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Data and scripts associated with a manuscript modeling microbial regulation of priming effects

This data package is associated with the publication “Modeling Microbial Regulatory Feedback in Organic Matter Decomposition Identifies Copiotrophic Traits as Key Drivers of Positive Priming” published as a preprint on BioRXiv by Ahamed et al. (2026); https://doi.org/10.1101/2024.08.11.607483. The package contains MATLAB scripts and saved simulation outputs used to implement a cybernetic model of microbial regulation during complex organic matter (OM) decomposition governing priming effects. It includes models of (i) single microbial functional groups (copiotrophic or oligotrophic degraders) and (ii) binary consortia composed of degraders and non-degraders with contrasting or common growth traits. Simulation results were generated using Monte Carlo analyses, with randomized key model parameters across a range of environmental mixing fractions of complex and labile OM. The dataset was created to provide a transparent and reusable computational framework for systematically exploring how microbial growth traits, metabolic regulation, and community composition influence OM decomposition dynamics and priming effects. For details on how to navigate data packages generated by this project, see https://data.ess-dive.lbl.gov/portals/PNNLRiverCorridorSFA/About. In addition to a readme, this data package also includes a file-level metadata (FLMD) file that describes each file and a data dictionary (DD) that describes the variable definitions. This package includes: (1) annotated MATLAB code implementing the system of ordinary differential equations and cybernetic control laws; (2) saved output files containing data (e.g., biomass, substrates, enzyme levels, priming metrics); and (3) scripts for processing saved outputs and regenerating figures. Specifically, the data package contains three main MATLAB scripts: runPrimingModel.m, runPlotData.m, and runPlotSuppFigS1.m, along with this readme and supporting documentation. Users should begin with runPrimingModel.m, which contains the annotated code implementing the system of ordinary differential equations and cybernetic control laws. This script runs the Monte Carlo simulations of microbial OM decomposition and allows users to modify microbial trait definitions, adjust parameter distributions, or define new community configurations. Simulation outputs are automatically saved as .mat files in the folder named SavedData, which stores all pre-generated results included in this package. The second script, runPlotData.m, reads files from the SavedData folder and processes them to regenerate the figures presented in the manuscript. The third script, runPlotSuppFigS1.m, specifically generates Figure S1 in the Supplementary Material of the manuscript. The package also includes the aforementioned files in non-proprietary .txt format. If users intend to use them, they should first save the files in their respective .m or .mat formats prior to execution in MATLAB.

Biomass concentration↗

Sensitivity Analysis of Genome-Scale Metabolic Flux Prediction

TRIMER, Transcription Regulation Integrated with MEtabolic Regulation, is a genome-scale modeling pipeline targeting at metabolic engineering applications. Using TRIMER, regulated metabolic reactions can be effectively predicted by integrative modeling of metabolic reactions with a Transcription Factor (TF)-gene regulatory network (TRN), which is modeled via a Bayesian network (BN). In this paper, we focus on sensitivity analysis of metabolic flux prediction for uncertainty quantification of BN structures for TRN modeling in TRIMER. We propose a computational strategy to construct the uncertainty class of TRN models based on the inferred regulatory order uncertainty given transcriptomic expression data. With that, we analyze the prediction sensitivity of the TRIMER pipeline for the metabolite yields of interest. The obtained sensitivity analyses can guide Optimal Experimental Design (OED) to help acquire new data that can enhance TRN modeling and achieve specific metabolic engineering objectives, including metabolite yield alterations. Here we have performed small- and large-scale simulated experiments, demonstrating the effectiveness of our developed sensitivity analysis strategy for BN structure learning to quantify the edge importance in terms of metabolic flux prediction uncertainty reduction and its potential to effectively guide OED.

