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DOE OSTI2024

Application of functional genomics for domestication of novel non-model microbes

Abstract With the expansion of domesticated microbes producing biomaterials and chemicals to support a growing circular bioeconomy, the variety of waste and sustainable substrates that can support microbial growth and production will also continue to expand. The diversity of these microbes also requires a range of compatible genetic tools to engineer improved robustness and economic viability. As we still do not fully understand the function of many genes in even highly studied model microbes, engineering improved microbial performance requires introducing genome-scale genetic modifications followed by screening or selecting mutants that enhance growth under prohibitive conditions encountered during production. These approaches include adaptive laboratory evolution, random or directed mutagenesis, transposon-mediated gene disruption, or CRISPR interference (CRISPRi). Although any of these approaches may be applicable for identifying engineering targets, here we focus on using CRISPRi to reduce the time required to engineer more robust microbes for industrial applications. One-Sentence Summary The development of genome scale CRISPR-based libraries in new microbes enables discovery of genetic factors linked to desired traits for engineering more robust microbial systems.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Plant–microbe networks in soil are weakened by century-long use of inorganic fertilizers

Understanding the changes in plant-microbe interactions is critically important for predicting ecosystem functioning in response to human-induced environmental changes such as nitrogen (N) addition. In this study, the effects of a century-long fertilization treatment (> 150 years) on the networks between plants and soil microbial functional communities, detected by GeoChip, in grassland were determined in the Park Grass Experiment at Rothamsted Research, UK. Our results showed that plants and soil microbes have a consistent response to long-term fertilization-both richness and diversity of plants and soil microbes are significantly decreased, as well as microbial functional genes involved in soil carbon (C), nitrogen (N) and phosphorus (P) cycling. The network-based analyses showed that long-term fertilization decreased the complexity of networks between plant and microbial functional communities in terms of node numbers, connectivity, network density and the clustering coefficient. Similarly, within the soil microbial community, the strength of microbial associations was also weakened in response to long-term fertilization. Mantel path analysis showed that soil C and N contents were the main factors affecting the network between plants and microbes. Our results indicate that century-long fertilization weakens the plant-microbe networks, which is important in improving our understanding of grassland ecosystem functions and stability under long-term agriculture management.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Microbes and Climate Change: a Research Prospectus for the Future

Climate change is the most serious challenge facing humanity. Microbes produce and consume three major greenhouse gases—carbon dioxide, methane, and nitrous oxide—and some microbes cause human, animal, and plant diseases that can be exacerbated by climate change. Hence, microbial research is needed to help ameliorate the warming trajectory and cascading effects resulting from heat, drought, and severe storms. We present a brief summary of what is known about microbial responses to climate change in three major ecosystems: terrestrial, ocean, and urban. We also offer suggestions for new research directions to reduce microbial greenhouse gases and mitigate the pathogenic impacts of microbes. These include performing more controlled studies on the climate impact on microbial processes, system interdependencies, and responses to human interventions, using microbes and their carbon and nitrogen transformations for useful stable products, improving microbial process data for climate models, and taking the One Health approach to study microbes and climate change.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2015

Engineered microbes and methods for microbial oil production

Some aspects of this invention provide engineered microbes for oil production. Methods for microbe engineering and for use of engineered microbes are also provided herein. In some embodiments, microbes are provided that are engineered to modulate a combination of rate-controlling steps of lipid synthesis, for example, a combination of a step generating metabolites, acetyl-CoA, ATP or NADPH for lipid synthesis (a push step), and a step sequestering a product or an intermediate of a lipid synthesis pathway that mediates feedback inhibition of lipid synthesis (a pull step). Such push-and-pull engineered microbes exhibit greatly enhanced conversion yields and TAG synthesis and storage properties.

