Results for “microbial metabolites” — Page 9

DOE OSTI2021

WA-Omic_LA.1.0 - Quantitative Lipidomics, Metabolomics, and Sequencing (16S/ITS) Publication Data DOI Package

Corresponding Data Publication: "Rapid remodeling of the soil lipidome in response to a drying-rewetting event." This study reveals specific changes in lipids and metabolites that are indicative of stress adaptation, substrate use, and cellular recovery during soil drying and subsequent rewetting. Drought induced nutrient limitation was reflected in the lipidome and polar metabalome, both of which rapidly shifted (within hours) upon rewet. Reduced nutrient access in dry soil caused the replacement of glycerophospholipids with phosphorus-free lipids and impeded resource-expensive osmolyte accumulation. Elevated levels of ceramides and lipids with long chain polyunsaturated fatty acids, in dry soil suggests that lipids play an important role in fungal drought tolerance. Increasing abundance of bacterial glycerophospholipids and triacylglycerols with fatty acids typical of bacteria and polar metabolites suggest metabolic recovery in representative bacteria once the environmental conditions are conducive for growth. These results underscore the importance of the soil lipidome as a robust indicator of microbial community responses, especially at the short time scales of cell-environment reactions. Data package contents reported here are the first version and contain pre- and post-processed data acquisition and subsequent downstream analysis files using various data source instrument method techniques and Mass Spectroscopy (MS) EMSL capabilities. This publication data package DOI is a comprehensive high-throughput multi-omics data lifecycle collection containing processed data method metadata. Support files include additional data download “Read Me” file containing data descriptor information and data source application ontologies (see data dictionary). Reported data download contents are structured for compliance with project data sharing guidelines, community standards initiatives, and sponsor stakeholder policies supporting FAIR data principles. For increased data availability and interoperability, GC-MS/LC-MS mass spectrometry datasets (Thermo .raw ) were deposited at the MassIVE database repository under the related data accession MSV000086931 and can be accessed by using the API. Statistical data processing software, analysis tools, and data workflows are listed below corresponding to the host repository long-term location.

Amplicon sequencing 16S ITS LC-MS/MS lipidomics mu↗

DOE OSTI2021

WA-Omic_LA.1.0 - Quantitative Lipidomics, Metabolomics, and Sequencing (16S/ITS) Publication Data DOI Package

Corresponding Data Publication: "Rapid remodeling of the soil lipidome in response to a drying-rewetting event." This study reveals specific changes in lipids and metabolites that are indicative of stress adaptation, substrate use, and cellular recovery during soil drying and subsequent rewetting. Drought induced nutrient limitation was reflected in the lipidome and polar metabalome, both of which rapidly shifted (within hours) upon rewet. Reduced nutrient access in dry soil caused the replacement of glycerophospholipids with phosphorus-free lipids and impeded resource-expensive osmolyte accumulation. Elevated levels of ceramides and lipids with long chain polyunsaturated fatty acids, in dry soil suggests that lipids play an important role in fungal drought tolerance. Increasing abundance of bacterial glycerophospholipids and triacylglycerols with fatty acids typical of bacteria and polar metabolites suggest metabolic recovery in representative bacteria once the environmental conditions are conducive for growth. These results underscore the importance of the soil lipidome as a robust indicator of microbial community responses, especially at the short time scales of cell-environment reactions. Data package contents reported here are the first version and contain pre- and post-processed data acquisition and subsequent downstream analysis files using various data source instrument method techniques and Mass Spectroscopy (MS) EMSL capabilities. This publication data package DOI is a comprehensive high-throughput multi-omics data lifecycle collection containing processed data method metadata. Support files include additional data download “Read Me” file containing data descriptor information and data source application ontologies (see data dictionary). Reported data download contents are structured for compliance with project data sharing guidelines, community standards initiatives, and sponsor stakeholder policies supporting FAIR data principles. For increased data availability and interoperability, GC-MS/LC-MS mass spectrometry datasets (Thermo .raw ) were deposited at the MassIVE database repository under the related data accession MSV000086931 and can be accessed by using the API. Statistical data processing software, analysis tools, and data workflows are listed below corresponding to the host repository long-term location.

