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All sourcesNASA NTRSDOE OSTI
NASA NTRS1991

The biogeochemistry of microbial mats, stromatolites and the ancient biosphere

Stromatolites offer an unparalleled geologic record of early life, because they constitute the oldest and most abundant recognizable remains of microbial ecosystems. Microbial mats are living homologs of stromatolites; thus, the physiology of the microbiota as well as the processes which create those features of mats (e.g., biomarker organic compounds, elemental and stable isotopic compositions) which are preserved in the ancient record. Observations of the carbon isotopic composition (delta C-13) of stromatolites and microbial mats were made and are consistent with the hypothesis that atmospheric CO2 concentrations have declined by at least one to two orders of magnitude during the past 2.5 Ga. Whereas delta C-13 values of carbonate carbon average about 0 permil during both the early and mid-Proterozoic, the delta C-13 values of stromatolitic organic matter increase from an average of -35 between 2.0 and 2.6 Ga ago to an average of about -28 about 1.0 Ga ago. Modern microbial mats in hypersaline environments have delta C-13 values typically in the range of -5 to -9, relative to an inorganic bicarbonate source at 0 permil. Both microbial mats and pur cultures of cyanobacteria grown in waters in near equilibrium with current atmospheric CO2 levels exhibit minimal discrimination against C-13. In contrast, hot spring cyanobacterial mats or cyanobacterial cultures grown under higher CO2 levels exhibit substantially greater discrimination. If care is taken to compare modern mats with stromatolites from comparable environments, it might be possible to estimate ancient levels of atmospheric CO2. In modern microbial mats, a tight coupling exists between photosynthetic organic carbon production and subsequent carbon oxidation, mostly by sulfate reduction. The rate of one process fuels a high rate of the other, with much of the sulfate reduction occurring within the same depth interval as oxygenic photosynthesis. Other aspects of this study are presented.

Desmarais, D. J.↗
DOE OSTI2020

A Systems Biology Approach to Energy Flow in H 2 Producing Microbial Communities: Microbial Mats

