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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Integration of plant and microbial oil processing at oilcane biorefineries for more sustainable biofuel production

Oilcane—an oil-accumulating crop engineered from sugarcane—and microbial oil have the potential to improve renewable oil production and help meet the expected demand for bioderived oleochemicals and fuels. To assess the potential synergies of processing both plant and microbial oils, the economic and environmental implications of integrating microbial oil production at oilcane and sugarcane biorefineries were characterized. Due to decreased crop yields that lead to higher simulated feedstock prices and lower biorefinery capacities, current oilcane prototypes result in higher costs and carbon intensities than microbial oil from sugarcane. To inform oilcane feedstock development, we calculated the required biomass yields (as a function of oil content) for oilcane to achieve financial parity with sugarcane. At 10 dw% oil, oilcane can sustain up to 30% less yield than sugarcane and still be more profitable in all simulated scenarios. Assuming continued improvements in microbial oil production from cane juice, achieving this target results in a minimum biodiesel selling price of 1.34 [0.90, 1.85] USD∙L –1 (presented as median [5th, 95th] percentiles), a carbon intensity of 0.51 [0.47, 0.55] kg CO 2 e L –1 , and a total biodiesel yield of 2140 [1870, 2410] L ha –1 year –1 . Compared to biofuel production from soybean, this outcome is equivalent to 3.0–3.9 as much biofuel per hectare of land and a 57%–63% reduction in carbon intensity. While only 20% of simulated scenarios fell within the market price range of biodiesel (0.45–1.11 USD∙L –1 ), if the oilcane biomass yield would improve to 25.6 DMT∙ha –1 ∙y –1 (an equivalent yield to sugarcane) 87% of evaluated scenarios would have a minimum biodiesel selling price within or below the market price range.

09 BIOMASS FUELS↗
DOE OSTI2020

Functional and genetic markers of niche partitioning among enigmatic members of the human oral microbiome

Abstract Introduction Microbial residents of the human oral cavity have long been a major focus of microbiology due to their influence on host health and intriguing patterns of site specificity amidst the lack of dispersal limitation. However, the determinants of niche partitioning in this habitat are yet to be fully understood, especially among taxa that belong to recently discovered branches of microbial life. Results Here, we assemble metagenomes from tongue and dental plaque samples from multiple individuals and reconstruct 790 non-redundant genomes, 43 of which resolve to TM7, a member of the Candidate Phyla Radiation, forming six monophyletic clades that distinctly associate with either plaque or tongue. Both pangenomic and phylogenomic analyses group tongue-specific clades with other host-associated TM7 genomes. In contrast, plaque-specific TM7 group with environmental TM7 genomes. Besides offering deeper insights into the ecology, evolution, and mobilome of cryptic members of the oral microbiome, our study reveals an intriguing resemblance between dental plaque and non-host environments indicated by the TM7 evolution, suggesting that plaque may have served as a stepping stone for environmental microbes to adapt to host environments for some clades of microbes. Additionally, we report that prophages are widespread among oral-associated TM7, while absent from environmental TM7, suggesting that prophages may have played a role in adaptation of TM7 to the host environment. Conclusions Our data illuminate niche partitioning of enigmatic members of the oral cavity, including TM7, SR1, and GN02, and provide genomes for poorly characterized yet prevalent members of this biome, such as uncultivated Flavobacteriaceae.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Lost and Found: Rediscovering Microbiome-Associated Phenotypes that Reshape Agricultural Sustainability

Modern agriculture faces an urgent need to improve nutrient use efficiency while reducing environmental impacts. Here, we show that ancestral traits controlling rhizosphere microbiome functions can be reintroduced into elite maize through targeted teosinte introgressions. Using near-isogenic lines, we mapped microbiome-associated phenotypes (MAPs) derived from teosinte that suppress nitrification and denitrification—key microbial processes contributing to nitrogen loss. These introgressions altered root exudate chemistry, resulting in distinct microbial assemblies and enhanced nitrogen retention. We identified candidate loci and metabolites responsible for suppressive activity and demonstrated their functional effects in vitro. Our findings reveal a genetic and biochemical basis for rewilding microbiome-mediated ecosystem services in crops, offering a scalable path toward sustainable nutrient management in global agriculture. ---- These maize root exduate metabolomics data are a subset of this larger project and make up a phenotyping for candidate lines.