Amplicon sequencing 16S ITS LC-MS/MS lipidomics mu↗

DOE OSTI2021

WA-Omic_LA.1.0 - Quantitative Lipidomics, Metabolomics, and Sequencing (16S/ITS) Publication Data DOI Package

Corresponding Data Publication: "Rapid remodeling of the soil lipidome in response to a drying-rewetting event." This study reveals specific changes in lipids and metabolites that are indicative of stress adaptation, substrate use, and cellular recovery during soil drying and subsequent rewetting. Drought induced nutrient limitation was reflected in the lipidome and polar metabalome, both of which rapidly shifted (within hours) upon rewet. Reduced nutrient access in dry soil caused the replacement of glycerophospholipids with phosphorus-free lipids and impeded resource-expensive osmolyte accumulation. Elevated levels of ceramides and lipids with long chain polyunsaturated fatty acids, in dry soil suggests that lipids play an important role in fungal drought tolerance. Increasing abundance of bacterial glycerophospholipids and triacylglycerols with fatty acids typical of bacteria and polar metabolites suggest metabolic recovery in representative bacteria once the environmental conditions are conducive for growth. These results underscore the importance of the soil lipidome as a robust indicator of microbial community responses, especially at the short time scales of cell-environment reactions. Data package contents reported here are the first version and contain pre- and post-processed data acquisition and subsequent downstream analysis files using various data source instrument method techniques and Mass Spectroscopy (MS) EMSL capabilities. This publication data package DOI is a comprehensive high-throughput multi-omics data lifecycle collection containing processed data method metadata. Support files include additional data download “Read Me” file containing data descriptor information and data source application ontologies (see data dictionary). Reported data download contents are structured for compliance with project data sharing guidelines, community standards initiatives, and sponsor stakeholder policies supporting FAIR data principles. For increased data availability and interoperability, GC-MS/LC-MS mass spectrometry datasets (Thermo .raw ) were deposited at the MassIVE database repository under the related data accession MSV000086931 and can be accessed by using the API. Statistical data processing software, analysis tools, and data workflows are listed below corresponding to the host repository long-term location.

Amplicon sequencing 16S ITS LC-MS/MS lipidomics mu↗

DOE OSTI2022

Plant-based engineering for production of high-valued natural products

Plants are a unique source of complex specialized metabolites, many of which play significant roles in human society. In many cases, however, the availability of these metabolites from naturally occurring sources fails to meet current demands. Thus, there is much interest in expanding the production capacity of target plant molecules. Traditionally, plant breeding, chemical synthesis, and microbial fermentation are considered the primary routes towards large scale production of natural products. Here, in this paper, we explore the advances, challenges, and future of plant engineering as a complementary path. Although plants are an integral part of our food and agricultural systems and sustain an extensive array of chemical constituents, their complex genetics and physiology have prevented the optimal exploitation of plants as a production chassis. We highlight emerging engineering tools and scientific advances developed in recent years that have improved the prospects of using plants as a sustainable and scalable production platform. We also discuss technological limitations and overall economic outlook of plant-based production of natural products.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Siena College 2022 MRA Genome Announcement Narrative - 6 Isolates

Soil bacteria and endospore forming bacteria in particular play critical environmental roles in biomass degradation, plant health, and many have also been sources for commercially important secondary metabolites including antibiotics. Sequencing multiple isolates of known species also can identify new genes and gene clusters not found in other members of the same species, increasing our understanding of microbial diversity.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Genome-Resolved Metaproteomics Decodes the Microbial and Viral Contributions to Coupled Carbon and Nitrogen Cycling in River Sediments