This report describes the results of work conducted at NASA Ames Research Center, in collaboration with investigators at Lawrence Livermore National Laboratory, during the time period 12/15/2014 to 12/15/2017. The work was an extension of a collaboration with LLNL that began in 2008. Work this reporting period focused primarily on tasks related to microbial mats from both the Elkhorn Slough and Baja California field sites, including the analysis and publication of data produced prior to 12/15/2014. During this reporting period, in addition to the microbial mat work, we also conducted a series of experiments with a particular algal-bacterial association (C. sorokiniana and A. brasilense). A number of aspects of this well-characterized algal-bacterial association enabled us not only to further our understanding of interactions between these particular organisms, but to apply that knowledge and techniques to our collection of microorganisms isolated from microbial mats. Microbial mat experiments focused both on intact mat communities and on cultures of organisms isolated from the mats (the Microbial Mat Living Library). In a series of co-culture experiments, we documented both positive and negative interactions amongst heterotrophic and autotrophic isolates of microbial mat microorganisms. One particularly important and novel mat cyanobacterium (ESFC-1) was studied in detail. We developed techniques to pursue an investigation of functional redundancy (the idea that closely related organisms perform identical roles in a community) in cyanobacterial autotrophs in the Elkhorn Slough microbial mat. In those experiments, we documented numerous instances of increases in growth rate between co-cultures of two organisms (relative to their growth rate in isolation) performing what would seem to be identical functional roles. Simplified microbial mat communities consisting of organisms from the Microbial Mat Living Library were constructed and studied in laboratory experiments. The “constructed mats” were grown in standard laboratory glassware and plasticware, but cultures were also combined with artificial materials in an effort to re-create the three-dimensional structure of naturally occurring microbial mats. These constructed mats re-created a number of functions attributable to natural microbial mats (N-fixation, H2-production) and simplified their study using molecular ecological techniques due to the smaller number of microbial “species” present. Cyanobacteria which migrated in response to vertical variations in light intensity (irradiance) obtained characteristic positions in constructed mats that were similar to their positions in natural mats. A detailed investigation of nitrogen cycling in both the Baja California and Elkhorn Slough microbial mats was initiated during this reporting period. The nitrogen cycle in the microbial mats has not been studied in depth, despite the fact that nitrogen usually limits growth in marine environments. The organisms conducting nitrogen cycling in both microbial mats were studied using the genomes available from earlier work on these mats, as well as with a targeted approach using molecular ecological methods developed here. Quantitative measurements of rates of nitrogen transformations were made using stable isotopically labeled nitrogen. The processes quantified and examined using molecular ecological methods included: nitrogen fixation, ammonification, ammonium and nitrate assimilation, denitrification, anaerobic oxidation of ammonium, and dissimilatory nitrate reduction to ammonium. In a series of experiments designed to examine a potential application for microbial mats, we demonstrated that both Elkhorn Slough and Baja California microbial mats were very effective in removing large quantities of both nitrate and ammonium from waste streams containing these compounds at levels where they would be considered to be pollutants. Considerable effort was expended during this reporting period to analyze, and prepare for publication, metagenomes and metatranscriptomes collected from two experimental manipulations of microbial mats. The first, a diel study, was conducted in November 2011, while the second, an investigation of the effects of sulfate on community composition, was conducted during this reporting period. For the earlier study, reference-based and reference-free methods were used to assess microbial ecology and genetic partitioning in these complex microbial systems, and that work is available as a pre-print. Four metagenomes from the same experiment were published. In the second, longer (nearly 200 day) manipulation, rRNA iTag libraries for cDNA and DNA were analyzed to determine the effects of sulfate concentration on microbial mat community composition over the course of the experiment. Work on the algal-bacterial association (Chlorella sorokiniana and Azospirillum brasilense) focused initially on demonstrating the transfer of carbon and nitrogen between the partners, using stable isotopic labeling and the LLNL NanoSIMS. Subsequently, the potential role of diazotrophy (nitrogen fixation) by the bacterial partner in supporting the growth of the association was assessed in a series of experiments. It appears to be the case that, although the bacterial partner demonstrably increases the fitness of the algae under nitrogen limitation, and cells of C. sorokiniana receive nitrogen originally fixed from atmospheric nitrogen by A. brasilense, the amount of nitrogen obtained by diazotrophy is too small to explain the increase in fitness.

08 HYDROGEN↗
DOE OSTI2022

Metabolic activity and community structure of prokaryotes associated with particles in the twilight zone of the South China Sea

The twilight zone is an important depth of the ocean where particulate organic matter (POM) remineralization takes place, and prokaryotes contribute to more than 70% of the estimated remineralization. However, little is known about the microbial community and metabolic activity associated with different particles in the twilight zone. The composition and distribution of particle-attached prokaryotes in the twilight zone of the South China Sea (SCS) were investigated using high-throughput sequencing and quantitative PCR, together with the Biolog Ecoplate™ microplates culture to analyze the microbial metabolic activity. We found that α- and γ-Proteobacteria dominating at the lower and upper boundary of the twilight zone, respectively; Methanosarcinales and Halobacteriales of the Euyarchaeota occupied in the larger particles at the upper boundary. Similar microbial community existed between euphotic layer and the upper boundary. Higher amount of shared Operational Taxonomic Units (OTUs) in the larger particles along the water depths, might be due to the fast sinking and major contribution of carbon flux of the larger particles from the euphotic layer. In addition to polymers as the major carbon source, carbohydrates and amino acids were preferentially used by microbial community at the upper and lower boundary, respectively. This could potentially be attributed to the metabolic capabilities of attached microbial groups in different particles, and reflected the initial preference of the carbon source by the natural microbes in the twilight zone as well. The microbial structure and carbon metabolic profiles could be complemented with metatranscriptomic analysis in future studies to augment the understanding of the complex carbon cycling pathways in the twilight zone.