09 BIOMASS FUELS↗

DOE OSTI2018

Engineered microbes and methods for microbial oil production

09 BIOMASS FUELS↗

DOE OSTI2022

Modeling methane dynamics in three wetlands in Northeastern China by using the CLM-Microbe model

Wetlands account for up to 70% of the natural source of methane (CH 4 ) in terrestrial ecosystems on a global scale. Soil microbes are the ultimate producers and biological consumers of CH 4 in wetlands. Therefore, simulating microbial mechanisms of CH 4 production and consumptionwould improve the predictability of CH 4 flux in wetland ecosystems. In this study, we applied a microbial-explicit model, the CLM-Microbe, to simulate CH 4 flux in three major natural wetlands in northeastern China. The CLM-Microbe model was able to capture the seasonal variation of gross primary productivity (GPP), dissolved organic carbon (DOC), and CH 4 flux. The CLM-Microbe model explained more than 40% of the variation in GPP and CH 4 flux across sites. Marsh wetlands had higher CH 4 flux than mountain peatlands. Ebullition dominated the CH 4 transport pathway in all three wetlands. The methanogenesis dominates while methanotroph makes a minor contribution to the CH 4 flux, making all wetlands a CH 4 source. Sensitivity analysis indicated that microbial growth and death rates are the key factors governing CH 4 emission and vegetation physiological properties (flnr) and maintenance respiration predominate GPP variation. Explicitly simulating microbial processes allows genomic information to be incorporated, laying a foundation for better predicting CH 4 dynamics under the changing environment.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2016

A new theory of plant–microbe nutrient competition resolves inconsistencies between observations and model predictions

Terrestrial plants assimilate anthropogenic CO 2 through photosynthesis and synthesizing new tissues. However, sustaining these processes requires plants to compete with microbes for soil nutrients, which therefore calls for an appropriate understanding and modeling of nutrient competition mechanisms in Earth System Models (ESMs). In this paper, we survey existing plant–microbe competition theories and their implementations in ESMs. We found no consensus regarding the representation of nutrient competition and that observational and theoretical support for current implementations are weak. To reconcile this situation, we applied the Equilibrium Chemistry Approximation (ECA) theory to plant–microbe nitrogen competition in a detailed grassland 15 N tracer study and found that competition theories in current ESMs fail to capture observed patterns and the ECA prediction simplifies the complex nature of nutrient competition and quantitatively matches the 15 N observations. Finally, since plant carbon dynamics are strongly modulated by soil nutrient acquisition, we conclude that (1) predicted nutrient limitation effects on terrestrial carbon accumulation by existing ESMs may be biased and (2) our ECA-based approach may improve predictions by mechanistically representing plant–microbe nutrient competition.

15N tracer↗

DOE OSTI2022

Exploring the roles of microbes in facilitating plant adaptation to climate change

Plants benefit from their close association with soil microbes which assist in their response to abiotic and biotic stressors. Yet much of what we know about plant stress responses is based on studies where the microbial partners were uncontrolled and unknown. Under climate change, the soil microbial community will also be sensitive to and respond to abiotic and biotic stressors. Thus, facilitating plant adaptation to climate change will require a systems-based approach that accounts for the multi-dimensional nature of plant–microbe–environment interactions. In this perspective, we highlight some of the key factors influencing plant–microbe interactions under stress as well as new tools to facilitate the controlled study of their molecular complexity, such as fabricated ecosystems and synthetic communities. When paired with genomic and biochemical methods, these tools provide researchers with more precision, reproducibility, and manipulability for exploring plant–microbe–environment interactions under a changing climate.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Interactive Effects of Microbes and Nitrogen on Panicum virgatum Root Functional Traits and Patterns of Phenotypic Selection

