances can provide useful insights into chemical processes within the fireball. Data confirm that the uranium in the fallout glass population derives from two isotopically distinct endmembers: isotopically enriched uranium (presumably from the weapon), and natural composition uranium that may be a combination of anthropogenic and environmental materials from within the blast zone. The similarity between major and trace element concentrations in fallout and corresponding local soils from the event site confirm the local soils as the most probable source of entrained material into the fireball and the source of carrier material into which the bomb vapor was incorporated. The lack of correlation between major and trace element abundances with size indicates that volatility driven processes, such as condensation from the fireball, do not control the composition of macroscale fallout melt glass. Although the fallout has major and trace element chemical characteristics broadly similar to those of the local, associated soils, some systematic differences are observed between the two populations. Fallout melt glass is depleted in volatile elements such as K, Na, Tl and Pb, consistent with heating to temperatures above ~1000 °C for 3-10 s. This is supported by the results of laser heating experiments performed on rhyolitic soil at temperatures (1600-2200 °C) and timescales (1-120 s) that are broadly relevant to fallout formation conditions. Relative enrichments of metals such as Cu and Co do not correlate with the abundance of uranium, suggesting that fallout also records input of near field anthropogenic materials. Our observations suggest that major chemical features can be related to processing in the fireball and used to inform the thermal-chemical evolution of the system. Ultimately, these data are consistent with a fallout formation mechanism that involves rapid melting of surface materials to form carrier material melts with minor incorporation of bomb vapor and a degree of volumetric volatile loss due to heating.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

Sample Preparation Methods for Targeted Single-Cell Proteomics

We compared three cell isolation and two proteomic sample preparation methods for single-cell and near-single-cell analysis. Whole blood was used to quantify hemoglobin (Hb) and glycated-Hb (gly-Hb) in erythrocytes using targeted mass spectrometry and stable isotope-labeled standard peptides. Each method differed in cell isolation and sample preparation as follows: 1) FACS and automated preparation in one-pot for trace samples (autoPOTS); 2) limited dilution via microscopy and a novel rapid one-pot sample preparation method that circumvented the need for the solid-phase extraction, low-volume liquid handling instrumentation and humidified incubation chamber; and 3) CellenONE-based cell isolation and the same one-pot sample preparation method used for limited dilution. Only the CellenONE device routinely isolated single-cells from which Hb was measured to be 540–660 amol per red blood cell (RBC), which was comparable to the calculated SI reference range for mean corpuscular hemoglobin (390–540 amol/RBC). FACSAria sorter and limited dilution could routinely isolate single-digit cell numbers, to reliably quantify CMV-Hb heterogeneity. Finally, we observed that repeated measures, using 5–25 RBCs obtained from N = 10 blood donors, could be used as an alternative and more efficient strategy than single RBC analysis to measure protein heterogeneity, which revealed multimodal distribution, unique for each individual.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Shrub and sedge rhizosphere communities display distinct affinities toward exudates and soil organic matter degradation: a quantitative stable isotope probing analysis

Warming temperatures are accelerating permafrost thaw and changing tundra vegetation, where woody shrubs are displacing sedges. Shrubs, such as Betula nana, and sedges, such as Eriophorum vaginatum, exhibit distinct life strategies including unique root-associated, or rhizosphere microbial communities. As permafrost thaws it unlocks previously unavailable carbon and nutrient sources resulting in deeper roots and a translocation of rhizosphere communities. Because permafrost microbial communities contain lower diversity and biomass than rhizosphere communities, the coalescence of rhizosphere and permafrost microbial communities could alter soil organic matter (SOM) degradation rates and increase greenhouse gas emissions. To identify metabolic strategies across distinct rhizosphere and permafrost microbial communities we conducted an isotope tracing incubation experiment. We inoculated thawed permafrost with shrub and sedge rhizosphere communities while adding exudates or water daily and compared this to an uninoculated control. After 46 days, we spiked samples with 18O enriched water or 13C enriched exudates and measured isotope incorporation into microbial DNA with quantitative stable isotope probing (qSIP). Our results indicate that exudate additions had little effect on uninoculated permafrost communities but the addition of exudates and rhizosphere inoculants had a compounding effect on respiration rates. We found that soils inoculated with shrub rhizosphere communities contained a mixture of exudate and SOM degraders while soils inoculated with sedge rhizosphere communities contained mainly SOM degraders. Finally, we found that individual microbial taxa exhibited maximum growth rates under specific combinations of microbial inoculant communities and exudate addition treatments. Our results reveal that microbial niches are strongly influenced by substrate preferences and community context, and suggest that a reduction in sedges and an expansion of shrubs may provide a mechanism by which permafrost carbon losses are mitigated through corresponding shifts in microbial communities and their substrate preferences.