Liu, Hao↗
DOE OSTI2023

Combined effects of lipase and Lactiplantibacillus plantarum 1‐24‐LJ on physicochemical property, microbial succession and volatile compounds formation in fermented fish product

Abstract BACKGROUND Studies have shown that either the addition of starter culture or enzyme can improve fermentation in fish or other products. However, little research has been carried out on the effects of coupling starter cultures with lipase on the microbial community and product quality. Suanzhayu is a Chinese fermented fish product that mainly relies on spontaneous fermentation, resulting in an unstable flavor and quality. The present study investigated the impact of lipase and Lactiplantibacillus plantarum 1‐24‐LJ on the quality of Suanzhayu . RESULTS Inoculation decreased pH and 2‐thiobarbituric acid reactive substances (TBARS) values, and also helped the dominance of the strain in the ecosystem, whereas lipase addition raised TBARS values and had little effect on pH, water activity ( a w ) and microbiota. The addition of lipase and/or Lpb. plantarum increased the content of alcohols, aldehydes, ketones, esters and umami amino acids. The co‐additions with the most significant effect and the total contents of volatile compounds (VCs) and free amino acids (FAAs) were 1801.92 g per 100 g and 21 357.05 mg per 100 g, respectively. Former‐ Lactobacillus was negatively correlated with pH, a w and Prevotella , but positively with VCs (ethyl ester of heptanoic acid, ethyl ester of octanoic acid) and FAAs (Tyr, Phe). Furthermore, adding Lpb. plantarum 1‐24‐LJ alone or in combination with lipase shortened the fermentation process. CONCLUSION The present study provides a recommended Suanzhayu process approach for improving product quality and flavor, as well as shortening fermentation time, by adding Lpb. plantarum 1‐24‐LJ with or without lipase. © 2023 Society of Chemical Industry.

Zhang, Zuoli↗
DOE OSTI2020

Duckweed hosts a taxonomically similar bacterial assemblage as the terrestrial leaf microbiome

Culture-independent characterization of microbial communities associated with popular plant model systems have increased our understanding of the plant microbiome. However, the integration of other model systems, such as duckweed, could facilitate our understanding of plant microbiota assembly and evolution. Duckweeds are floating aquatic plants with many characteristics, including small size and reduced plant architecture, that suggest their use as a facile model system for plant microbiome studies. Here, we investigated the structure and assembly of the duckweed bacterial microbiome. First, a culture-independent survey of the duckweed bacterial microbiome from different locations in New Jersey revealed similar phylogenetic profiles. These studies showed that Proteobacteria is a dominant phylum in the duckweed bacterial microbiome. To observe the assembly dynamics of the duckweed bacterial community, we inoculated quasi-gnotobiotic duckweed with wastewater effluent from a municipal wastewater treatment plant. Our results revealed that duckweed strongly shapes its bacterial microbiome and forms distinct associations with bacterial community members from the initial inoculum. Additionally, these inoculation studies showed the bacterial communities of different duckweed species were similar in taxa composition and abundance. Analysis across the different duckweed bacterial communities collected in this study identified a set of “core” bacterial taxa consistently present on duckweed irrespective of the locale and context. Furthermore, comparison of the duckweed bacterial community to that of rice and Arabidopsis revealed a conserved taxonomic structure between the duckweed microbiome and the terrestrial leaf microbiome. Our results suggest that duckweeds utilize similar bacterial community assembly principles as those found in terrestrial plants and indicate a highly conserved structuring effect of leaf tissue on the plant microbiome.