Favela, Alonso [School of Plant Sciences, Universi↗
DOE OSTI2020

Long-Term Stochasticity Combines With Short-Term Variability in Assembly Processes to Underlie Rice Paddy Sustainability

Revealing temporal patterns of community assembly processes is important for understanding how microorganisms underlie the sustainability of agroecosystem. The ancient terraced rice paddies at Longji provide an ideal platform to study temporal dynamics of agroecosystem sustainability due to their chronosequential records of soil physicochemistry and well-archived microbial information along 630-year rice cultivation. We used statistical null models to evaluate microbial assembly processes along the soil chronosequences of Longji rice paddies through time. Stochastic and deterministic assembly processes jointly governed microbial community composition within successional eras (less than 250 years), and within-era determinism was mainly driven by soil fertility and redox conditions alone or in combination. Conversely, across successional eras (i.e., over 300 years), stochasticity linearly increased with increasing duration between eras and was eventually predominant for the whole 630 years. We suggest that the impact of stochasticity vs. determinism on assembly is timescale-dependent, and we propose that the importance of stochastic assembly of microbial community at longer timescales is due to the gradual changes in soil properties under long-term rice cultivation, which in turn contribute to the sustainability of paddy ecosystem by maintaining a diverse community of microorganisms with multi-functional traits. In total, our results indicate that knowledge on the timescales at which assembly processes govern microbial community composition is key to understanding the ecological mechanisms generating agroecosystem sustainability.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Inline small-angle X-ray scattering-coupled chromatography under extreme hydrostatic pressure

As continuing discoveries highlight the surprising abundance and resilience of deep ocean and subsurface microbial life, the effects of extreme hydrostatic pressure on biological structure and function have attracted renewed interest. Biological small-angle X-ray scattering (BioSAXS) is a widely used method of obtaining structural information from biomolecules in solution under a wide range of solution conditions. Due to its ability to reduce radiation damage, remove aggregates, and separate monodisperse components from complex mixtures, size-exclusion chromatography-coupled SAXS (SEC-SAXS) is now the dominant form of BioSAXS at many synchrotron beamlines. While BioSAXS can currently be performed with some difficulty under pressure with non-flowing samples, it has not been clear how, or even if, continuously flowing SEC-SAXS, with its fragile media-packed columns, might work in an extreme high-pressure environment. Here we show, for the first time, that reproducible chromatographic separations coupled directly to high-pressure BioSAXS can be achieved at pressures up to at least 100 MPa and that pressure-induced changes in folding and oligomeric state and other properties can be observed. Further, the apparatus described here functions at a range of temperatures (0°C–50°C), expanding opportunities for understanding biomolecular rules of life in deep ocean and subsurface environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

An Improved CRISPR Interference Tool to Engineer Rhodococcus opacus

Rhodococcus opacus is a non-model bacterium that is well suited for valorizing lignin. Despite recent advances in our systems-level understanding of its versatile metabolism, studies of its gene functions at a single gene level are still lagging. Elucidating gene functions in non-model organisms is challenging due to limited genetic engineering tools that are convenient to use. To address this issue, we developed a simple gene repression system based on CRISPR interference (CRISPRi). This gene repression system uses a T 7 RNA polymerase system to express a small guide RNA, demonstrating improved repression compared to the previously demonstrated CRISPRi system (i.e., the maximum repression efficiency improved from 58% to 85%). Additionally, our cloning strategy allows for building multiple CRISPRi plasmids in parallel without any PCR step, facilitating the engineering of this GC-rich organism. Using the improved CRISPRi system, we confirmed the annotated roles of four metabolic pathway genes, which had been identified by our previous transcriptomic analysis to be related to the consumption of benzoate, vanillate, catechol, and acetate. Furthermore, we showed our tool’s utility by demonstrating the inducible accumulation of muconate that is a precursor of adipic acid, an important monomer for nylon production. While the maximum muconate yield obtained using our tool was 30% of the yield obtained using gene knockout, our tool showed its inducibility and partial repressibility. In conclusion, our CRISPRi tool will be useful to facilitate functional studies of this non-model organism and engineer this promising microbial chassis for lignin valorization.