Schaefer, Sean R [Natural Resources and Earth Syst↗

DOE OSTI2024

Metal Organic Complexation in Seawater: Historical Background and Future Directions

The speciation of most biologically active trace metals in seawater is dominated by complexation by organic ligands. This review traces the history of work in this area, from the early observations that showed surprisingly poor recoveries using metal preconcentration protocols to the present day, where advances in mass spectroscopy and stable isotope geochemistry are providing new insights into the structure, origin, fate, and biogeochemical impact of organic ligands. Many long-standing hypotheses about the specific biological origin of ligands such as siderophores in seawater are finally being validated. This work has revealed the complexity of organic complexation, with multiple ligands and, in some cases, timescales of ligand exchange that are much slower than originally thought. The influence of organic complexation on scavenging is now a key parameter in biogeochemical models of biologically essential metals, especially iron. New insights about the sources and sinks of ligands are required to enhance the usefulness of these models.

Geochemistry & Geophysics↗

DOE OSTI2023

Flow Directions and Ages of Subsurface Water in a Salt Marsh System Constrained by Isotope Tracing

Salt marshes are dynamic hydrologic systems where terrestrial groundwater, terrestrial surface water, and seawater mix due to bi-directional flows and pressure gradients. Due to the counteracting terrestrial and marine forcings that control these environments, we do not comprehensively understand water fluxes in these complex coastal systems. To understand the water sources, flow directions, and velocities in salt marsh porewater, we employed a combination of geochemical tracers and analytical models across a hillslope-to-salt marsh continuum in a salt marsh experiencing daily inundation of estuarine surface water (SW) from tides and mixing of fresh seasonal groundwater. We used tritium ( 3 H) as a hydrologic tracer to assess porewater ages and stable water isotope ($\delta$ 2 H and $\delta$ 18 O) analyses to separate isotopically distinct estuarine and terrestrial groundwater across different depths and landscape positions in the study transect. We employed electrical conductivity to constrain the role of source mixing and evapotranspiration in salt marsh hydrology. Salinity and stable isotopes revealed that transpiration, rather than evaporation, increased subsurface water salinity to concentrations above estuarine SW during summer. Elevated salinity at depth indicated that salt marsh subsurface water is recharged during the dry growing season. Seasonal recharge patterns drive long-term deep subsurface water dynamics across the salt marsh, with 3 H ages of 3–7 years, and daily tidal cycles drive short-term shallow porewater dynamics with 3 H ages of 0 ± 3.6 years. Furthermore, our conceptual understanding of the spatiotemporal changes in SW-subsurface water interactions at the terrestrial-marine interface quantifies the hydrological constraints we are missing to improve our understanding of biogeochemical cycles within the salt marsh.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2025

Isotopic Fractionation and Kinetic Isotope Effects of a Purified Bacterial Nitric Oxide Reductase (NOR)