54 ENVIRONMENTAL SCIENCES↗
NASA NTRS2015

In-Flight Microbial Monitor

Previous research has shown that potential human pathogens have been detected on the International Space Station (ISS). New microorganisms are introduced with every exchange of crew and cargo. Microorganisms introduced to the ISS are readily transferred between crew and subsystems (i.e., ECLSS, environmental control and life support systems). Current microbial characterization methods require a culture-based enrichment of microorganisms and at least a 48-hour incubation time. This increases the microbial load while detecting only a limited number of microorganisms. The culture-based method detects approximately 1-10% of the total organisms present and provides no identification. To identify and enumerate ISS samples requires that the microbes be returned to Earth for complete analysis. Therefore, a more expedient, low-cost, inflight method of microbial detection, identification, and enumeration is needed. The RAZOR EX, a ruggedized, commercial off the shelf, real-time PCR field instrument was tested for its ability to detect microorganisms at low concentrations within one hour. Escherichia coli, Salmonella enterica Typhimurium, and Pseudomonas aeruginosa were detected at low levels using real-time DNA amplification. Total heterotrophic counts could also be detected using a 16S gene marker that can identify up to 98% of all bacteria. To reflect viable cells found in the samples, RNA was also detectable using a modified, single-step reverse transcription reaction.

Technology Portfolio System↗
NASA NTRS2011

PMA-PhyloChip DNA Microarray to Elucidate Viable Microbial Community Structure

Since the Viking missions in the mid-1970s, traditional culture-based methods have been used for microbial enumeration by various NASA programs. Viable microbes are of particular concern for spacecraft cleanliness, for forward contamination of extraterrestrial bodies (proliferation of microbes), and for crew health/safety (viable pathogenic microbes). However, a "true" estimation of viable microbial population and differentiation from their dead cells using the most sensitive molecular methods is a challenge, because of the stability of DNA from dead cells. The goal of this research is to evaluate a rapid and sensitive microbial detection concept that will selectively estimate viable microbes. Nucleic acid amplification approaches such as the polymerase chain reaction (PCR) have shown promise for reducing time to detection for a wide range of applications. The proposed method is based on the use of a fluorescent DNA intercalating agent, propidium monoazide (PMA), which can only penetrate the membrane of dead cells. The PMA-quenched reaction mixtures can be screened, where only the DNA from live cells will be available for subsequent PCR reaction and microarray detection, and be identified as part of the viable microbial community. An additional advantage of the proposed rapid method is that it will detect viable microbes and differentiate from dead cells in only a few hours, as opposed to less comprehensive culture-based assays, which take days to complete. This novel combination approach is called the PMA-Microarray method. DNA intercalating agents such as PMA have previously been used to selectively distinguish between viable and dead bacterial cells. Once in the cell, the dye intercalates with the DNA and, upon photolysis under visible light, produces stable DNA adducts. DNA cross-linked in this way is unavailable for PCR. Environmental samples suspected of containing a mixture of live and dead microbial cells/spores will be treated with PMA, and then incubated in the dark. Thereafter, the sample is exposed to visible light for five minutes, so that the DNA from dead cells will be cross-linked. Following this PMA treatment step, the sample is concentrated by centrifugation and washed (to remove excessive PMA) before DNA is extracted. The 16S rRNA gene fragments will be amplified by PCR to screen the total microbial community using PhyloChip DNA microarray analysis. This approach will detect only the viable microbial community since the PMA intercalated DNA from dead cells would be unavailable for PCR amplification. The total detection time including PCR reaction for low biomass samples will be a few hours. Numerous markets may use this technology. The food industry uses spore detection to validate new alternative food processing technologies, sterility, and quality. Pharmaceutical and medical equipment companies also detect spores as a marker for sterility. This system can be used for validating sterilization processes, water treatment systems, and in various public health and homeland security applications.