59 BASIC BIOLOGICAL SCIENCES↗

Differential DNA methylation and metabolite profiling of Atlantic killifish (Fundulus heteroclitus) from the New Bedford Harbor Superfund site

Atlantic killifish (Fundulus heteroclitus) is a valuable model in evolutionary toxicology to study how the interactions between genetic and environmental factors serve the adaptive ability of organisms to resist chemical pollution. Killifish populations inhabiting environmental toxicant-contaminated New Bedford Harbor (NBH) show phenotypes tolerant to polychlorinated biphenyls (PCBs) and differences at the transcriptional and genomic levels. However, limited research has explored epigenetic alterations and metabolic effects in NBH killifish. To identify the involvement of epigenetic and metabolic regulation in the adaptive response of killifish, we investigated tissue- and sex-specific differences in global DNA methylation and metabolomic profiles of NBH killifish populations, compared to sensitive populations from a non-polluted site, Scorton Creek (SC). The results revealed that liver-specific global DNA hypomethylation and differential metabolites were evident in fish from NBH compared with those from SC. The sex-specific differences were not greater than the tissue-specific differences. We demonstrated liver-specific enriched metabolic pathways (e.g., amino acid metabolic pathways converged into the urea cycle and glutathione metabolism), suggesting possible crosstalk between differential metabolites and DNA hypomethylation in the livers of NBH killifish. Additional investigation of methylated gene regions is necessary to understand the functional role of DNA hypomethylation in the regulation of enzyme-encoding genes associated with metabolic processes and physiological changes in NBH populations.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Endothelial cells control vascular smooth muscle cell cholesterol levels by regulating 24-dehydrocholesterol reductase expression

Highlights: • Suspended endothelial and vascular smooth muscle cells organize to form spherical vascular organoids in hanging drops. • Vascular smooth muscle cells downregulate the expression of metabolism regulating genes in presence of endothelial cells. • Endothelial cells decrease the levels of 24-dehydrocholesterol reductase and cholesterol in vascular smooth muscle cells. • Diminished cholesterol biosynthesis inhibits vascular smooth muscle cell activity. Communication of vascular cells is essential for the control of organotypic functions of blood vessels. In this context, vascular endothelial cells (EC) act as potent regulators of vascular smooth muscle cell (VSMC) functions such as contraction and relaxation. However, the impact of ECs on the gene expression pattern of VSMCs is largely unknown. Here, we investigated changes of the VSMC transcriptome by utilizing 3D human vascular organoids organized as a core of VSMCs enclosed by a monolayer of ECs. Microarray-based analyses indicated that interaction with ECs for 48 h down-regulates expression of genes in VSMCs controlling rate-limiting steps of the cholesterol biosynthesis such as HMGCR, HMGCS1, DHCR24 and DHCR7. Protein analyses revealed a decrease in the abundance of DHCR24 (24-dehydrocholesterol reductase) and lower cholesterol levels in VSMCs co-cultured with ECs. On the functional level, the blockade of the DHCR24 activity impaired adhesion, migration and proliferation of VSMCs. Collectively, these findings indicate that ECs have the capacity to instruct VSMCs to shut down the expression of DHCR24 thereby limiting their cholesterol biosynthesis, which may support their functional steady state.

60 APPLIED LIFE SCIENCES↗

Metabolomics analysis reveals differences in evolution between maize and rice

SUMMARY Metabolites are the intermediate and final products of metabolism, which play essential roles in plant growth, evolution and adaptation to changing climates. However, it is unclear how evolution contributes to metabolic variation in plants. Here, we investigated the metabolomics data from leaf and seed tissues in maize and rice. Using principal components analysis based on leaf metabolites but not seed metabolites, metabolomics data could be clearly separated for rice Indica and Japonica accessions, while two maize subgroups, temperate and tropical, showed more visible admixture. Rice and maize seed exhibited significant interspecific differences in metabolic variation, while within rice, leaf and seed displayed similar metabolic variations. Among 10 metabolic categories, flavonoids had higher variation in maize than rice, indicating flavonoids are a key constituent of interspecific metabolic divergence. Interestingly, metabolic regulation was also found to be reshaped dramatically from positive to negative correlations, indicative of the differential evolutionary processes in maize and rice. Moreover, perhaps due to this divergence significantly more metabolic interactions were identified in rice than maize. Furthermore, in rice, the leaf was found to harbor much more intense metabolic interactions than the seed. Our result suggests that metabolomes are valuable for tracking evolutionary history, thereby complementing and extending genomic insights concerning which features are responsible for interspecific differentiation in maize and rice.