Nitrous oxide (N 2 O) is a serious concern due to its role in global warming and ozone destruction. Agricultural practices account for ~80% of all anthropogenic N 2 O produced in the US, due in large part to the stimulation of microbial denitrification. Stable isotopes are uniquely suited to examine both microbial N 2 O sources and the mechanism of N 2 O biosynthesis through the use of 2 Site Preference (δ 15 N SP ; the difference in δ 15 N between the central and outer N atoms in N 2 O) and kinetic isotope effects (KIEs), respectively. Using trace gas isotope ratio mass spectrometry (TG-IRMS), we determined the δ 15 N, δ 15 N α , δ 15 N β , and δ 18 O of N 2 O produced by a purified cytochrome c nitric oxide reductase (cNOR) from Paracoccus denitrificans. We also calculated δ 15 N SP , the KIEs, and associated isotopic enrichment factors (ε) for N bulk , N α , and N β . A normal isotope effect was observed for bulk 15 N, with a KIE value of 1.0086 ± 0.0009 (ε = -8.6 ± 0.9‰). The isotope effects for both 15 N α and 15 N β were also normal, with position-specific KIEs of 1.0072 ± 0.0010 (ε = -7.2 ± 1.0‰) and 1.0100 ± 0.0010 (ε = -9.9 ± 1.0‰), respectively, and δ 15 N SP values ranged from 0.5 to 8.7‰ with no significant trend as the reaction proceeded. Values of δ 18 O increased with N 2 O production (slope of δ 18 O against [-flnf/(1-f)] = -19.9 ± 1.9‰). We present implications for the mechanism of N 2 O production from cNOR based on our data.

Rivett, Elise D. [Michigan State Univ., East Lansi↗

DOE OSTI2022

Versatile soil gas concentration and isotope monitoring: optimization and integration of novel soil gas probes with online trace gas detection

Abstract. Gas concentrations and isotopic signatures can unveil microbial metabolisms and their responses to environmental changes in soil. Currently, few methods measure in situ soil trace gases such as the products of nitrogen and carbon cycling or volatile organic compounds (VOCs) that constrain microbial biochemical processes like nitrification, methanogenesis, respiration, and microbial communication. Versatile trace gas sampling systems that integrate soil probes with sensitive trace gas analyzers could fill this gap with in situ soil gas measurements that resolve spatial (centimeters) and temporal (minutes) patterns. We developed a system that integrates new porous and hydrophobic sintered polytetrafluoroethylene (sPTFE) diffusive soil gas probes that non-disruptively collect soil gas samples with a transfer system to direct gas from multiple probes to one or more central gas analyzer(s) such as laser and mass spectrometers. Here, we demonstrate the feasibility and versatility of this automated multiprobe system for soil gas measurements of isotopic ratios of nitrous oxide (δ18O, δ15N, and the 15N site preference of N2O), methane, carbon dioxide (δ13C), and VOCs. First, we used an inert silica matrix to challenge probe measurements under controlled gas conditions. By changing and controlling system flow parameters, including the probe flow rate, we optimized recovery of representative soil gas samples while reducing sampling artifacts on subsurface concentrations. Second, we used this system to provide a real-time window into the impact of environmental manipulation of irrigation and soil redox conditions on in situ N2O and VOC concentrations. Moreover, to reveal the dynamics in the stable isotope ratios of N2O (i.e., 14N14N16O, 14N15N16O, 15N14N16O, and 14N14N18O), we developed a new high-precision laser spectrometer with a reduced sample volume demand. Our integrated system – a tunable infrared laser direct absorption spectrometry (TILDAS) in parallel with Vocus proton transfer reaction mass spectrometry (PTR-MS), in line with sPTFE soil gas probes – successfully quantified isotopic signatures for N2O, CO2, and VOCs in real time as responses to changes in the dry–wetting cycle and redox conditions. Broadening the collection of trace gases that can be monitored in the subsurface is critical for monitoring biogeochemical cycles, ecosystem health, and management practices at scales relevant to the soil system.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

The impact of helium on plasma-driven hydrogen permeation and implications for direct internal recycling in the fusion fuel cycle

