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DOE OSTI2023

Arabidopsis stromal carbonic anhydrases exhibit non–overlapping roles in photosynthetic efficiency and development

Carbonic anhydrases (CAs) are ubiquitous enzymes that accelerate the reversible conversion of CO 2 to HCO 3 – . The Arabidopsis genome encodes members of the α-, β- and γ-CA families, and it has been hypothesized that βCA activity has a role in photosynthesis. In this work, we tested this hypothesis by characterizing the two plastidial βCAs, βCA1 and βCA5, in physiological conditions of growth. We conclusively established that both proteins are localized in the chloroplast stroma and that the loss of βCA5 induced the expression of βCA1, supporting the existence of regulatory mechanisms to control the expression of stromal βCAs. We also established that βCA1 and βCA5 have markedly different enzymatic kinetics and physiological relevance. Specifically, we found that βCA5 had a first-order rate constant ~10-fold lower than βCA1, and that the loss of βCA5 is detrimental to growth and could be rescued by high CO 2 . Furthermore, we established that, while a βCA1 mutation showed near wild-type growth and no significant impact on photosynthetic efficiency, the loss of βCA5 markedly disrupted photosynthetic efficiency and light-harvesting capacity at ambient CO 2 . Therefore, we conclude that in physiological autotrophic growth, the loss of the more highly expressed βCA1 does not compensate for the loss of a less active βCA5, which in turn is involved in growth and photosynthesis at ambient CO 2 levels. These results lend support to the hypothesis that, in Arabidopsis,βCAs have non-overlapping roles in photosynthesis and identify a critical activity of stromal βCA5 and a dispensable role for βCA1.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

High-throughput genetics enables identification of nutrient utilization and accessory energy metabolism genes in a model methanogen

Archaea are widespread in the environment and play fundamental roles in diverse ecosystems; however, characterization of their unique biology requires advanced tools. This is particularly challenging when characterizing gene function. Here, we generate randomly barcoded transposon libraries in the model methanogenic archaeon Methanococcus maripaludis and use high-throughput growth methods to conduct fitness assays (RB-TnSeq) across over 100 unique growth conditions. Using our approach, we identified new genes involved in nutrient utilization and response to oxidative stress. We identified novel genes for the usage of diverse nitrogen sources in M. maripaludis including a putative regulator of alanine deamination and molybdate transporters important for nitrogen fixation. Furthermore, leveraging the fitness data, we inferred that M. maripaludis can utilize additional nitrogen sources including $\tiny{L}$-glutamine, $\tiny{D}$-glucuronamide, and adenosine. Under autotrophic growth conditions, we identified a gene encoding a domain of unknown function (DUF166) that is important for fitness and hypothesize that it has an accessory role in carbon dioxide assimilation. Finally, comparing fitness costs of oxygen versus sulfite stress, we identified a previously uncharacterized class of dissimilatory sulfite reductase-like proteins (Dsr-LP; group IIId) that is important during growth in the presence of sulfite. When overexpressed, Dsr-LP conferred sulfite resistance and enabled use of sulfite as the sole sulfur source. The high-throughput approach employed here allowed for generation of a large-scale data set that can be used as a resource to further understand gene function and metabolism in the archaeal domain.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Separate, separated, and together: the transcriptional program of the Clostridium acetobutylicum-Clostridium ljungdahlii syntrophy leading to interspecies cell fusion