Venkateswaran, Kasthuri J.↗
DOE OSTI2020

FeGenie: a comprehensive tool for the identification of iron genes and iron gene neighborhoods in genomes and metagenome assemblies

Iron is a micronutrient for nearly all life on Earth. It can be used as an electron donor and electron acceptor by iron-oxidizing and iron-reducing microorganisms and is used in a variety of biological processes, including photosynthesis and respiration. While it is the fourth most abundant metal in the Earth’s crust, iron is often limiting for growth in oxic environments because it is readily oxidized and precipitated. Much of our understanding of how microorganisms compete for and utilize iron is based on laboratory experiments. However, the advent of next-generation sequencing and surge in publicly available sequence data has made it possible to probe the structure and function of microbial communities in the environment. To bridge the gap between our understanding of iron acquisition, iron redox cycling, iron storage, and magnetosome formation in model microorganisms and the plethora of sequence data available from environmental studies, we have created a comprehensive database of hidden Markov models (HMMs) based on genes related to iron acquisition, storage, and reduction/oxidation in Bacteria and Archaea. Along with this database, we present FeGenie, a bioinformatics tool that accepts genome and metagenome assemblies as input and uses our comprehensive HMM database to annotate provided datasets with respect to iron-related genes and gene neighborhood. An important contribution of this tool is the efficient identification of genes involved in iron oxidation and dissimilatory iron reduction, which have been largely overlooked by standard annotation pipelines. We validated FeGenie against a selected set of 28 isolate genomes and showcase its utility in exploring iron genes present in 27 metagenomes, 4 isolate genomes from human oral biofilms, and 17 genomes from candidate organisms, including members of the candidate phyla radiation. We show that FeGenie accurately identifies iron genes in isolates. Furthermore, analysis of metagenomes using FeGenie demonstrates that the iron gene repertoire and abundance of each environment is correlated with iron richness. While this tool will not replace the reliability of culture-dependent analyses of microbial physiology, it provides reliable predictions derived from the most up-to-date genetic markers. FeGenie’s database will be maintained and continually updated as new genes are discovered.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Effect of tectonic processes on biosphere–geosphere feedbacks across a convergent margin

The subsurface is among Earth’s largest biomes, but the extent to which microbial communities vary across tectonic plate boundaries or interact with subduction-scale geological processes remains unknown. Here we compare bacterial community composition with deep-subsurface geochemistry from 21 hot springs across the Costa Rican convergent margin. We find that cation and anion compositions of the springs reflect the dip angle and position of the underlying tectonic structure and also correlate with the bacterial community. Co-occurring microbial cliques related to cultured chemolithoautotrophs that use the reverse tricarboxylic acid cycle (rTCA) as well as abundances of metagenomic rTCA genes correlate with concentrations of slab-volatilized carbon. This, combined with carbon isotope evidence, suggests that fixation of slab-derived CO2 into biomass may support a chemolithoautotrophy-based subsurface ecosystem. We calculate that this forearc subsurface biosphere could sequester 1.4 × 10 9 to 1.4 × 10 10 mol of carbon per year, which would decrease estimates of the total carbon delivered to the mantle by 2 to 22%. Furthermore, based on the observed correlations, we suggest that distribution and composition of the subsurface bacterial community are probably affected by deep tectonic processes across the Costa Rican convergent margin and that, by sequestering carbon volatilized during subduction, these chemolithoautotrophic communities could in turn impact the geosphere.

54 ENVIRONMENTAL SCIENCES↗
NASA NTRS2002

From Field to Laboratory: A New Database Approach for Linking Microbial Field Ecology with Laboratory Studies

The Ames Exobiology Culture Collection Database (AECC-DB) has been developed as a collaboration between microbial ecologists and information technology specialists. It allows for extensive web-based archiving of information regarding field samples to document microbial co-habitation of specific ecosystem micro-environments. Documentation and archiving continues as pure cultures are isolated, metabolic properties determined, and DNA extracted and sequenced. In this way metabolic properties and molecular sequences are clearly linked back to specific isolates and the location of those microbes in the ecosystem of origin. Use of this database system presents a significant advancement over traditional bookkeeping wherein there is generally little or no information regarding the environments from which microorganisms were isolated. Generally there is only a general ecosystem designation (i.e., hot-spring). However within each of these there are a myriad of microenvironments with very different properties and determining exactly where (which microenvironment) a given microbe comes from is critical in designing appropriate isolation media and interpreting physiological properties. We are currently using the database to aid in the isolation of a large number of cyanobacterial species and will present results by PI's and students demonstrating the utility of this new approach.