Abstract Metal foil pumps (MFPs) are the leading technology for direct internal recycling (DIR) of hydrogen isotopes from the plasma exhaust in future fusion plants. MFPs rely on the concept of superpermeation, where superthermal H atoms directly absorb into the metal foil, rapidly diffuse, and desorb downstream. To date, studies of superpermeation have predominantly employed either pure hydrogen or in some cases trace levels of impurities. The plasma exhaust is expected to contain just ∼1% helium, but in DIR the source gas would be enriched in helium as hydrogen isotopes are extracted. In this work, we explore the impact of helium on hydrogen superpermeation at low temperature (75 °C–200 °C) using Pd-based foils. To first order, the flux scaled linearly with the hydrogen mole fraction. Stable permeation was observed until the helium fraction reached ∼80%, where the flux began to decline slowly with time. In addition, short term (1–5 min) exposure to pure helium plasma significantly attenuated subsequent hydrogen plasma permeation, and the degree was more dramatic at elevated temperature. This attenuation was correlated with He retention in the foils, which was detected by time-of-flight secondary ion mass spectrometry at low levels (<0.1 at. %) and limited to the near surface (<10 nm). Similar trends were observed among all alloys (Pd, PdAg, PdCu), and the foils were restored to full performance with an Ar + sputter clean. The potential for helium plasma exposure to impact MFP performance under these conditions has not been previously reported, and these findings have significant implications to the design and implementation of practical DIR systems.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗

DOE OSTI2022

Examining the effects of chemical cleaning, leaching, and partial dissolution on zinc and cadmium isotope fractionation in marine carbonates

The application of zinc (Zn) and cadmium (Cd) isotopes as palaeo-proxies in carbonate sediment is rapidly expanding due to their potential for tracing changes in biological productivity in the modern and past oceans. However, there are limited investigations into the chemical cleaning methods required to produce the most consistent and accurate data for these novel isotope systems. This could impact their use as palaeo-proxies to reconstruct ocean-atmosphere-climate interactions throughout Earth's history. To address this concern and expand the utility of the Zn and Cd stable isotope systems as palaeo-productivity tracers, the performance of two standard chemical cleaning protocols for acquiring robust and reliable Zn and Cd isotope datasets were assessed. These include (i) the Cd-cleaning method that uses a reductive step to selectively leach contaminating secondary iron (Fe)-manganese (Mn) (oxyhydr)oxide coatings from the carbonate surface, and an oxidative step that is used to remove post-depositional organic matter and sulphide precipitates; and (ii) the magnesium/calcium cleaning protocol that includes an oxidative step only, leaving secondary Fesingle bondMn (oxyhydr)oxide coatings largely intact. Well-preserved Holocene-, and Mesozoic-aged carbonate sediments were used to test the reliability of these two chemical cleaning methods. The Holocene samples comprised not only aliquots of bulk sediment, but also individual species of planktic and benthic foraminifera. Our results show that the best practice chemical cleaning method for retrieving consistent and accurate Zn and Cd isotope, and Zn/Ca and Cd/Ca datasets for carbonate sediments, requires both reductive and oxidative cleaning following the Cd-cleaning method. This differs from most methodological approaches applied to date that remove the reductive step from the chemical cleaning protocol and apply an oxidative step only, or no chemical cleaning at all. Inclusion of the reductive step in the chemical cleaning method typically shifts δ 66 Zn by ~0.1‰ lower and δ 114 Cd by 0.3‰ higher in the solid phase, while Zn/Ca and Cd/Ca typically decrease 2-fold. The benthic foraminifera, C. wuellerstorfi, that live in ocean bottom waters where the seawater Zn and Cd isotope composition is homogeneous display evidence of Zn and Cd isotope fractionation between seawater and carbonate on the order of 0.08 ± 0.08‰ (2SE, n = 4) and -0.25 ± 0.13‰ (2SE, n = 4), respectively, in agreement with experimental constraints. Furthermore, evidence of Zn isotope fractionation effects are recorded in a naturally-dissolved carbonate sediment, together with laboratory-controlled carbonate dissolution experiments. Based on these results, we recommend the Cd-cleaning method and the application of Zn and Cd isotope fractionation factors to accurately reconstruct past seawater Zn and Cd isotope compositions from carbonate sediments.