ABSTRACT Syntrophic cocultures (hitherto assumed to be commensalistic) of Clostridium acetobutylicum and Clostridium ljungdahlii , whereby CO 2 and H 2 produced by the former feed the latter, result in interspecies cell fusion involving large-scale exchange of protein, RNA, and DNA between the two organisms. Although mammalian cell fusion is mechanistically dissected, the mechanism for such microbial-cell fusions is unknown. To start exploring this mechanism, we used RNA sequencing to identify genes differentially expressed in this coculture using two types of comparisons. One type compared coculture to the two monocultures, capturing the combined impact of interactions through soluble signals in the medium and through direct cell-to-cell interactions. The second type compared membrane-separated versus -unseparated cocultures, isolating the impact of interspecies physical contact. While we could not firmly identify specific genes that might drive cell fusion, consistent with our hypothesized model for this interspecies microbial cell fusion, we observed differential regulation of genes involved in C. ljungdahlii’s autotrophic Wood-Ljungdahl pathway metabolism and genes of the motility machinery. Unexpectedly, we also identified differential regulation of biosynthetic genes of several amino acids, and notably of arginine and histidine. We verified that they are produced by C. acetobutylicum and are metabolized by C. ljungdahlii to its growth advantage. These and other findings, and notably upregulation of C. acetobutylicum ribosomal-protein genes, paint a more complex syntrophic picture and suggest a mutualistic relationship, whereby beyond CO 2 and H 2 , C. acetobutylicum feeds C. ljungdahlii with growth-boosting amino acids, while benefiting from the H 2 utilization by C. ljungdahlii . IMPORTANCE The construction and study of synthetic microbial cocultures is a growing research area due to the untapped potential of defined multi-species industrial bioprocesses and the utility of defined cocultures for generating insight into complex, undefined, natural microbial consortia. Our previous work showed that coculturing C. acetobutylicum and C. ljungdahlii leads to a unique metabolic phenotype (production of isopropanol) and heterologous cell fusion events. Here, we used RNAseq to explore genes involved in and impacted by these fusions. First, we compared gene expression in coculture to each monoculture. Second, we utilized a transwell system to compare gene expression in mixed cocultures to cocultures with both species physically separated by a permeable membrane, isolating the impact of interspecies “touching” on the transcriptome. This study deepens our mechanistic understanding of the C. acetobutylicum-C. ljungdahlii coculture phenotype, laying the groundwork for reverse genetic studies of heterologous cell fusion in Clostridium cocultures.

Willis, Noah B. (ORCID:0009000689365955)↗

DOE OSTI2024

Clostridium autoethanogenum alters cofactor synthesis, redox metabolism, and lysine-acetylation in response to elevated H 2 :CO feedstock ratios for enhancing carbon capture efficiency

Clostridium autoethanogenum is an acetogenic bacterium that autotrophically converts carbon monoxide (CO) and carbon dioxide (CO 2 ) gases into bioproducts and fuels via the Wood–Ljungdahl pathway (WLP). To facilitate overall carbon capture efficiency, the reaction stoichiometry requires supplementation of hydrogen at an increased ratio of H 2 :CO to maximize CO 2 utilization; however, the molecular details and thus the ability to understand the mechanism of this supplementation are largely unknown. In order to elucidate the microbial physiology and fermentation where at least 75% of the carbon in ethanol comes from CO 2 , we established controlled chemostats that facilitated a novel and high (11:1) H 2 :CO uptake ratio. We compared and contrasted proteomic and metabolomics profiles to replicate continuous stirred tank reactors (CSTRs) at the same growth rate from a lower (5:1) H 2 :CO condition where ~ 50% of the carbon in ethanol is derived from CO 2 . Our hypothesis was that major changes would be observed in the hydrogenases and/or redox-related proteins and the WLP to compensate for the elevated hydrogen feed gas. Our analyses did reveal protein abundance differences between the two conditions largely related to reduction–oxidation (redox) pathways and cofactor biosynthesis, but the changes were more minor than we would have expected. While the Wood–Ljungdahl pathway proteins remained consistent across the conditions, other post-translational regulatory processes, such as lysine-acetylation, were observed and appeared to be more important for fine-tuning this carbon metabolism pathway. Metabolomic analyses showed that the increase in H 2 :CO ratio drives the organism to higher carbon dioxide utilization resulting in lower carbon storages and accumulated fatty acid metabolite levels. This research delves into the intricate dynamics of carbon fixation in C. autoethanogenum, examining the influence of highly elevated H 2 :CO ratios on metabolic processes and product outcomes. The study underscores the significance of optimizing gas feed composition for enhanced industrial efficiency, shedding light on potential mechanisms, such as post-translational modifications (PTMs), to fine-tune enzymatic activities and improve desired product yields.