Bebout, Leslie↗
DOE OSTI2020

A long-term pilot-scale algal cultivation on power plant flue gas – Cultivation stability and biomass accumulation

A long-term culture of a selected freshwater microalgal assemblage on power plant flue gas was realized using a pilot-scale photobioreactor. Different culture conditions of light cycle, harvesting amount, pH stabilization, and sulfur addition were tested during a 12-month continuous operation at a natural gas fired power plant. The results concluded that the daily harvesting amount of 60% with no light-cycle and NH 4 NO 3 as the nitrogen source and pH stabilization agent, resulted in optimal cultivation performance. Biomass concentration and biomass productivity reached 1.07 ± 0.23 g L -1 and 0.43 ± 0.14 g L -1 d -1 , respectively. During the year-long cultivation period, the algal assemblage was relatively stable with average abundances of 88 ± 4% and 12 ± 4% for algae and bacteria, respectively, and no significant differences between individual case scenarios. The stable culture fostered a stable biomass composition. Protein, carbohydrate, and lipid contents of the algal biomass were 58.60 ± 2.41%, 19.5 ± 2.44%, and 9.70 ± 1.68%, respectively.

09 BIOMASS FUELS↗
DOE OSTI2021

Experimental and theoretical determinations of hydrogen isotopic equilibrium in the system CH 4 —H 2 —H 2 O from 3 to 200°C

The stable isotopic composition of methane (CH 4 ) is commonly used to fingerprint natural gas origins. Over the past 50 years, there have been numerous proposals that both microbial and thermogenic CH 4 can form in or later attain hydrogen isotopic equilibrium with water (H 2 O) and carbon isotopic equilibrium with carbon dioxide (CO 2 ). Evaluation of such proposals requires knowledge of the equilibrium fractionation factors between CH 4 and H 2 O or CO 2 at the temperatures where microbial and thermogenic CH 4 form in or are found in the environment, which is generally less than 200°C. Experimental determinations of these fractionation factors are only available above 200°C, requiring extrapolation of these results beyond the calibrated range or the use of theoretical calculations at lower temperatures. Here, we provide a calibration of the equilibrium hydrogen isotopic fractionation factor for CH 4 and hydrogen gas (H 2 ) ( D α CH4(g)–H2(g) ) based on experiments using γ-Al 2 O 3 and Ni catalysts from 3 to 200°C. Results were regressed as a 2 nd order polynomial of 1000 × ln D α CH4(g)–H2(g) vs. 1/T (K -1 ) yielding: 1000 × l n D α C H 4 ( g ) - H 2 ( g ) = 3.5317 × 10 7 T 2 + 2.7749 × 10 5 T - 179.48 We combine this calibration with previous experimental determinations of hydrogen isotope equilibrium between H 2 , H 2 O(g), and H 2 O(l) and we provide an interpolatable experimental calibration of 1000 × ln D α CH4(g)–H2O(l) from 3 to 200°C. Our resulting 4th order polynomial is the following equation: 1000 × l n D α C H 4 ( g ) - H 2 O l = - 7.9443 × 10 12 T 4 + 8.7772 × 10 10 T 3 - 3.4973 × 10 8 T 2 + 5.4398 × 10 5 T - 382.05 At 3°C, the value from our calibration differs by 93‰ relative to what would be calculated based on the extrapolation of the only experimental calibration currently available to temperatures below its calibrated range (lowest temperature of 200°C; Horibe and Craig, 1995). We additionally provide new theoretical estimates of hydrogen isotopic equilibrium between CH 4 (g), H 2 (g), and H 2 O(g) and carbon isotopic equilibrium between CH 4 (g) and CO 2 (g) using Path Integral Monte Carlo (PIMC) calculations. Our PIMC calculations for hydrogen isotopic equilibrium between CH 4 and H 2 agree 1:1 with our experiments. Finally, we compile carbon and hydrogen isotopic measurements of CH 4 , CO 2 , and H 2 O from various environmental systems and compare observed differences between carbon and hydrogen isotopes to those expected based on isotopic equilibrium. We find that isotopic compositions of some microbial gases from marine sedimentary, coalbed, and shale environments are consistent with those expected for CH 4 H 2 O(l) hydrogen and CH 4 CO 2 carbon isotopic equilibrium. In contrast, microbial terrestrial and pure culture gases are not consistent with both CH 4 H 2 O(l) hydrogen and CH 4 CO 2 carbon isotopic equilibrium. Overall, these results are explained qualitatively using previously developed conceptual models that link free energy gradients available to microorganisms to the degree that their enzymes can promote isotope-exchange reactions between CH 4 , CO 2 , and H 2 O.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Metaoptics Enabled Multifunctional Imaging