Deng, Min↗

Ampk alpha2 T172 activation dictates exercise performance and energy transduction in skeletal muscle

Adenosine 5′-monophosphate–activated protein kinase (AMPK) is an energetic sensor for metabolic regulation and integration. Here, we used CRISPR-Cas9 to generate nonactivatable Ampkα knock-in (KI) mice with mutation of threonine-172 phosphorylation site to alanine (T172A), circumventing the limitations of previous genetic interventions that disrupt the protein stoichiometry. KI mice of Ampkα2, but not Ampkα1, demonstrated phenotypic changes with increased fat-to-lean mass, impaired endurance exercise capacity, and diminished mitochondrial maximal respiration and conductance in skeletal muscle. Integrated temporal multiomics analysis (proteomics/phosphoproteomics/metabolomics) in skeletal muscle at rest and during exercise establishes a pleiotropic yet imperative role of Ampkα2 T172 activation for glycolytic and oxidative metabolism, mitochondrial respiration, and contractile function. There is a substantial overlap of skeletal muscle proteomic changes in Ampkα2 T172A KI mice with that of patients with type 2 diabetes. Our findings suggest that Ampkα2 T172 activation is critical for exercise performance and energy transduction in skeletal muscle and may serve as a therapeutic target for type 2 diabetes.

Bioenergetics↗

Vip1 is a kinase and pyrophosphatase switch that regulates inositol diphosphate signaling

Inositol diphosphates (PP-IPs), also known as inositol pyrophosphates, are high-energy cellular signaling codes involved in nutrient and regulatory responses. We report that the evolutionarily conserved gene product, Vip1, possesses autonomous kinase and pyrophosphatase domains capable of synthesis and destruction of D-1 PP-IPs. Our studies provide atomic-resolution structures of the PP-IP products and unequivocally define that the Vip1 gene product is a highly selective 1-kinase and 1-pyrophosphatase enzyme whose activities arise through distinct active sites. Kinetic analyses of kinase and pyrophosphatase parameters are consistent with Vip1 evolving to modulate levels of 1-IP 7 and 1,5-IP 8 . Individual perturbations in kinase and pyrophosphatase activities in cells result in differential effects on vacuolar morphology and osmotic responses. Analogous to the dual-functional key energy metabolism regulator, phosphofructokinase 2, Vip1 is a kinase and pyrophosphatase switch whose 1-PP-IP products play an important role in a cellular adaptation.

59 BASIC BIOLOGICAL SCIENCES↗

Engineering Clostridium Tyrobutyricum for High Butanol Production through Induction Expression of Exogenous NADPH-Dependent HBD

Clostridium tyrobutyricum Δ cat 1:: adh E2 is a promising cell factory for butanol production because of its robustness, high butanol tolerance, and minimal butyrate production. However, excessive acetate and ethanol production remains a major bottleneck limiting its butanol yield. Coexpressing an exogenous hbd ( Ck ) encoding the NADPH-dependent 3-hydroxybutyryl-CoA dehydrogenase (HBD) from Clostridium kluyveri with adh E2 could increase the C4 carbon flux, resulting in increased butanol and decreased acetate and ethanol production. However, constitutively overexpressing hbd ( Ck ) in Δ cat 1:: adh E2 shows little improvement in butanol yield, productivity, and selectivity, which might be caused by redox imbalance and growth inhibition. To alleviate this problem, C. tyrobutyricum MΔ cat 1:: adh E2- Pbgal-hbd ( Ck ) with a dynamic expression of hbd ( Ck ) controlled by an inducible promoter was developed. In serum bottle fermentation at 37 °C, when the hbd ( Ck ) expression was induced at 12 h or in the early exponential phase, butanol production increased ∼20% in yield (from 0.22 to 0.27 g/g glucose), 87.5% in productivity (from 0.16 to 0.30 g/L·h), and 52% in selectivity (from 0.46 to 0.70 g/g total products) compared to the control strain without expressing any hbd(Ck), whereas hbd ( Ck ) expression induced at 0 or 24 h in MΔ cat 1:: adh E2-P bgal - hbd ( Ck ) or constitutively in MΔ cat 1:: adh E2-P cat 1- hbd ( Ck ) showed significantly lower butanol yield and productivity. At 25 °C, MΔ cat 1:: adh E2-P bgal - hbd ( Ck ) with 12 h induction produced the highest butanol titer of 23 g/L with 0.32 g/g yield, 0.16 g/L·h productivity, and 0.83 g/g product selectivity due to much reduced acetate formation. Subsequent scale-up to a stirred-tank bioreactor at 37 °C increased productivity to 0.39 g/L·h while also achieving high butanol titer (21.8 g/L), yield (0.30 g/g), and selectivity (0.67 g/g). The optimized induction timing resulted in a balanced NAD(P)H pool, effectively channeling substrates toward butanol biosynthesis. It was concluded that the timing for hbd ( Ck ) expression was critical as it affected glucose catabolism, cell growth, redox balance, and carbon flux distribution. These findings underscore the potential of dynamic metabolic regulation to overcome bottlenecks in biobutanol production, providing a scalable and economically viable bioprocess for industrial application.