Rivers have a significant role in global carbon and nitrogen cycles, serving as a nexus for nutrient transport between terrestrial and marine ecosystems. Although rivers have a small global surface area, they contribute substantially to worldwide greenhouse gas emissions through microbially mediated processes within the river hyporheic zone. Despite this importance, research linking microbial and viral communities to specific biogeochemical reactions is still nascent in these sediment environments. To survey the metabolic potential and gene expression underpinning carbon and nitrogen biogeochemical cycling in river sediments, we collected an integrated data set of 33 metagenomes, metaproteomes, and paired metabolomes. We reconstructed over 500 microbial metagenome-assembled genomes (MAGs), which we dereplicated into 55 unique, nearly complete medium- and high-quality MAGs spanning 12 bacterial and archaeal phyla. We also reconstructed 2,482 viral genomic contigs, which were dereplicated into 111 viral MAGs (vMAGs) of >10 kb in size. As a result of integrating gene expression data with geochemical and metabolite data, we created a conceptual model that uncovered new roles for microorganisms in organic matter decomposition, carbon sequestration, nitrogen mineralization, nitrification, and denitrification. We show how these metabolic pathways, integrated through shared resource pools of ammonium, carbon dioxide, and inorganic nitrogen, could ultimately contribute to carbon dioxide and nitrous oxide fluxes from hyporheic sediments. Further, by linking viral MAGs to these active microbial hosts, we provide some of the first insights into viral modulation of river sediment carbon and nitrogen cycling.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

Discovering type I cis-AT polyketides through computational mass spectrometry and genome mining with Seq2PKS

Type 1 polyketides are a major class of natural products used as antiviral, antibiotic, antifungal, antiparasitic, immunosuppressive, and antitumor drugs. Analysis of public microbial genomes leads to the discovery of over sixty thousand type 1 polyketide gene clusters. However, the molecular products of only about a hundred of these clusters are characterized, leaving most metabolites unknown. Characterizing polyketides relies on bioactivity-guided purification, which is expensive and time-consuming. To address this, we present Seq2PKS, a machine learning algorithm that predicts chemical structures derived from Type 1 polyketide synthases. Seq2PKS predicts numerous putative structures for each gene cluster to enhance accuracy. The correct structure is identified using a variable mass spectral database search. Benchmarks show that Seq2PKS outperforms existing methods. Applying Seq2PKS to Actinobacteria datasets, we discover biosynthetic gene clusters for monazomycin, oasomycin A, and 2-aminobenzamide-actiphenol.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

Effects of ingested nanocellulose on intestinal microbiota and homeostasis in Wistar Han rats

Micron scale crystalline and fibrillar cellulose materials are “generally regarded as safe” (GRAS) as binders and thickeners in food products. Yet, partly due to a lack of relevant toxicological data, nanocellulose (NC) materials, which have unique properties that can be exploited to improve food quality and safety, have yet to receive FDA approval as food ingredients. Results of in vitro and in vivo toxicological studies of ingested NC, detailed in a recent companion report, revealed minimal acute in vitro cytotoxicity, and no toxicity in a subacute rat gavage model, suggesting that NC materials are non-hazardous. However, ingested materials may also modulate gut microbial populations, or alter aspects of intestinal function not evaluated in standard totoxicity testing, which could have important health implications. Here, we report the results of studies of the effects of ingested cellulose nanofibrils (CNF) on the fecal microbiome and metabolome, intestinal (ileal) epithelial cell mRNA expression of cell junction genes, and ileal cytokine production. Feces, plasma, and ileum samples were collected from Wistar Han rats before and after five weeks of biweekly gavages of water or cream, with or without 1% w/w CNF. Analysis of fecal microbial populations revealed that CNF caused changes in both genus and species diversity, with specific effects on species that produce short chain fatty acids, and that have been associated with increased IgA production and elevation of insulin levels. Fecal metabolomic analysis revealed relatively few effects of CNF, with significant changes in the levels of only ten metabolites out of 366 measured. Exposure to CNF also caused differential regulation of mRNA expression of several genes involved in epithelial cell junctions, and increased production of cytokines that both promote and inhibit proliferation of CD8 T cells. These perturbations in intestinal homeostasis contrast somewhat with the absence of in vitro and in vivo toxicity observed previously but would appear to represent minor effects. Additionally, the gut microbiome is highly sensitive to ingested substances, and such disturbances of the intestinal microbial ecosystem do not necessarily represent or predict meaningful pathology. Further studies, including chronic feeding studies, are needed to assess the real health implications, if any, of these changes.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2022