Despite the fact that most microorganisms occupy two- and three-dimensional space in heterogeneous arrangements and in proximity to other microorganisms of different species, much of our current knowledge about their metabolic processes is derived from homogenizing, then analyzing, the chemical content of cultures containing a single microbial species. This project addressed this fundamental dichotomy by developing a set of optical imaging principles combining multiple new technologies. The imaging strategies developed in this project combine metaoptics structures with active in situ nanoscale control of the chemical environment and applied them to a microbial system, Myxococcus xanthus, with particular relevance to the DoE mission. The combination of metaoptical architectures and nanoscale control over the molecular environment enables: (a) precise control over the electromagnetic (EM) field at length scales smaller than the wavelength of light; (b) control of the interaction of the EM field with critical molecular systems in DOE relevant microbes; (c) control over the chemical environment – especially the presence and quantity of reactive oxygen species (ROS) that can affect redox homeostasis; and (d) the ability to ask new kinds of questions not accessible to ‘omics’ approaches or standard methods of biological imaging. These capabilities are applicable to detailed studies of metabolic pathways in microbes and to lignocellulosic biomass deconstruction. To accomplish these objectives, we pursued two over-arching technical goals: (1) the development of new metaoptics-enabled approaches to imaging and spectroscopic characterization; and (2) the development of tools to control the chemical environment of a microbial sample with nanometer-scale precision. Goal 1 was addressed through the design, fabrication, and characterization of new metasurfaces capable of super-resolution imaging through extreme confinement of the optical field. Goal 2 was addressed by controlling the redox potential on the nanoscale in microbial communities and characterizing their effect on intrinsic bacterial fluorophores which act as molecular sentinels and through characterization of soluble factors secreted by Myxococcus xanthus by confocal Raman imaging. The optical imaging/sensing approaches developed here make it possible to use these powerful new imaging and sensing modalities in metabolic studies by making it possible to visualize and track the spatial and temporal expression patterns of natural or engineered pathways in microorganisms.

09 BIOMASS FUELS↗
DOE OSTI2021

Community Modeling and Flux Balance Analysis of Synthetic Bacterial Community in MOPS Medium

This narrative was used for the 4-member community modeling using MOPS medium for the bacteria isolated from Populus deltoides (PD). 10 bacterial strains isolated from PD were co-cultured as a synthetic microbial community in MOPS minimal medium. After 15 passages there were 4 members survived in MOPS medium. The genome of each community member was sequenced and uploaded into KBase for genome annotation and metabolic modeling. To simulate the metabolic interactions among the finally surviving community members, the individual metabolic models of each strain were merged into a compartmentalized community model, and then the community model was gapfilled using MOPS medium. The FBA modeling was performed and the metabolites exchange among community members was proposed. The publication by Jia Wang, Dana L. Carper, Leah H. Burdick, Him Shrestha, Manasa Appidi, Paul E. Abraham, Collin M. Timm, Robert L. Hettich, Dale A. Pelletier, Mitchel J. Doktycz can be found here: https://doi.org/10.​1016/​j.​csbj.​2021.​03.​034