09 BIOMASS FUELS↗

DOE OSTI2021

Stem CO2 Efflux measurements from Manaus, Brazil, 2017

This data was collected in order to test the effects of temperatures on the autotrophic respiratory process in the tropics. This data package contains raw real-time stem CO2 efflux files during the night and day from 3 different species near the K34 in Manaus during the day and the night. Raw and derived data files are included in the format of .csv and .xlsx and information about the canopy trees and temperatures recorded can be found in the field event log using microsoft excel. The data was collected from three canopy dominant trees in the central Amazon near the K34 tower. Real-time stem CO2 efflux was determined using a Li7000 gas analyzer configured in differential mode and connected to a dynamic stem chamber secured to the stem at breast height using straps to generate a reasonable seal. Ambient air was continuously pumped into the stem chamber and CO2 concentrations of the air entering and exiting the chamber were continuously monitored. CO2 efflux was calculated based on the CO2 concentration difference between ambient and stem chambers, the flow rate of air through the chamber, and the area of the enclosed stem. Additional auxiliary data, including sap flow and crown temperature were also measured. No data processing or QA/QC was done on the raw data packages.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2021

Progress Towards a New Model Chemolithoautotrophic Host (CRADA Final Report)

Some of the largest scale chemical processes for the production of important commodity chemicals and fuels involve an H 2 -containing gas mixture as an intermediate [e.g. ammonia, urea, methanol, Fischer-Tropsch (F-T) diesel, and hydrotreatment of crude petroleum to refined fuels]. The successful development of a model H 2 -oxidizing chemoautotrophic host could expand the range of fuels and chemicals produced from H 2 and CO 2 intermediates in the chemical, oil, and gas sectors in the near term, as well as from renewable and waste-derived sources of these gases that are expected to greatly expand in coming years. Among non-photosynthetic bacteria that can utilize H 2 and CO 2 , Cupriavidus necator (C. necator, formerly Ralstonia eutropha), is the best studied. C. necator is an excellent microbial host for the production of a variety of chemicals because it grows extremely quickly to very high cell densities autotrophically on H 2 and CO 2 , is genetically tractable, and has the ability to accumulate polymers, such as polyhydroxybutyrate, at industrial levels. Despite having great potential as a platform bioproduction host, genetic tools are limited, making metabolic engineering of this organism slow and laborious. In this CRADA project, we developed a number of genetic tools for C. necator: 1) Improvement of C. necator genetic transformation efficiency 2) Integration of heterologous genes into the C. necator chromosome and development of promoter library 3) Development of graded RBSs to control heterologous protein expression 4) Use of RBSs to demonstrate fatty alcohol production in C. necator 5) Demonstration of CRISPR-Cas9 gene editing in C. necator

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

Microalgae Hydrothermal Liquefaction and Biocrude Upgrading: 2022 State of Technology

A preliminary techno-economic analysis (TEA) was developed for the fiscal year (FY) 2022 state of technology (SOT) assessment to evaluate the benefits and risks of using demonstrated, high-productivity algae strains for fuels generation, including sustainable aviation fuel (SAF). In 2022, the marine algal strain, Picochlorum celeri, which demonstrated the highest outdoor biomass productivities reported to date in the DOE-funded open-pond raceway testbed at the Arizona Center for Algae Technology and Innovation (AzCATI), was tested for continuous hydrothermal liquefaction (HTL) processing at PNNL. HTL testing results demonstrated a biocrude yield of 0.33 g/g algae on an ash-free dry weight (AFDW) basis from P. celeri. The hydrotreatment testing of the HTL biocrude from P. celeri was also conducted to investigate the production of jet fuel from marine algal biomass. To the best of our knowledge, this is the first report of jet fuel production from autotrophically grown marine algal biomass. The current hydrotreating testing demonstrated approximately 22.7 wt% of the hydrotreated oil within the typical boiling-point range of jet fuel (150–250 °C). Initial testing of the jet fuel cut (JFC) showed that the physical properties under investigation were within typical ranges for petroleum-based jet fuels. The experimental work of this study closes the gap between outdoor algae cultivation and algae conversion to critical transportation fuels using the same algae strain for both cultivation and conversion testing. The continuous HTL and the upgrading testing described herein demonstrate the potential of producing sustainable aviation fuel (SAF) from algae cultivated in open-pond systems using the primary inputs of sunlight and carbon dioxide.