09 BIOMASS FUELS↗
DOE OSTI2024

Phylogenetic distribution and experimental characterization of corrinoid production and dependence in soil bacterial isolates

Abstract Soil microbial communities impact carbon sequestration and release, biogeochemical cycling, and agricultural yields. These global effects rely on metabolic interactions that modulate community composition and function. However, the physicochemical and taxonomic complexity of soil and the scarcity of available isolates for phenotypic testing are significant barriers to studying soil microbial interactions. Corrinoids—the vitamin B12 family of cofactors—are critical for microbial metabolism, yet they are synthesized by only a subset of microbiome members. Here, we evaluated corrinoid production and dependence in soil bacteria as a model to investigate the ecological roles of microorganisms involved in metabolic interactions. We isolated and characterized a taxonomically diverse collection of 161 soil bacteria from a single study site. Most corrinoid-dependent bacteria in the collection prefer B12 over other corrinoids, while all tested producers synthesize B12, indicating metabolic compatibility between producers and dependents in the collection. Furthermore, a subset of producers release B12 at levels sufficient to support dependent isolates in laboratory culture at estimated ratios of up to 1000 dependents per producer. Within our isolate collection, we did not find strong phylogenetic patterns in corrinoid production or dependence. Upon investigating trends in the phylogenetic dispersion of corrinoid metabolism categories across sequenced bacteria from various environments, we found that these traits are conserved in 47 out of 85 genera. Together, these phenotypic and genomic results provide evidence for corrinoid-based metabolic interactions among bacteria and provide a framework for the study of nutrient-sharing ecological interactions in microbial communities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Metagenomes and Metagenome-Assembled Genomes from Microbiomes Metabolizing Thin Stillage from an Ethanol Biorefinery

Here, we report the metagenomes from five anaerobic bioreactors, operated under different conditions, that were fed carbohydrate-rich thin stillage from a corn starch ethanol plant. The putative functions of the abundant taxa identified here will inform future studies of microbial communities involved in valorizing this and other low-value agroindustrial residues.

Fortney, Nathaniel W.↗
NASA NTRS2005

"Ames Research Center: Linking our Origins to our Future"

Our research traces a path from interstellar materials to inhabited worlds and beyond. We examine how protoplanetary disks evolve and form terrestrial planets, the evolutionary paths of habitable planets, and how external factors (e.g., orbital eccentricity) and internal factors (atmospheric circulation) affect habitability. We trace, spectroscopically and chemically, the evolution of organic molecules from the interstellar medium onto habitable bodies. We examine how membranes might form under prebiotic planetary conditions. We evolve proteins capable of sustaining early metabolism, such as synthesis of biopolymers and transport of ions across membranes. We estimate the frequency of finding a functional prebiotic protein that formed spontaneously. We characterize the formation of diagnostic microbial biosignatures in rock-hosted ecosystems in ophiolite springs as an analog for subsurface life within our solar system, and photosynthetic microbial mats as biota that could be detected on extrasolar planets. We develop quantitative models that simulate energy relationships, biogeochemical cycling, trace gas exchange, and biodiversity. We examine the effects of climate variability on a vegetation-rich biosphere over intermediate time scales, using South American ecosystems as a model. We address natural transport of life beyond its planet of origin, such as on a meteorite, where survivors must withstand radiation, desiccation, and time in transit. We fly organisms and ecosystems in low Earth orbit to test their resistance to space. The Ames E&PO program disseminates these themes to national- and international-scale audiences through partnerships with the California Academy of Sciences, Yellow stone National Park, New York Hall of Science, and several K-14 educational organizations.