Clostridium tyrobutyricum↗

Arabidopsis cytochrome b 5 proteins support fatty acid ω-3 but not ω-6 desaturation

Fatty acids are primary components of lipids, which serve as major energy sources in cells and play essential roles in membrane structure, signaling, and metabolic regulation (Shanklin and Cahoon 1998). The degree of fatty acid unsaturation critically influences lipid physicochemical properties, thereby affecting membrane fluidity and biological function (Nguyen et al. 2019). In Arabidopsis thaliana, fatty acid desaturation occurs via 2 parallel pathways: the “prokaryotic pathway” in plastids, involving glycosylglycerides, such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), and phospholipid phosphatidylglycerol (PG); and the “eukaryotic pathway” in the endoplasmic reticulum (ER), involving phosphatidylcholine (PC) (Lou et al. 2014) (Supplementary Figure S1). Seven fatty acid desaturases (FADs) in Arabidopsis differentially desaturate each glycerolipid class in the plastid and ER (Nguyen et al. 2019). FAD2, an ER-resident ω-6 fatty acid desaturase, catalyzes the conversion of oleic acid (18:1) to linoleic acid (18:2), which can be further desaturated to α-linolenic acid (18:3) by FAD3, an ER-resident ω-3 fatty acid desaturase. In plastids, FAD6 catalyzes the desaturation of 18:1/16:1 to produce 18:2/16:2, while FAD7 and FAD8 redundantly convert 18:2/16:2 to 18:3/16:3 (Li-Beisson et al. 2013; Nguyen et al. 2019). Additionally, fatty acids synthesized in the ER can also be reimported into plastids to their site of de novo synthesis (Xu et al. 2010). All FADs require reducing power, in the form of 2 electrons, for catalysis, but the sources of the electrons vary between their subcellular localizations. In the ER, FAD2 and FAD3 receive electrons from a cytochrome b 5 (CB5)-based electron transfer chain comprising cytochrome b 5 reductase (CBR) and CB5. In contrast, ferredoxin serves as the electron donor for plastid-localized FAD6, FAD7, and FAD8 (Ohlrogge and Browse 1995; Andreu et al. 2007). While the relative contributions of the 2 pathways to total cellular desaturation products vary across tissues and species, most polyunsaturated FA biosynthesis in seeds occurs via ER-resident FAD2 and FAD3 (Miquel and Browse 1992; Ohlrogge and Browse 1995).

59 BASIC BIOLOGICAL SCIENCES↗

pnnl-predictive-phenomics/csc031cyc

Organism-specific Pathway/Genome databases enable the analysis, visualization and interrogation of metabolism, regulation, and genetics. Licensed under the CC-BY-4.0 license

Zucker, Jeremy [Pacific Northwest National Laborat↗

pnnl-predictive-phenomics/csc043cyc

Organism-specific Pathway/Genome databases enable the analysis, visualization and interrogation of metabolism, regulation, and genetics. Licensed under the CC-BY-4.0 license

Zucker, Jeremy [Pacific Northwest National Laborat↗

Ghrelin Modulates Differential Expression of Genes Relevant to Immune Activities and Antimicrobial Peptides in Primary Head Kidney Cells of Rainbow Trout ( Oncorhynchus mykiss )

Ghrelin is a peptide hormone/cytokine that regulates metabolic processes and plays essential roles in the immune system. To evaluate the immunomodulatory actions of ghrelin isoforms in rainbow trout (RT), an in vitro model was utilized with primary cells isolated from fish head kidney (HKD). These RT-HKD cells were treated with synthetic rainbow trout ghrelin and its truncated isoform, desVRQ-ghrelin, over time (0, 2, 4, and 24 h). Reverse transcriptase-coupled qPCR was used to measure the differential expression patterns of genes relevant to various immune processes and genes of antimicrobial peptides. Ghrelin isoform treatments resulted in functional perturbations that displayed overlapping and divergent patterns of gene expression. The differing actions between the two ghrelin isoforms on various assessed genes, and at differing time points, suggested that the two analogs may activate unique pathways, thereby eliciting distinct responses in fish immunity.