09 BIOMASS FUELS↗

DOE OSTI2024

Using a systems biology approach to describe the role of dissimilatory phosphite oxidation in the global phosphorus cycle

We investigated the role of redox reactions in the global phosphorous (P) cycle. We identified novel microbial processes responsible for oxidation of reduced phosphorous in the form of phosphite producing bioavailable phosphate. We identified central tenets that define microbial dissimilatory phosphite oxidation (DPO) and the breadth of the taxonomy of DPO microorganisms (DPOM). We demonstrated that this metabolism is vertically evolved from 3.8 GYA contemporaneously with anoxygenic phototrophy. We further demonstrate its occurrence on extant Earth as a primary mechanism driving a phosphorous redox cycle. Finally, we showed that this metabolism is environmentally maintained by the newly described autotrophic Phosphitivorax genus which rely on the reductive glycine pathway for carbon fixation. Our results demonstrate that these species are involved in a complex nutrient-dependent symbiotic relationship with their community in which they provide the fixed carbon to support the heterotrophic community and in return receive prerequisite corrinoids to maintain their activities.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2026

Integrating CO2 Electrolysis with Gas Fermentation to Produce Valuable Fuels and Chemicals

Many industrial activities squander CO2, decreasing process yield. We envision a future where this waste carbon is instead captured, upgraded, and valorized directly at the point of emission. Within the CO2 Reduction and Upgrading Consortium (a collaboration of seven US national laboratories and industrial partners), we are pursuing this goal by developing and de-risking new technologies for low temperature CO2 electrolysis, coupled with biological upgrading of intermediates into more valuable compounds. One such process involves electrocatalytic reduction of CO2 to generate carbon monoxide (CO). As both a carbon and energy source, CO represents an attractive feedstock for microbial upgrading by certain syngas-fermenting species, such as the autotrophic bacterium Clostridium autoethanogenum. Our team has developed new genetic tools and optimized cultivation techniques to enhance C. autoethanogenum as a platform host for the biological conversion of syngas into value-added products. For example, we have created novel CRISPR-based genetic engineering techniques to build new, genome-reduced, platform strains of C. autoethanogenum with improved growth rates. Further, we ve introduced heterologous biochemical pathways into C. autoethanogenum to enable the production of high-value compounds from syngas, such as the isoprenoid precursor mevalonic acid. With the tools of electrochemistry and synthetic biology, there is virtually no limit to the spectrum of products that could be sustainably manufactured from CO2.

09 BIOMASS FUELS↗

DOE OSTI2022

Belowground respiration, root traits, and soil characteristics of an East Tennessee deciduous forest, 2019-2020

This dataset contains empirical physiological, morphological, and chemical data of root systems, and elemental, nutrient content for soils collected on forty individuals of eight temperate tree species, between June 2019 and July 2020 at The University of Tennessee Forest Research Center and Arboretum in Oak Ridge, Tennessee. The project used a novel methodology to empirically derive estimates of the autotrophic and heterotrophic components of soil respiration in-situ. The project consists of two measurement approaches. The first set of measurements uses a standard approach for measuring specific root respiration on excised root systems. The second used “in-situ root trays” This dataset includes 10 data files in comma separate (*.csv) ASCII format. Data include measurements of leaf and root functional traits for excised root systems and for living root systems housed within in-situ root trays, data on soil carbon and nitrogen pools, in-situ measurements of soil moisture and temperature, data on soil respiration rates for in-situ root trays (both as soil mass-based fluxes, and soil-area based fluxes), and data on the geographic coordinates and tree sizes of study trees. Forty study trees of eight temperate tree species were studied (five individuals per species). Two in-situ root trays were installed per species, each housing one entire root system comprising < 3 root orders, and still being attached to the tree via transportive root. All respiration measurements were conducted with the Li-6800 portable photosynthesis system (Li-COR, Lincoln, NE, USA). Root respiration measurements of excised root tissues were made using the Li-6800 and the Walz 3010-GWK1 gas exchange chamber (Heinz Walz GmbH, Effeltrich, Germany). Root scan images were analyzed using WinRHIZO. These images are companion files to this dataset and are contained in two compressed (*.zip) folders. Additional metadata are provided: 10 data dictionaries and a file-level metadata file in comma separate (*.csv) format and a user guide in PDF (*.pdf) format.