58 GEOSCIENCES↗

DOE OSTI2022

Quantifying fish otolith mineralogy for trace-element chemistry studies

Otoliths are frequently used to infer environmental conditions or fish life history events based on trace-element concentrations. However, otoliths can be comprised of any one or combination of the three most common polymorphs of calcium carbonate - aragonite, calcite, and vaterite - which can affect the ecological interpretation of otolith trace-element results. Previous studies have reported heterogeneous calcium carbonate compositions between left and right otoliths but did not provide quantitative assessments of polymorph abundances. In this study, neutron diffraction and Raman spectroscopy were used to identify and quantify mineralogical compositions of Chinook salmon Oncorhynchus tshawytscha otolith pairs. We found mineralogical compositions frequently differed between otoliths in a pair and accurate calcium carbonate polymorph identification was rarely possible by visual inspection alone. The prevalence of multiple polymorphs in otoliths is not well-understood, and future research should focus on identifying otolith compositions and investigate how variations in mineralogy affect trace-element incorporation and potentially bias environmental interpretations.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

Associative nitrogen fixation linked with three perennial bioenergy grasses in field and greenhouse experiments

Abstract Associative nitrogen (N 2 )‐fixation (ANF) by bacteria in the root‐zone of perennial bioenergy grasses has the potential to replace or supplement N fertilizer and support sustainable production of biomass, but its application in marginal ecosystems requires further evaluation. In this study, we first combined both greenhouse and field experiments, to explore the N 2 fixation effects of three temperate feedstocks Miscanthus × giganteus (giant miscanthus, Freedom), Panicum virgatum (switchgrass, Alamo), and Saccharum sp. (energycane, Ho 02‐147). In field studies across three growing seasons, plant and soil pools of candidate feedstocks were partially composed of N derived from the atmosphere (Ndfa). Energycane, giant miscanthus, and switchgrass were estimated to derive >30%, %Ndfa. Greenhouse studies were also performed to trace isotopically labeled 15 N 2 into plant biomass and soil pools. Evidence for Ndfa was detected in all three feedstock grasses (using reference 15 N of soil, chicory, and sorghum, δ 15 N~+7.0). Isotopically labeled 15 N 2 was traced into biomass (during grass elongation stage) and soil pools. Extrapolation of rates during the 24 hr labeling period to 50 days estimated 30%–55% of plant Ndfa, with the greatest Ndfa for energycane. The findings of the field natural abundance and greenhouse 15 N 2 feeding experiments provided complementary evidence that perennial bioenergy grasses have the potential to support relatively high rates of ANF, and accumulate diazotroph‐derived N into biomass when grown on non‐fertilized soil.

09 BIOMASS FUELS↗

DOE OSTI2023

Water vapor isotopic composition during TRACER IOP

The TRACER-Iso project took place from June 1 to September 30, 2022 as part of the intensive observation period (IOP) of the Tracking Aerosol Convection Interactions (TRACER) project at the main project site at the La Porte, Texas, municipal airport. The project sought to use measurements of the stable isotopic composition of water vapor to better understand cloud-aerosol interactions in deep convection in a polluted urban setting. Water vapor isotopologue and humidity measurements were determined using a Picarro L2130 Water Vapor Isotope Analyzer. The instrument was deployed in the guest van and consists of three main components: a water vapor isotope analyzer, and a Los Gatos Research Water Vapor Isotope Standard Source (WVISS), and a Los Gatos Research Dry Air Source. Ambient air samples were collected through an inlet located about one meter above roof of the shipping container. Samples were then delivered to the analyzer through teflon tubing by use of an external pump to minimize the transport time between the inlet and analyzer. The inlet tubing was surrounded by a Briskheat heat trace and insulating material to ensure ambient air samples did not fall below the dew point and result in condensation. The analyzer uses cavity ringdown spectroscopy to report isotopic ratios of ambient air samples at a frequency of 1 Hz. The calibration unit of the instrument was used in conjunction with the dry air source to periodically measure the δ values of standard waters. It uses a nebulizer to push small water droplets into a hot chamber that vaporizes the water without fractionation. This vapor was then transported to the analyzer using a built-in compressor and the dry air source, which allows each standard with known δ values to be measured at a wide range of humidity values for post-measurement calibration of ambient air samples. There are multiple sources of uncertainty introduced at different stages during the data collection and processing. These include (1) instrument precision, (2) uncertainty in the secondary standards, (3) humidity-correction uncertainty, and (4) VSMOW-SLAP calibration uncertainty. Uncertainty from each step is propagated in quadrature to calculate a total uncertainty of each isotopologue. Uncertainty was determined to be 2.3‰ for δD and 0.4‰ for δ18O.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2025