DesMarais, David J.↗
NASA NTRS2015

Omics Research on the International Space Station

The International Space Station (ISS) is an orbiting laboratory whose goals include advancing science and technology research. Completion of ISS assembly ushered a new era focused on utilization, encompassing multiple disciplines such as Biology and Biotechnology, Physical Sciences, Technology Development and Demonstration, Human Research, Earth and Space Sciences, and Educational Activities. The research complement planned for upcoming ISS Expeditions 45&46 includes several investigations in the new field of omics, which aims to collectively characterize sets of biomolecules (e.g., genomic, epigenomic, transcriptomic, proteomic, and metabolomic products) that translate into organismic structure and function. For example, Multi‐Omics is a JAXA investigation that analyzes human microbial metabolic cross‐talk in the space ecosystem by evaluating data from immune dysregulation biomarkers, metabolic profiles, and microbiota composition. The NASA OsteoOmics investigation studies gravitational regulation of osteoblast genomics and metabolism. Tissue Regeneration uses pan‐omics approaches with cells cultured in bioreactors to characterize factors involved in mammalian bone tissue regeneration in microgravity. Rodent Research‐3 includes an experiment that implements pan‐omics to evaluate therapeutically significant molecular circuits, markers, and biomaterials associated with microgravity wound healing and tissue regeneration in bone defective rodents. The JAXA Mouse Epigenetics investigation examines molecular alterations in organ specific gene expression patterns and epigenetic modifications, and analyzes murine germ cell development during long term spaceflight. Lastly, Twins Study ("Differential effects of homozygous twin astronauts associated with differences in exposure to spaceflight factors"), NASA's first foray into human omics research, applies integrated analyses to assess biomolecular responses to physical, physiological, and environmental stressors associated with spaceflight.

Love, John↗
DOE OSTI2019

Synthetic Methane-Consuming Communities from a Natural Lake Sediment

The factors and processes that influence the behavior and functionality of ecosystems inhabited by complex microbiomes are still far from being clearly understood. Synthetic microbial communities provide reduced-complexity models that allow an examination of ecological theories under defined and controlled conditions. In this study, we applied a multiphasic approach to study synthetic methane-oxidizing communities and species interactions as proxies to the natural communities. Our results confirm that, under selective pressures, natural-sediment communities of high complexity simplify rapidly, selecting for several major functional guilds, the major partners in methane oxidation being the Methylococcaceae methanotrophs and the Methylophilaceae methylotrophs, along with minor but persistent partners, members of Burkholderiales and Flavobacteriales. As a proof of concept, we established minimalist synthetic communities that were representative of the four functional guilds to demonstrate the dependency of the non-methane-utilizing species on the methanotrophs as the primary carbon-providing species. We observed that in communities consisting of multiple representatives of the key guilds, members of the same guild appeared to compete for resources. For example, when two methanotrophs of the same family were present, the two expressed similar key methanotrophy pathways and responded similarly to changing environmental conditions, suggesting that they perform a similar keystone function in situ. Similar observations were made for the Methylophilaceae. However, differences were noted in the expression of auxiliary and unique genes among strains of the same functional guild, reflecting differential adaptation and suggesting mechanisms for competition. At the same time, differences were also noted in the performances of partners with specific metabolic schemes. For example, a mutant of Methylotenera mobilis impaired in nitrate utilization behaved as a more efficient cooperator in methane consumption, suggesting that the loss of function may lead to changes in communal behavior. Overall, we demonstrate the robust nature of synthetic communities built of native lake sediment strains and their utility in addressing important ecological questions while using a simplified model.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Time-series RNA metabarcoding of the active Populus tremuloides root microbiome reveals hidden temporal dynamics and dormant core members

The rhizosphere is a critical interface between plant roots and soil, harboring diverse microbial communities that are essential to plant and ecosystem health. Although these communities exhibit stark temporal dynamics, their dormancy/activity transitions remain poorly understood. Such transitions may enable microbes to rapidly adjust functional contributions faster than community turnover alone would allow. Here, we used RNA metabarcoding to characterize the active fraction of microbial communities on the roots of quaking aspen (Populus tremuloides) in a time-series study across a natural environmental gradient. We explore cryptic temporal microbial community dynamics of rhizosphere communities at the ecosystem scale. The active rhizosphere bacterial and fungal communities were more temporally dynamic than total communities, while total communities exhibited a stronger response to site-specific conditions. Notably, some core microbiome members were often inactive, yielding a smaller “active core” subset. The fungal endophyte Hyaloscypha finlandica was the only microbe that was both present and active in all plots across all timepoints. Soil temperature strongly influenced both total and active community composition, with the fungal class Eurotiomycetes showing a temperature-dependent seasonal decline in abundance. Together, these results reveal that modulation of microbial activity levels is a key mechanism by which the plant root holobiont responds to environmental variation, and that even dominant symbionts may frequently persist in dormancy within the rhizosphere.