EARTH SCIENCE > BIOSPHERE > VEGETATION > LEAF CHAR↗

DOE OSTI2022

Chemotaxonomic patterns in intracellular metabolites of marine microbial plankton

Most biological diversity on Earth is contained within microbial communities. In the ocean, these communities dominate processes related to carbon fixation and nutrient recycling. Yet, specific factors that determine community composition and metabolic activity are difficult to resolve in complex microbial populations, complicating predictions of microbial processes in a changing ocean. Microbial metabolism generates small organic molecules that reflect both the biochemical and physiological diversity as well as the taxonomic specificity of these biological processes. These small molecules serve as the conduit for taxon-specific signaling and exchange. Here, we use liquid chromatography-mass spectrometry (LC-MS)-based metabolomics to taxonomically categorize 111 metabolites that include small molecules in central and secondary metabolism across 42 taxa representing numerically dominant and metabolically important lineages of microbial autotrophs and heterotrophs. Patterns in metabolite presence-absence broadly reflected taxonomic lineages. A subset of metabolites that includes osmolytes, sulfur-containing metabolites, sugars, and amino acid derivatives provided chemotaxonomic information among phytoplankton taxa. A variety of phytohormones and signaling molecules were predominantly found in the heterotrophic bacteria and archaea, expanding knowledge of metabolites implicated in modulating interactions between microbes. This chemotaxonomic inventory of marine microbial metabolites is a key step in deciphering metabolic networks that influence ocean biogeochemical cycles.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Daylight-driven carbon exchange through a vertically structured microbial community

Interactions between autotrophs and heterotrophs are central to carbon (C) exchange across trophic levels in essentially all ecosystems and metabolite exchange is a frequent mechanism for distributing C within spatially structured ecosystems. Yet, despite the importance of C exchange, the timescales at which fixed C is transferred in microbial communities is poorly understood. We employed a stable isotope tracer combined with spatially resolved isotope analysis to quantify photoautotrophic uptake of bicarbonate and track subsequent exchanges across a vertical depth gradient in a stratified microbial mat over a light-driven diel cycle. We observed that C mobility, both across the vertical strata and between taxa, was highest during periods of active photoautotrophy. Parallel experiments with 13 C-labeled organic substrates (acetate and glucose) showed comparably less exchange of C within the mat. Metabolite analysis showed rapid incorporation of 13 C into molecules that can both comprise a portion of the extracellular polymeric substances in the system and serve to transport C between photoautotrophs and heterotrophs. Stable isotope proteomic analysis revealed rapid C exchange between cyanobacterial and associated heterotrophic community members during the day with decreased exchange at night. We observed strong diel control on the spatial exchange of freshly fixed C within tightly interacting mat communities suggesting a rapid redistribution, both spatially and taxonomically, primarily during daylight periods.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Genome-Resolved Metagenomics of Nitrogen Transformations in the Switchgrass Rhizosphere Microbiome on Marginal Lands

Switchgrass (Panicum virgatum L.) remains the preeminent American perennial (C4) bioenergy crop for cellulosic ethanol, that could help displace over a quarter of the US current petroleum consumption. Intriguingly, there is often little response to nitrogen fertilizer once stands are established. The rhizosphere microbiome plays a critical role in nitrogen cycling and overall plant nutrient uptake. We used high-throughput metagenomic sequencing to characterize the switchgrass rhizosphere microbial community before and after a nitrogen fertilization event for established stands on marginal land. We examined community structure and bulk metabolic potential, and resolved 29 individual bacteria genomes via metagenomic de novo assembly. Community structure and diversity were not significantly different before and after fertilization; however, the bulk metabolic potential of carbohydrate-active enzymes was depleted after fertilization. We resolved 29 metagenomic assembled genomes, including some from the ‘most wanted’ soil taxa such as Verrucomicrobia, Candidate phyla UBA10199, Acidobacteria (rare subgroup 23), Dormibacterota, and the very rare Candidatus Eisenbacteria. The Dormibacterota (formally candidate division AD3) we identified have the potential for autotrophic CO utilization, which may impact carbon partitioning and storage. Our study also suggests that the rhizosphere microbiome may be involved in providing associative nitrogen fixation (ANF) via the novel diazotroph Janthinobacterium to switchgrass.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