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Community Modeling and Flux Balance Analysis of Synthetic Bacterial Community in R2A Medium

This narrative was used for the 3-member community modeling using R2A medium for the bacteria isolated from Populus deltoides (PD). 10 bacteiral strains isolated from PD were co-cultured as a synthetic microbial community in R2A complex medium. After 15 passages, there were 3 members survived in R2A medium. The genome of each community member was sequenced and uploaded into KBase for genome annotation and metabolic modeling. To simulate the metabolic interactions among the finally surviving community members, the individual metabolic models of each strain were merged into a compartmentalized community model, and then the community model was gapfilled using R2A medium. The FBA modeling was performed and the metabolites exchange among community members was proposed. The publication by Jia Wang, Dana L. Carper, Leah H. Burdick, Him Shrestha, Manasa Appidi, Paul E. Abraham, Collin M. Timm, Robert L. Hettich, Dale A. Pelletier, Mitchel J. Doktycz can be found here: https://doi.org/10.​1016/​j.​csbj.​2021.​03.​034

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1999

Gentamicin: effect on E. coli in space

Previous investigations have shown that liquid bacterial cultures grown in space flight were not killed as effectively by antibiotic treatments as were cultures grown on Earth. However, the cause for the decreased antibiotic effectiveness remains unknown. Possible explanations include modified cell proliferation and modified antibiotic transport in the culture medium. Escherichia coli cultures were grown in space flight (STS-69 and STS-73), with and without gentamicin, on a solid agar substrate thus eliminating fluid effects and reducing the unknowns associated with space-flight bacterial cultures in suspension. This research showed that E. coli cultures grown in flight on agar for 24 to 27 hours experienced a heightened growth compared to simultaneous controls. However, addition of gentamicin to the agar killed the bacteria such that both flight and ground control E. coli samples had similar final cell concentrations. Therefore, while the reported existence of a decrease in antibiotic effectiveness in liquid cultures remains unexplained, these data suggest that gentamicin in space flight was at least as effective as, if not more effective than, on Earth, when E. coli cells were grown on agar.

STS-69 Shuttle Project↗
DOE OSTI2021

Reduction and bacterial adsorption of dissolved mercuric ion by indigenous bacteria at the Oak Ridge Reservation site

Mercury exists in various forms in the environment and the indigenous bacteria mediated processes have the potential to be used for mercury remediation. In this study, two mixed cultures of indigenous bacteria at the Oak Ridge Reservation site (i.e., ORR soil culture and ORR sediment culture) were selected to study the microbial mediated mercuric reduction under an aerobic condition as well as mercury adsorption onto bacterial surfaces. PCR analysis was performed to provide insights into the microbial community. The mercuric volatilizing experiment demonstrated the mercuric reducing capacity for both ORR cultures, in which the Pseudomonas genus was the dominating Hg 0 producer. The investigation of the impact of the sole carbon source revealed the energy-dependent characteristics of the mercuric reduction in this study. Namely, the mercuric reduction was nearly not impacted by the type of carbon source but positively related to the energy that a unit amount of substrate could provide. The study also indicated that the mercury adsorption competed with the reduction. According to the fitting of the Langmuir isotherm, the ORR soil culture was found to have a higher mercury adsorption capacity (i.e., 67.5 mg Hg/g dry biomass) than the ORR sediment culture (i.e., 53.1 mg Hg/g dry biomass). The negative correlation between the reduced mercury mass and adsorbed mercury mass was identified for both ORR cultures.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