Community Structure and Diversity↗
DOE OSTI2019

A New Microbial Pathway for Organophosphonate Degradation Catalyzed by Two Previously Misannotated Non-Heme-Iron Oxygenases

The assignment of biochemical functions to hypothetical proteins is challenged by functional diversification within many protein structural superfamilies. This diversification, which is particularly common for metalloenzymes, renders functional annotations that are founded solely on sequence and domain similarities unreliable and often erroneous. Definitive biochemical characterization to delineate functional subgroups within these superfamilies will aid in improving bioinformatic approaches for functional annotation. We describe here the structural and functional characterization of two non-heme-iron oxygenases, TmpA and TmpB, which are encoded by a genomically clustered pair of genes found in more than 350 species of bacteria. TmpA and TmpB are functional homologues of a pair of enzymes (PhnY and PhnZ) that degrade 2-aminoethylphosphonate but instead act on its naturally occurring, quaternary ammonium analogue, 2-(trimethylammonio)ethylphosphonate (TMAEP). TmpA, an iron(II)- and 2-(oxo)glutarate-dependent oxygenase misannotated as a γ-butyrobetaine (γbb) hydroxylase, shows no activity toward γbb but efficiently hydroxylates TMAEP. The product, (R)-1-hydroxy-2-(trimethylammonio)ethylphosphonate [(R)-OH-TMAEP], then serves as the substrate for the second enzyme, TmpB. By contrast to its purported phosphohydrolytic activity, TmpB is an HD-domain oxygenase that uses a mixed-valent diiron cofactor to enact oxidative cleavage of the C–P bond of its substrate, yielding glycine betaine and phosphate. The high specificities of TmpA and TmpB for their N-trimethylated substrates suggest that they have evolved specifically to degrade TMAEP, which was not previously known to be subject to microbial catabolism. Here, this study thus adds to the growing list of known pathways through which microbes break down organophosphonates to harvest phosphorus, carbon, and nitrogen in nutrient-limited niches.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

In silico evaluation of a targeted metaproteomics strategy for broad screening of cellulolytic enzyme capacities in anaerobic microbiome bioreactors

Microbial-driven solubilization of lignocellulosic material is a natural mechanism that is exploited in anaerobic digesters (ADs) to produce biogas and other valuable bioproducts. Glycoside hydrolases (GHs) are the main enzymes that bacterial and archaeal populations use to break down complex polysaccharides in these reactors. Methodologies for rapidly screening the physical presence and types of GHs can provide information about their functional activities as well as the taxonomical diversity within AD systems but are largely unavailable. Targeted proteomic methods could potentially be used to provide snapshots of the GHs expressed by microbial consortia in ADs, giving valuable insights into the functional lignocellulolytic degradation diversity of a community. Such observations would be essential to evaluate the hydrolytic performance of a reactor or potential issues with it.

09 BIOMASS FUELS↗
DOE OSTI2019

Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi

We present that the fast-growing Gram-negative bacterium Vibrio natriegens is an attractive microbial system for molecular biology and biotechnology due to its remarkably short generation time and metabolic prowess. However, efforts to uncover and utilize the mechanisms underlying its rapid growth are hampered by the scarcity of functional genomic data. Here, we develop a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) screen to identify a minimal set of genes required for rapid wild-type growth. Targeting 4,565 (99.7%) of predicted protein-coding genes, our screen uncovered core genes comprising putative essential and growth-supporting genes that are enriched for respiratory pathways. We found that 96% of core genes were located on the larger chromosome 1, with growth-neutral duplicates of core genes located primarily on chromosome 2. Lastly, our screen also refines metabolic pathway annotations by distinguishing functional biosynthetic enzymes from those predicted on the basis of comparative genomics. Taken together, this work provides a broadly applicable platform for high-throughput functional genomics to accelerate biological studies and engineering of V. natriegens.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Deforestation impacts network co-occurrence patterns of microbial communities in Amazon soils