The Effect of Rapid Development on Soil CO2 Efflux in a Cellulosic Biofuel Stand

As awareness of climate change increases, the need for carbon neutral fuel sources is growing. Lignocellulosic biofuel derived from pine trees has been suggested as one potential energy source; however, it requires more research before its efficacy for climate change mitigation can be determined. Due to the large share of forest carbon held in soils and the extensive area of pine plantations in the southeast U.S., a better understanding of plantation soil carbon dynamics is critical for biofuel carbon accounting. This study evaluated the effects of canopy development and productivity on soil CO2 efflux, a proxy for soil respiration (Rs), in an intensively managed loblolly pine (Pinus taeda) stand over a period from May 2015 to December 2019. We found that leaf area index (LAI) and gross ecosystem production (GEP), as well as meteorological variables, had significant effects on Rs, but that both overall Rs and soil carbon pools did not increase over the course of the study. We thus hypothesize that GEP and LAI had intra-annual effects on Rs, and that the lack of change in Rs is the result of an increase in autotrophic respiration (Ra) that offset a decrease in decomposition of the previous stand’s organic matter.

09 BIOMASS FUELS↗

DOE OSTI2020

EMS-Induced Mutagenesis of Clostridium carboxidivorans for Increased Atmospheric CO 2 Reduction Efficiency and Solvent Production

Clostridium carboxidivorans (P7) is one of the most important solvent-producing bacteria capable of fermenting syngas (CO, CO 2 , and H 2 ) to produce chemical commodities when grown as an autotroph. This study aimed to develop ethyl methanesulfonate (EMS)-induced P7 mutants that were capable of growing in the presence of CO 2 as a unique source of carbon with increased solvent formation and atmospheric CO 2 reduction to limit global warming. Phenotypic analysis including growth and end product characterization of the P7 wild type (WT) demonstrated that this strain grew better at 25 °C than 37 °C when CO 2 served as the only source of carbon. In the current study, 55 mutagenized P7-EMS mutants were developed by using 100 mM and 120 mM EMS. Interestingly, using a forward genetic approach, three out of the 55 P7-EMS mutants showed a significant increase in ethanol, butyrate, and butanol production. The three P7-EMS mutants presented on average a 4.68-fold increase in concentrations of ethanol when compared to the P7-WT. Butyric acid production from 3 P7-EMS mutants contained an average of a 3.85 fold increase over the levels observed in the P7-WT cultures under the same conditions (CO 2 only). In addition, one P7-EMS mutant presented butanol production (0.23 ± 0.02 g/L), which was absent from the P7-WT under CO 2 conditions. Most of the P7-EMS mutants showed stability of the obtained end product traits after three transfers. Most importantly, the amount of reduced atmospheric CO 2 increased up to 8.72 times (0.21 g/Abs) for ethanol production and up to 8.73 times higher (0.16 g/Abs) for butyrate than the levels contained in the P7-WT. Additionally, to produce butanol, the P7-EMSIII-J mutant presented 0.082 g/Abs of CO 2 reduction. This study demonstrated the feasibility and effectiveness of employing EMS mutagenesis in generating solvent-producing anaerobic bacteria mutants with improved and novel product formation and increased atmospheric CO 2 reduction efficiency.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Using respiration quotients to track changing sources of soil respiration seasonally and with experimental warming

Developing a more mechanistic understanding of soil respiration is hampered by the difficulty in determining the contribution of different organic substrates to respiration and in disentangling autotrophic-versus-heterotrophic and aerobic-versus-anaerobic processes. Here, we use a relatively novel tool for better understanding soil respiration: the apparent respiration quotient (ARQ). The ARQ is the amount of CO 2 produced in the soil divided by the amount of O 2 consumed, and it changes according to which organic substrates are being consumed and whether oxygen is being used as an electron acceptor. We investigated how the ARQ of soil gas varied seasonally, by soil depth, and by in situ experimental warming ( +4 ° C) in a coniferous-forest whole-soil-profile warming experiment over 2 years. We then compared the patterns in ARQ to those of soil δ 13 CO 2 . Our measurements showed strong seasonal variations in ARQ, from ≈0.9 during the late spring and summer to ≈0.7 during the winter. This pattern likely reflected a shift from respiration being fueled by oxidized substrates like sugars and organic acids derived from root and root respiration during the growing season to more reduced substrates such as lipids and proteins derived from microbial necromass during the winter. This interpretation was supported by δ 13 CO 2 values, which were lower, like lipids, in the winter and higher, like sugars, in the summer. Furthermore, experimental warming significantly changed how both ARQ and δ 13 CO 2 responded to soil temperature. Wintertime ARQ and δ 13 CO 2 values were higher in heated than in control plots, probably due to the warming-driven increase in microbial activity that may have utilized oxidized carbon substrates, while growing-season values were lower in heated plots. Experimental warming and phenology change the sources of soil respiration throughout the soil profile. The sensitivity of ARQ to these changes demonstrates its potential as a tool for disentangling the biological sources contributing to soil respiration.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Rapidly changing high-latitude seasonality: Implications for the 21st century carbon cycle in Alaska: Modeling Archive