Adaptations of Endolithic Communities to Abrupt Environmental Changes in A Hyper-Arid Desert

The adaptation mechanisms of microbial communities to natural perturbations remain relatively unexplored, particularly in extreme environments. The extremophilic communities of halite (NaCl) nodules from the hyper-arid core of the Atacama Desert are self-sustained and represent a unique opportunity to study functional adaptations and community dynamics with changing environmental conditions. We transplanted halite nodules to different sites in the desert and investigated how their taxonomic, cellular, and biochemical changes correlated with water availability, using environmental data modeling and metagenomic analyses. Salt-in strategists, mainly represented by haloarchaea, significantly increased in relative abundance at sites characterized by extreme dryness, multiple wet/dry cycles, and colder conditions. The functional analysis of metagenome-assembled genomes (MAGs) revealed site-specific enrichments in archaeal MAGs encoding for the uptake of various compatible solutes and for glycerol utilization. These findings suggest that opportunistic salt-in strategists took over the halite communities at the driest sites. They most likely benefited from metabolites newly released in the environment by the death of microorganisms least adapted to the new conditions. The observed changes were consistent with the need to maximize cellular bioenergetics when confronted with lower water availability and higher salinity, providing valuable information on microbial community adaptations and resilience to climate change.

Atacama↗

DOE OSTI2025

Exploring single-cell biosynthetic noise and dynamics for enhanced betaxanthin production in Escherichia coli

Cell-to-cell variability often limits the efficiency of microbial bioproduction, yet how individual cells fluctuate over time and how these fluctuations shape population-level output remain unclear. To address this issue, we tracked a heterologous betaxanthin pathway in Escherichia coli using microfluidics-assisted time-lapse microscopy, allowing simultaneous measurement of fluctuations in betaxanthin, its biosynthetic enzyme DOD and growth across generations. Here we show that over 50% of high betaxanthin producers become medium or low producers after two divisions. Betaxanthin variation primarily originates from DOD noise, with a smaller contribution from growth rate fluctuations. We further develop a stochastic model to explore various control circuits and find that pathway enzyme or metabolite-based growth selection strategies are most effective in enhancing production. We experimentally validate the model by coupling enzyme expression to nutrient availability, which enriches high producers and boosts titer by 4.4-fold. Our results highlight key sources of metabolic heterogeneity and provide a framework for designing robust microbial processes.

Bacterial systems biology↗

DOE OSTI2023

Decomposition decreases molecular diversity and ecosystem similarity of soil organic matter

Soil organic matter (SOM) is comprised of a diverse array of reactive carbon molecules, including hydrophilic and hydrophobic compounds, that impact rates of SOM formation and persistence. Despite clear importance to ecosystem science, little is known about broad-scale controls on SOM diversity and variability in soil. Here, we show that microbial decomposition drives significant variability in the molecular richness and diversity of SOM between soil horizons and across a continental-scale gradient in climate and ecosystem type (arid shrubs, coniferous, deciduous, and mixed forests, grasslands, and tundra sedges). The molecular dissimilarity of SOM was strongly influenced by ecosystem type (hydrophilic compounds: 17%, P < 0.001; hydrophobic compounds: 10% P < 0.001) and soil horizon (hydrophilic compounds: 17%, P < 0.001; hydrophobic compounds: 21%, P < 0.001), as assessed using metabolomic analysis of hydrophilic and hydrophobic metabolites. While the proportion of shared molecular features was significantly higher in the litter layer than subsoil C horizons across ecosystems (12 times and 4 times higher for hydrophilic and hydrophobic compounds, respectively), the proportion of site-specific molecular features nearly doubled from the litter layer to the subsoil horizon, suggesting greater differentiation of compounds after microbial decomposition within each ecosystem. Together, these results suggest that microbial decomposition of plant litter leads to a decrease in SOM α-molecular diversity, yet an increase in β-molecular diversity across ecosystems. The degree of microbial degradation, determined by the position in the soil profile, exerts a greater control on SOM molecular diversity than environmental factors, such as soil texture, moisture, and ecosystem type.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Cell to Ecosystem: Understanding Methane and Associated Nutrient Cycling by Sediment Hosted Syntrophic Consortia and Their Viral Predators