Co-occurrence networks allow for the identification of potential associations among species, which may be important for understanding community assembly and ecosystem functions. We employed this strategy to examine prokaryotic co-occurrence patterns in the Amazon soils and the response of these patterns to land use change to pasture, with the hypothesis that altered microbial composition due to deforestation will mirror the co-occurrence patterns across prokaryotic taxa. In this study, we calculated Spearman correlations between operational taxonomic units (OTUs) as determined by 16S rRNA gene sequencing, and only robust correlations were considered for network construction (-0.80 ≥ ρ ≥ 0.80, adjusted P <0.01). The constructed network represents distinct forest and pasture components, with altered compositional and topological features. A comparative analysis between two representative modules of these contrasting ecosystems revealed novel information regarding changes to metabolic pathways related to nitrogen cycling. Our results showed that soil physicochemical properties such as temperature, C/N and H + +Al 3+ had a significant impact on prokaryotic communities, with alterations to network topologies. Taken together, changes in co-occurrence patterns and physicochemical properties may contribute to ecosystem processes including nitrification and denitrification, two important biogeochemical processes occurring in tropical forest systems.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Metaproteomics reveals insights into microbial structure, interactions, and dynamic regulation in defined communities as they respond to environmental disturbance

Abstract Background Microbe-microbe interactions between members of the plant rhizosphere are important but remain poorly understood. A more comprehensive understanding of the molecular mechanisms used by microbes to cooperate, compete, and persist has been challenging because of the complexity of natural ecosystems and the limited control over environmental factors. One strategy to address this challenge relies on studying complexity in a progressive manner, by first building a detailed understanding of relatively simple subsets of the community and then achieving high predictive power through combining different building blocks (e.g., hosts, community members) for different environments. Herein, we coupled this reductionist approach with high-resolution mass spectrometry-based metaproteomics to study molecular mechanisms driving community assembly, adaptation, and functionality for a defined community of ten taxonomically diverse bacterial members of Populus deltoides rhizosphere co-cultured either in a complex or defined medium. Results Metaproteomics showed this defined community assembled into distinct microbiomes based on growth media that eventually exhibit composition and functional stability over time. The community grown in two different media showed variation in composition, yet both were dominated by only a few microbial strains. Proteome-wide interrogation provided detailed insights into the functional behavior of each dominant member as they adjust to changing community compositions and environments. The emergence and persistence of select microbes in these communities were driven by specialization in strategies including motility, antibiotic production, altered metabolism, and dormancy. Protein-level interrogation identified post-translational modifications that provided additional insights into regulatory mechanisms influencing microbial adaptation in the changing environments. Conclusions This study provides high-resolution proteome-level insights into our understanding of microbe-microbe interactions and highlights specialized biological processes carried out by specific members of assembled microbiomes to compete and persist in changing environmental conditions. Emergent properties observed in these lower complexity communities can then be re-evaluated as more complex systems are studied and, when a particular property becomes less relevant, higher-order interactions can be identified.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Lost and found: Rediscovering microbiome-associated phenotypes that reshape agricultural sustainability

Modern agriculture faces an urgent need to improve nutrient use efficiency while reducing environmental impacts. Here, we show that ancestral traits controlling rhizosphere microbiome functions can be reintroduced into elite maize through targeted teosinte introgressions. Using near-isogenic lines, we mapped microbiome-associated phenotypes (MAPs) derived from teosinte that suppress nitrification and denitrification—key microbial processes contributing to nitrogen loss. These introgressions altered root exudate chemistry, resulting in distinct microbial assemblies and enhanced nitrogen retention. We identified candidate loci and exudate metabolites responsible for suppressive activity and demonstrated their functional effects in vitro. These findings reveal a genetic and biochemical basis for rewilding microbiome-mediated ecosystem services in crops, offering a scalable path toward sustainable nutrient management in global agriculture.

60 APPLIED LIFE SCIENCES↗