This Modeling Archive is in support of an NGEE Arctic publication "Rapidly changing high-latitude seasonality: Implications for the 21st century carbon cycle in Alaska" DOI: 10.1088/1748-9326/ac4362.The dataset contains "ecosys" model outputs reported in Shirley et al. (2022) that explored how climate warming will shift carbon cycle seasonality in Alaska. The study used analysis of seasonal dynamics to support the prediction that high-latitude ecosystems are carbon sinks and will continue to accumulate carbon throughout the century. The study predicts surprisingly large increases in spring net carbon uptake that result in larger net carbon uptake in spring than in summer by year 2100. Included as *.nc files are modeled daily net ecosystem exchange (NEE), net primary productivity (NPP), heterotrophic respiration (Rh), autotrophic respiration (Ra), gross primary productivity (GPP), and plant nitrogen uptake. The output is provided (1) at 25 km resolution across Alaska; (2) for years 2010 through 2100. Scripts to generate publication figures included as text files (*.m) from MATLAB. A user guide included as *.pdf.The Next-Generation Ecosystem Experiments: Arctic (NGEE Arctic), was a research effort to reduce uncertainty in Earth System Models by developing a predictive understanding of carbon-rich Arctic ecosystems and feedbacks to climate. NGEE Arctic was supported by the Department of Energy's Office of Biological and Environmental Research. The NGEE Arctic project had two field research sites: 1) located within the Arctic polygonal tundra coastal region on the Barrow Environmental Observatory (BEO) and the North Slope near Utqiagvik (Barrow), Alaska and 2) multiple areas on the discontinuous permafrost region of the Seward Peninsula north of Nome, Alaska. Through observations, experiments, and synthesis with existing datasets, NGEE Arctic provided an enhanced knowledge base for multi-scale modeling and contributed to improved process representation at global pan-Arctic scales within the Department of Energy's Earth system Model (the Energy Exascale Earth System Model, or E3SM), and specifically within the E3SM Land Model component (ELM).

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

BSEC VPRM 10m Hourly Biogenic Fluxes in Baltimore (2021)

Model outputs from the Vegetation Photosynthesis and Respiration Model (VPRM: version from Horne et al. in prep). Model remote sensing inputs come from Sential 2-derived EVI and LSWI. Model meteorological inputs for two-meter air temperature and shortwave incoming come from the BSEC WRF 2021 Control Run (Foust, W. 2023). Plant functional Types (PFTs) are spatially classified using the Chesapeake Bay Program 2018 land use land cover product. The final biogenic flux (µmol CO2 m^-2 s^-1) outputs of NEE, RESP, and GEE are a weighted average based on the portion of PFTs within the cell. Individual PFT outputs are saved inside PFT directories (e.g., Crops, Grass, etc.) inside the specific month directory. Model outputs are denoted as a negative flux into the land system (i.e., photosynthesis) and a positive flux as a net release into the overlying atmosphere. Respiration (RESP) fluxes are positive and combine heterotrophic (only soil) and autotrophic sources. Gross ecosystem exchange (GEE) is a negative flux driven by only photosynthetic activity from vegetation, and the Net ecosystem exchange (NEE) is the sum of the two (i.e., NEE=RESP+GEE). Data Characteristics Spatial Resolution: 10m Temporal Resolution: Hourly File Format: VPRM_ _BSEC. .tif (Hour is in UTC) For more information on the model results, please email Jason Horne (jph6488@psu.edu). References: Foust, W. (2023). BSEC WRF 2021 Control Run Output (v0.1.0) [Data set]. MSD-LIVE Data Repository. https://data.msdlive.org/records/m0e6m-vvq17

Baltimore↗