59 BASIC BIOLOGICAL SCIENCES↗

Pathology and Therapeutic Significance of Fibroblast Growth Factors

The fibroblast growth factor (FGF) family includes 22 proteins in humans. Based on their mode of action, there are three families of FGFs: paracrine FGFs (FGF 1–10, 16, 17, 18, 20, and 22), intracrine FGFs (FGF 11–14), and endocrine FGFs (FGF 19, 21, and 23). FGF signaling plays critical roles in embryonic development, tissue repair, regeneration, angiogenesis, and metabolic regulation. They exert their cellular functions by binding, dimerization, and activation of transmembrane FGF receptors (FGFRs). Aberrant FGF signaling is associated with various human diseases. Thus, understanding the unique properties of FGF signaling will help to explore new therapeutic interventions against FGF-mediated pathological conditions. This review will discuss the differential expression and regulation of each FGF under normal human physiological and pathological conditions. Moreover, we will outline current therapeutics and treatment strategies that have been developed against FGF-related pathology.

Edirisinghe, Oshadi↗

Aligned FTICR-MS, Respiration, and Biochemical Transformation Data from Columbia River Sediment Incubations Associated with: “Carbon Limitation Leads to Thermodynamic Regulation of Aerobic Metabolism”

Columbia River hyporheic zone sediments from the Hanford Site 300 Area in eastern Washington were incubated in a full factorial design with different organic matter (OM) amendments. The aim of the experiment was to test the influence of OM thermodynamics on aerobic respiration across different organic carbon pools and concentrations. This dataset is comprised of (1) a csv with high resolution characterization of dissolved organic matter via 12 Tesla Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) analyzed through the Environmental Molecular Sciences Laboratory (EMSL; https://www.pnnl.gov/environmental-molecular-sciences-laboratory), (2) a csv with sample IDs, replicates, amendments, dissolved oxygen (DO) measurements, and sampling time points, (3) a csv with sample IDs, replicates, respiration rates, FTICR summary metrics, and relative abundance of chemical classes; and (4) a csv with the number of chemical transformations in each sample replicate (microcosm). The data are associated with the publication “Carbon Limitation Leads to Thermodynamic Regulation of Aerobic Metabolism” published in ES&T Letters (Garayburu-Caruso et al., 2020). Please use the data package’s DOI to cite the data package. We ask that you acknowledge the U.S. Department of Energy (DOE) Biological and Environmental Research (BER) Subsurface Biogeochemical Research (SBR) program when using the data. All data are free to be used for any purpose, such as for manuscripts, presentations, and grant proposals. There is no obligation to include data package authors as co-authors.

54 ENVIRONMENTAL SCIENCES↗

Engineering transcriptional regulation of pentose metabolism in Rhodosporidium toruloides for improved conversion of xylose to bioproducts

Abstract Efficient conversion of pentose sugars remains a significant barrier to the replacement of petroleum-derived chemicals with plant biomass-derived bioproducts. While the oleaginous yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides ) has a relatively robust native metabolism of pentose sugars compared to other wild yeasts, faster assimilation of those sugars will be required for industrial utilization of pentoses. To increase the rate of pentose assimilation in R. toruloides, we leveraged previously reported high-throughput fitness data to identify potential regulators of pentose catabolism. Two genes were selected for further investigation, a putative transcription factor (RTO4_12978, Pnt1) and a homolog of a glucose transceptor involved in carbon catabolite repression (RTO4_11990). Overexpression of Pnt1 increased the specific growth rate approximately twofold early in cultures on xylose and increased the maximum specific growth by 18% while decreasing accumulation of arabitol and xylitol in fast-growing cultures. Improved growth dynamics on xylose translated to a 120% increase in the overall rate of xylose conversion to fatty alcohols in batch culture. Proteomic analysis confirmed that Pnt1 is a major regulator of pentose catabolism in R. toruloides. Deletion of RTO4_11990 increased the growth rate on xylose, but did not relieve carbon catabolite repression in the presence of glucose. Carbon catabolite repression signaling networks remain poorly characterized in R. toruloides and likely comprise a different set of proteins than those mainly characterized in ascomycete fungi.