Premise of research. Plants in natural and agricultural systems are influenced in myriad ways by their microbial communities, particularly by providing goods and services that change plant functional traits. Microbes are considered an influential part of the environmental context that change plant trait expression, but often, microbe-mediated effects are contingent on local resources, such as nitrogen. Here, we ask how microbes and nitrogen affect belowground functional traits and patterns of phenotypic selection. Methodology. We performed a fully factorial greenhouse experiment with switchgrass (Panicum virgatum), manipulating microbial community composition and nitrogen availability. We measured plant performance and belowground functional traits and performed 16S amplicon sequencing of the root-associated microbial communities. We looked for correlations between microbial taxa and root functional traits, and we performed phenotypic selection analysis on five belowground functional traits to determine how traits affect plant relative performance across biotic and abiotic contexts. Pivotal results. All belowground plant functional traits except root tissue density were affected by adding nitrogen. We found that a microbial taxon (amplicon sequence variant [ASV]) in the genus Micromonospora correlated with shorter root lengths. We also found strong positive selection for longer roots regardless of the abiotic or biotic environment. In contrast, selection favored lower root-to-shoot ratios in high-nitrogen conditions, and selection on root tissue density was highest in treatments that had high nitrogen levels and perturbed microbial communities. Conclusions. We did not detect microbial effects on the expression of plant traits (ecological effects); however, patterns of phenotypic selection (evolutionary effects) on root tissue density differed depending on the biotic and abiotic environment. Additionally, we detected strong selection for increased root length across treatments; we also found that one ASV correlated with decreased root length, indicating potential conflict between root microbiome components and plant fitness. Future work would be to include microbial taxa in phenotypic selection analysis and to conduct manipulations of the microbes correlated with functional traits to determine causality.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2015

Microbes to Biomes at Berkeley Lab

Microbes are the Earth's most abundant and diverse form of life. Berkeley Lab's Microbes to Biomes initiative -- which will take advantage of research expertise at the Joint Genome Institute, Advanced Light Source, Molecular Foundry, and the new computational science facility -- is designed to explore and reveal the interactions of microbes with one another and with their environment. Microbes power our planet’s biogeochemical cycles, provide nutrients to our plants, purify our water and are integral components in keeping the human body free of disease and may hold the key to the Earth’s future.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2018

Building a genome engineering toolbox in nonmodel prokaryotic microbes

The realization of a sustainable bioeconomy requires our ability to understand and engineer complex design principles for the development of platform organisms capable of efficient conversion of cheap and sustainable feedstocks (e.g. sunlight, CO 2 , non-food biomass) to biofuels and bioproducts at sufficient titers and costs. For model microbes such as E. coli , advances in DNA reading and writing technologies are driving adoption of new paradigms for engineering biological systems. Unfortunately, microbes with properties of interest for the utilization of cheap and renewable feedstocks such as photosynthesis, autotrophic growth, and cellulose degradation have very few, if any, genetic tools for metabolic engineering. Therefore, it is important to begin to develop 'design rules' for building a genetic toolbox for novel microbes. Furthermore, we present an overview of our current understanding of these rules for the genetic manipulation of prokaryotic microbes and available genetic tools to expand our ability to genetically engineer non-model systems.

09 BIOMASS FUELS↗

DOE OSTI2019

Microbe Encapsulation for Selective Rare-Earth Recovery from Electronic Waste Leachates

Rare earth elements (REEs) are indispensable components of many green technologies and of increasing demand globally. However, refining REEs from raw materials using current technologies is energy intensive and enviromentally damaging. Here, we describe the development of a novel biosorption-based flow-through process for selective REE recovery from electronic wastes. An Escherichia coli strain previously engineered to display lanthanide-binding tags on the cell surface was encapsulated within a permeable polyethylene glycol diacrylate (PEGDA) hydrogel at high cell density using an emulsion process. This microbe bead adsorbent contained a homogenous distribution of cells whose surface functional groups remained accessible and effective for selective REE adsorption. The microbe beads were packed into fixed-bed columns, and breakthrough experiments demonstrated effective Nd extraction at a flow velocity of up to 3 m/h at pH 4–6. The microbe bead columns were stable for reuse, retaining 85% of the adsorption capacity after nine consecutive adsorption/desorption cycles. A bench-scale breakthrough curve with a NdFeB magnet leachate revealed a two-bed volume increase in breakthrough points for REEs compared to non-REE impurities and 97% REE purity of the adsorbed fraction upon breakthrough. These results demonstrate that the microbe beads are capable of repeatedly separating REEs from non-REE metals in a column system, paving the way for a biomass-based REE recovery system.