Spatially structured bacterial interactions alter algal carbon flow to bacteria

Phytoplankton account for nearly half of global photosynthetic carbon fixation, and the fate of that carbon is regulated in large part by microbial food web processing. We currently lack a mechanistic understanding of how interactions among heterotrophic bacteria impact the fate of photosynthetically fixed carbon. Here, we used a set of bacterial isolates capable of growing on exudates from the diatom Phaeodactylum tricornutum to investigate how bacteria-bacteria interactions affect the balance between exudate remineralization and incorporation into biomass. With exometabolomics and genome-scale metabolic modeling, we estimated the degree of resource competition between bacterial pairs. In a sequential spent media experiment, we found that pairwise interactions were more beneficial than predicted based on resource competition alone, and 30% exhibited facilitative interactions. To link this to carbon fate, we used single-cell isotope tracing in a custom cultivation system to compare the impact of different "primary" bacterial strains in close proximity to live P. tricornutum on a distal "secondary" strain. We found that a primary strain with a high degree of competition decreased secondary strain carbon drawdown by 51% at the single-cell level, providing a quantitative metric for the "cost" of competition on algal carbon fate. Additionally, a primary strain classified as facilitative based on sequential interactions increased total algal-derived carbon assimilation by 7.6 times, integrated over all members, compared to the competitive primary strain. Our findings suggest that the degree of interaction between bacteria along a spectrum from competitive to facilitative is directly linked to algal carbon drawdown.

genome-scale metabolic model↗

DOE OSTI2024

SAIL-Isotopologue (SAIL-iso) Field Campaign Report

The SAIL-iso project took place from November 19, 2022 to June 3, 2023 as part of the U.S. Department of Energy Atmospheric Radiation Measurement (ARM) user facility’s Surface Atmosphere Integrated Field Laboratory (SAIL) project at the main project site in Gothic, Colorado. The project sought to use measurements of the stable isotopic composition of water vapor to better understand cloud-aerosol interactions in a remote mountainous watershed. Water vapor isotopologue and humidity measurements were determined using a Los Gatos Research Triple Water Vapor Isotope Analyzer (TWVIA). The instrument was deployed in the guest van and consists of three main components: a water vapor isotope analyzer, a Los Gatos Research Water Vapor Isotope Standard Source (WVISS), and a Los Gatos Research Dry Air Source. Ambient air samples were collected through an inlet located about one meter above roof of the shipping container. Samples were then delivered to the analyzer through Teflon tubing by use of an external pump to minimize the transport time between the inlet and analyzer. The inlet tubing was surrounded by a Briskheat heat trace and insulating material to ensure ambient air samples did not fall below the dew point and result in condensation. The analyzer uses cavity ringdown spectroscopy to report isotopic ratios of ambient air samples at a frequency of 1 Hz. The calibration unit of the instrument was used in conjunction with the dry air source to periodically measure the δ values of standard waters. It uses a nebulizer to push small water droplets into a hot chamber that vaporizes the water without fractionation. This vapor was then transported to the analyzer using a built-in compressor and the dry air source, which allows each standard with known δ values to be measured at a wide range of humidity values for post-measurement calibration of ambient air samples. Multiple sources of uncertainty are introduced at different stages during the data collection and processing. These include (1) instrument precision, (2) uncertainty in the secondary standards, (3) humidity-correction uncertainty, and (4) Vienna Standard Mean Ocean Water – Standard Light Antarctic Precipitation (VSMOW-SLAP) calibration uncertainty. Uncertainty from each step is propagated in quadrature to calculate a total uncertainty of each isotopologue. Uncertainty was determined to be 2.3‰ for δD and 0.4‰ for δ 18 O.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Single-cell isotope tracing reveals functional guilds of bacteria associated with the diatom Phaeodactylum tricornutum