59 BASIC BIOLOGICAL SCIENCES↗

Grape ASR Regulates Glucose Transport, Metabolism and Signaling

To decipher the mediator role of the grape Abscisic acid, Stress, Ripening (ASR) protein, VvMSA, in the pathways of glucose signaling through the regulation of its target, the promoter of hexose transporter VvHT1, we overexpressed and repressed VvMSA in embryogenic and non-embryogenic grapevine cells. The embryogenic cells with organized cell proliferation were chosen as an appropriate model for high sensitivity to the glucose signal, due to their very low intracellular glucose content and low glycolysis flux. In contrast, the non-embryogenic cells displaying anarchic cell proliferation, supported by high glycolysis flux and a partial switch to fermentation, appeared particularly sensitive to inhibitors of glucose metabolism. By using different glucose analogs to discriminate between distinct pathways of glucose signal transduction, we revealed VvMSA positioning as a transcriptional regulator of the glucose transporter gene VvHT1 in glycolysis-dependent glucose signaling. The effects of both the overexpression and repression of VvMSA on glucose transport and metabolism via glycolysis were analyzed, and the results demonstrated its role as a mediator in the interplay of glucose metabolism, transport and signaling. The overexpression of VvMSA in the Arabidopsis mutant abi8 provided evidence for its partial functional complementation by improving glucose absorption activity.

59 BASIC BIOLOGICAL SCIENCES↗

F‐Box Protein‐Mediated Proteolytic Regulation of Phenylpropanoid Metabolism in Response to Biotic and Abiotic Stresses

Protein ubiquitination is a central regulatory mechanism governing plant growth, development and environmental adaptation. Ubiquitylomic studies have revealed that many enzymes in phenylpropanoid biosynthetic pathways are subject to ubiquitination. Increasing evidence indicates that specific F-box proteins target key enzymes in these pathways, including PAL, CCR, CAD, COMT and peroxidases in the lignin biosynthetic branch, and CHS in the flavonoid biosynthetic branch, thereby promoting their ubiquitination and selective degradation. These F-box proteins act in response to diverse developmental and environmental cues, including cellular carbon status, light quality and intensity, and biotic stresses (e.g., pathogen and insect attack). By regulating the stability and activity of both enzymes and regulatory proteins involved in phenylpropanoid biosynthesis, F-box proteins modulate the accumulation of simple phenolics and lignin polymers, ultimately contributing to plant resilience. This review summarizes recent advances in the characterization of F-box proteins involved in phenylpropanoid metabolism and their regulatory roles in response to biotic and abiotic stresses and identifies key knowledge gaps that limit mechanistic understanding of F-box protein-mediated proteolytic regulation of phenylpropanoid metabolism. In conclusion, insights into ubiquitin-mediated proteolytic control of phenylpropanoid metabolism offer promising avenues for enhancing bioactive phenolic production, advancing biofuel feedstock engineering and improving crop stress tolerance.

(A)biotic stress↗

Carbon Limitation Leads to Thermodynamic Regulation of Aerobic Metabolism

Organic matter (OM) metabolism in freshwater ecosystems is a critical source of uncertainty in global biogeochemical cycles, yet the processes regulating aerobic respiration in aquatic environments remain poorly understood. Aerobic respiration is typically predicted through kinetic controls such as organic carbon and oxygen concentrations, while assuming thermodynamic regulation has no influence. The role of OM thermodynamics has been mainly explored under anaerobic conditions; however, recent observations have suggested that thermodynamic properties of OM may influence aerobic respiration rates. Here, we explicitly test OM thermodynamics as a key regulator of aerobic respiration. We pair controlled microcosm experiments with ultrahigh-resolution OM characterization to show that aerobic respiration increases with OM thermodynamic favorability under carbon limitation. We also demonstrate a shift in the regulation of aerobic respiration from OM thermodynamics to nitrogen content when carbon is in excess, highlighting a central role for OM thermodynamics particularly in carbon-limited ecosystems. This illuminates a structural gap in aquatic biogeochemical models by demonstrating that both kinetic and thermodynamic constraints can modulate aerobic respiration. Our work therefore proposes a new conceptual model in which thermodynamic and nutrient limitations dually control aerobic respiration.

54 ENVIRONMENTAL SCIENCES↗