The anaerobic oxidation of methane (AOM) is a significant worldwide microbial process in anoxic lake and ocean sediments, responsible for sequestering up to 80% of this greenhouse gas. Often considered a metabolism on the edge of thermodynamic probability, the impact of ANaerobic MEthane-oxidizing ‘ANME’ archaea on carbon and nutrient cycling in sediment ecosystems is far reaching. They not only serve as a sink for methane coupled to diverse electron acceptors, but also catalyze the transformation of many important nutrients including nitrogen, phosphate, and iron. While information about the potential mechanisms supporting metabolism in AOM is now available, remarkably little has been learned about their nutritional requirements, their dependencies on bacterial partners, and consequentially their ultimate impact on nutrient transformation and bioavailability within sedimentary ecosystems, and beyond. Further, the role of viruses within sediment ecosystems represents an essential but vastly understudied aspect of the transformation of carbon and nutrients by AOM. Viruses are now widely appreciated as central players in biogeochemical cycles across diverse ecosystems. These nanoscale predators have been shown to enhance the turnover of essential nutrients, thus stimulating microbial growth and environmental viruses themselves may constitute an important reservoir of nitrogen and phosphorous. Little is known about the role of viruses in methane-impacted sedimentary ecosystems, however prior genomic and microscopy evidence suggests that AOM consortia are susceptible to phage infection. The overarching scientific goal of this multi-disciplinary research proposal is to build on these recent discoveries and expand our understanding of interactions and fundamental activities involved in cycling of carbon and nutrients by syntrophic methanotrophic archaeal-bacterial consortia and associated viruses in anoxic sedimentary environments. Our three specific objectives are to 1) Quantify energy and nutrient exchange (e.g. N, P, Fe and vitamins) within AOM consortia and between ANME-bacterial partners; 2) Identify virus-host interactions associated with AOM and assess C and N transfer through viruses in methane-impacted sediment ecosystems; 3) Model energy and nutrient exchange in AOM consortia and viral-host interactions (i.e. viral activity), and their environmental distribution patterns. Our experimental emphasis cuts across scales that are important for understanding microbial and viral interactions and activities within their habitats, as well as community wide biogeochemical transformations. These research goals will be accomplished through the application of novel molecular techniques targeting DNA, RNA, proteins, and metabolites combined with a unique multi-modal analytical imaging pipeline. We will then model the ecophysiological capabilities of diverse sediment-hosted methanotrophic consortia to develop a more comprehensive understanding of the energetic and nutritional interactions between different AOM partner couplings that occur in sediments.

03 NATURAL GAS↗

NASA NTRSDate not supplied

Microbial Mutualism in (Modeled) Microgravity: Measuring the Effects of A Spaceflight-Like Fluid Environment on Cross-Feeding Communities

Microorganisms grow differently in spaceflight than they do on Earth. While numerous factors likely contribute, one ubiquitous feature of the space environment is altered gravity - but because microbes are generally considered too small to detect gravity directly, they likely experience the effects of gravity via changes in their fluid environment, primarily a reduction in convective mixing. This theory is supported by evidence that individual microbial strains experience starvation and acid stress in microgravity. If it is true, then microgravity would also have measurable effects on microbes in mixed communities, because many interspecies interactions involve the exchange of soluble metabolites through their medium (cross-feeding). Specifically, cooperative cross-feeding communities would grow more slowly in microgravity, and this effect should be reproducible in ground-based rotational culture systems (rotating wall vessels, RWVs) designed to simulate the quiescent environment of microgravity. Here we present preliminary results of our efforts to test this hypothesis using using a model system of Eschericia coli and Salmonella enterica that grow only when they can exchange methionine and acetate through the medium. We report on the development of a novel laboratory system that allows us measure growth rates and species ratios in real-time using fluorescence spectrometry as the organisms grow in RWVs. We are able to track growth rates of individual species and the co-culture continuously with high temporal resolution for several hours, a substantial improvement over prior RWV systems that require destructive sampling. We vary the fidelity of the microgravity simulation by varying the rotation rate, and find that rotation rate affects growth rates of the community more than that of individual strains, consistent with the claim that RWVs can simulate a microgravity-like quiescent environment. While more work needs to be done to probe the mechanisms of this effect, we hope that our system will ultimately allow us to generate quantitative predictions about the effects of spaceflight on organisms that are tiny but essential to sustaining long-term human space exploration.