Abstract Bacterial remineralization of algal organic matter fuels algal growth but is rarely quantified. Consequently, we cannot currently predict whether some bacterial taxa may provide more remineralized nutrients to algae than others. Here, we quantified bacterial incorporation of algal-derived complex dissolved organic carbon and nitrogen and algal incorporation of remineralized carbon and nitrogen in fifteen bacterial co-cultures growing with the diatom Phaeodactylum tricornutum at the single-cell level using isotope tracing and nanoSIMS. We found unexpected strain-to-strain and cell-to-cell variability in net carbon and nitrogen incorporation, including non-ubiquitous complex organic nitrogen utilization and remineralization. We used these data to identify three distinct functional guilds of metabolic interactions, which we termed macromolecule remineralizers, macromolecule users, and small-molecule users, the latter exhibiting efficient growth under low carbon availability. The functional guilds were not linked to phylogeny and could not be elucidated strictly from metabolic capacity as predicted by comparative genomics, highlighting the need for direct activity-based measurements in ecological studies of microbial metabolic interactions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Ambient Mass Spectrometry Rapid Analysis Methods for Trace Inorganics

The ability to rapidly detect and characterize the chemistry of trace quantities of inorganic species has multiple potential applications, including nuclear safeguards, nuclear forensics, and environmental monitoring. Some mass spectrometric methods currently exist, but they are costly and time-intensive, often destroying chemical information in the process of ionization. Ambient mass spectrometry is a relatively new analytical method in which samples are introduced to the mass spectrometer from atmospheric pressure, rather than from high vacuum. Ambient MS can operate with gentler ionization methods such as electrospray ionization (ESI) and paper-spray ionization (PSI), reducing fragmentation and preserving the chemical information of the analyte. This may be useful for determining the intended purpose of material in a sample. Strontium, part of nuclear fallout, is analyzed here to test these new analytical methods for viability. Electrospray ionization mass spectrometry functions by spraying the sample as a solution through a needle charged to a few kV. A nebulizing gas flows out along with the sample to create the spray before droplets enter a drying chamber with heated drying gas to remove the solvent molecules. From there bare analyte ions enter the mass spectrometer. This method has the advantage of relatively fast sample preparation and stable ionization. More stable ionization produces higher-quality data. Paper-spray ionization mass spectrometry operates by applying sample to a paper triangle. The paper triangle is then electrified in a metal clamp to a few kV before spray solvent is applied. The spray solvent transports analyte in the sample to the tip, where it is ionized and enters the mass spectrometer. One advantage of this method is reduced sample preparation - an analyst can even swipe a surface and cut a triangle directly from the swipe. The data analysis serves to rapidly process tens of samples all at once. The mass spectrometer outputs a file containing a few hundred spectra for each sample. The pipeline processes each one to identify peaks of interest. A full palette of data analysis metrics are generated, including plots of the raw data and plots of calculated values such as isotopic ratios. The pipeline filters the raw data to remove noise and background peaks, as well as peaks representing detector saturation. A heat map is generated to show the overall spectrum in mass-time- intensity space. Post-processing, we obtained the desired calibration curves, demonstrating that both ESI-MS and PSI-MS are viable methods for quantifying trace amounts of inorganics while retaining their chemical speciation information. ESI-MS demonstrated a linear response region of 100 ppb to 20 ppm, with a limit-of-detection of around 100 ppb. At top left is an example spectrum with the {sup 88}SrNO{sub 3} peak identified to show the retention of chemical speciation. PSI-MS showed a linear response region of around 1 ppb to 1 ppm, with a limit-of-detection of near 1 ppb. PSI-MS produced more erratic results than ESI-MS, likely due to the variability in ionization resulting from manual positioning of the paper triangle near the MS inlet. Future efforts will focus on improving the repeatability of PSI-MS as well as analyzing other species of interest such as cerium and uranium. Improvements will also be made to create more reliable data extraction and intelligent data filtering.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