microgravity↗

DOE OSTI2023

Construction and Modeling of a Coculture Microplate for Real-Time Measurement of Microbial Interactions

The dynamic structures of microbial communities emerge from the complex network of interactions between their constituent microorganisms. Quantitative measurements of these interactions are important for understanding and engineering ecosystem structure. Here, we present the development and application of the BioMe plate, a redesigned microplate device in which pairs of wells are separated by porous membranes. BioMe facilitates the measurement of dynamic microbial interactions and integrates easily with standard laboratory equipment. We first applied BioMe to recapitulate recently characterized, natural symbiotic interactions between bacteria isolated from the Drosophila melanogaster gut microbiome. Specifically, the BioMe plate allowed us to observe the benefit provided by two Lactobacillus strains to an Acetobacter strain. We next explored the use of BioMe to gain quantitative insight into the engineered obligate syntrophic interaction between a pair of Escherichia coli amino acid auxotrophs. We integrated experimental observations with a mechanistic computational model to quantify key parameters associated with this syntrophic interaction, including metabolite secretion and diffusion rates. This model also allowed us to explain the slow growth observed for auxotrophs growing in adjacent wells by demonstrating that, under the relevant range of parameters, local exchange between auxotrophs is essential for efficient growth. The BioMe plate provides a scalable and flexible approach for the study of dynamic microbial interactions.

3D printed device↗

NASA NTRSDate not supplied

Microbial Communities in Microgravity: Simulation in Lab and on the Computer

Microorganisms grow differently in spaceflight than they do on Earth. While much remains unexplained about how microgravity affects microbial growth, one dominant hypothesis is that the lack of density-driven convection in the liquid growth environment makes mixing diffusion-limited, and therefore slower. This is supported by evidence that individual microbial strains experience starvation and acid stress in microgravity. However, if it is true, then microgravity would also have measurable effects on microbes in mixed communities, because many interspecies interactions involve the exchange of soluble metabolites through the medium (cross-feeding). Specifically, cooperative cross-feeding communities would grow more slowly in microgravity, and cooperation would be less stable on evolutionary timescales. Here we describe our efforts to test this hypothesis by simulating microgravity in silico and in the lab, using a model system of Eschericia coli and Salmonella enterica that grow only when they can exchange methionine and acetate. We created CAMDLES (CFD-DEM Artificial Microgravity Developments for Living Ecosystem Simulation) as an extension of CFDEM®coupling software, to carry out computational modeling of biological flows, growth, and mass transfer in microgravity and also in laboratory artificial microgravity devices (rotating wall vessels, RWV). Using CAMDLES, we found distinct differences in growth rates between RWV and true microgravity, and we were able to identify several features, such as spatial distribution, biofilm formation, and product yield parameters, that influence the degree to which RWV growth recapitulates microgravity growth. In addition, we report on the development of a laboratory system for monitoring growth rates and species ratios of the community in RWVs, using fluorescent strains. Pairing CAMDLES with the laboratory model system allows us to generate quantitative predictions about the effects of spaceflight on organisms that will be essential to sustaining human space exploration in the long term.

microbiology↗

DOE OSTI2020

Simple Analysis of Primary and Secondary Bile Salt Hydrolysis in Mouse and Human Gut Microbiome Samples by Using Fluorogenic Substrates

Animals produce bile to maximize absorption of lipophilic nutrients in the gut. The physical properties of bile are largely dictated by amphipathic bile salt molecules, which also act as signaling molecules that bind host receptors and modulate physiological processes. Upon excretion of bile salts into the gut, the gut microbiome can create metabolites that have modified signaling capabilities. The category and magnitude of bile salt metabolism can affect the host either positively or negatively. A key modification is bile salt hydrolysis, which is a prerequisite step for all additional microbial modification. Here, we have synthesized five different fluorogenic bile salts that report on hydrolysis activity using a simple, continuous assay. We profile activity in both murine and human fecal samples. Our data demonstrate that most gut microbiomes have the highest capacity for hydrolysis of host-produced bile salts, but some microbially modified bile salts also display significant turnover.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Redox cycling-based detection of phenazine metabolites secreted from Pseudomonas aeruginosa in nanopore electrode arrays

The opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) produces several redox-active phenazine metabolites, including pyocyanin (PYO) and phenazine-1-carboxamide (PCN), which are electron carrier molecules that also aid in virulence. In particular, PYO is an exclusive metabolite produced by P. aeruginosa, which acts as a virulence factor in hospital-acquired infections and is therefore a good biomarker for identifying early stage colonization by this pathogen. Here, we describe the use of nanopore electrode arrays (NEAs) exhibiting metal–insulator–metal ring electrode architectures for enhanced detection of these phenazine metabolites. The size of the nanopores allows phenazine metabolites to freely diffuse into the interior and access the working electrodes, while the bacteria are excluded. Consequently, highly efficient redox cycling reactions in the NEAs can be accessed by free diffusion unhindered by the presence of bacteria. This strategy yields low limits of detection, i.e. 10.5 and 20.7 nM for PYO and PCN, respectively, values far below single molecule pore occupancy, e.g. at 10.5 nM < n pore > ~ 0.082 per nanopore – a limit which reflects the extraordinary signal amplification in the NEAs. Furthermore, experiments that compared results from minimal medium and rich medium show that P. aeruginosa produces the same types of phenazine metabolites even though growth rates and phenazine production patterns differ in these two media. Here, the NEA measurement strategy developed here should be useful as a diagnostic for pathogens generally and for understanding metabolism in clinically important microbial communities.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2022

Metabolome patterns identify active dechlorination in bioaugmentation consortium SDC-9™

Ultra-high performance liquid chromatography–high-resolution mass spectrometry (UPHLC–HRMS) is used to discover and monitor single or sets of biomarkers informing about metabolic processes of interest. The technique can detect 1000’s of molecules (i.e., metabolites) in a single instrument run and provide a measurement of the global metabolome, which could be a fingerprint of activity. Despite the power of this approach, technical challenges have hindered the effective use of metabolomics to interrogate microbial communities implicated in the removal of priority contaminants. Herein, our efforts to circumvent these challenges and apply this emerging systems biology technique to microbiomes relevant for contaminant biodegradation will be discussed. Chlorinated ethenes impact many contaminated sites, and detoxification can be achieved by organohalide-respiring bacteria, a process currently assessed by quantitative gene-centric tools (e.g., quantitative PCR). This laboratory study monitored the metabolome of the SDC-9™ bioaugmentation consortium during cis-1,2-dichloroethene (cDCE) conversion to vinyl chloride (VC) and nontoxic ethene. Untargeted metabolomics using an UHPLC-Orbitrap mass spectrometer and performed on SDC-9™ cultures at different stages of the reductive dechlorination process detected ~10,000 spectral features per sample arising from water-soluble molecules with both known and unknown structures. Multivariate statistical techniques including partial least squares-discriminate analysis (PLSDA) identified patterns of measurable spectral features (peak patterns) that correlated with dechlorination (in)activity, and ANOVA analyses identified 18 potential biomarkers for this process. Statistical clustering of samples with these 18 features identified dechlorination activity more reliably than clustering of samples based only on chlorinated ethene concentration and Dhc 16S rRNA gene abundance data, highlighting the potential value of metabolomic workflows as an innovative site assessment and bioremediation monitoring tool.

environmental monitoring↗