Biological and medical sciences↗

DOE OSTI2020

Dynamics of Fungal and Bacterial Biomass Carbon in Natural Ecosystems: Site-level Applications of the CLM-Microbe Model

Explicitly representing microbial processes has been recognized as a key improvement to Earth system models for the realistic projections of soil carbon (C) and climate dynamics. The CLM-Microbe model builds upon the CLM4.5 and explicitly represents two major soil microbial groups, fungi and bacteria. Based on the compiled time-series data of fungal (FBC) and bacterial (BBC) biomass C from nine biomes, we parameterized and validated the CLM-Microbe model, and further conducted sensitivity analysis and uncertainty analysis for simulating C cycling. The model performance was evaluated with mean absolute error (MAE), root mean square error (RMSE), and coefficient of determination (R2) for relative change in FBC and BBC. The CLM-Microbe model is able to reasonably capture the seasonal dynamics of FBC and BBC across biomes, particularly for tropical/subtropical forest, temperate broadleaf forest, and grassland, with MAE < 0.49 for FBC and <0.36 for BBC and RMSE <0.52 FBC and <0.39 for BBC, while R2 values are relatively smaller in some biomes (e.g., shrub) due to small sample sizes. We found good consistencies between simulated and observed FBC (R 2 =0.70, P<0.001) and BBC (R 2 =0.26, P<0.05) on average across biomes, but the model is not able to fully capture the large variation in observed FBC and BBC. Sensitivity analysis shows the most critical parameters are turnover rate, carbon-to-nitrogen ratio of fungi and bacteria, and microbial assimilation efficiency. This study confirms that the explicit representation of soil microbial mechanisms enhances model performance in simulating C variables such as heterotrophic respiration and soil organic C density. The further application of the CLM-Microbe model would deepen our understanding of microbial contributions to the global C cycle.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Ecological strategies of microbes: Thinking outside the triangle

Abstract I asked whether Grime's triangle of competitive, stress tolerance and ruderal ecological strategies—which was originally developed for plants—applies to microbes. I conducted a synthesis of empirical studies that tested relationships among microbial traits presumed to define the competitive, stress tolerance and ruderal, and other ecological strategies. There was broad support for Grime's triangle. However, the ecological strategies were inconsistently linked to shifts in microbial communities under environmental changes like nitrogen and phosphorus addition, warming, drought, etc. We may be missing important ecological strategies that more closely influence microbial community composition under shifting environmental conditions. We may need to start by documenting changes in microbial communities in response to environmental conditions at fine spatiotemporal scales relevant for microbes. We can then develop empirically based ecological strategies, rather than modifying those based on plant ecology. Synthesis . Microbes appear to sort into similar ecological strategies as plants. However, these microbial ecological strategies do not consistently predict how community composition will shift under environmental change. By starting ‘from the ground up’, we may be able to delineate ecological strategies more relevant for microbes.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

Identification and characterization of a skin microbiome on Caenorhabditis elegans suggests environmental microbes confer cuticle protection

ABSTRACT In the wild, C. elegans are emersed in environments teeming with a veritable menagerie of microorganisms. The C. elegans cuticular surface serves as a barrier and first point of contact with their microbial environments. In this study, we identify microbes from C. elegans natural habitats that associate with its cuticle, constituting a simple “skin microbiome.” We rear our animals on a modified CeMbio, mCeMbio, a consortium of ecologically relevant microbes. We first combine standard microbiological methods with an adapted micro skin-swabbing tool to describe the skin-resident bacteria on the C. elegans surface. Furthermore, we conduct 16S rRNA gene sequencing studies to identify relative shifts in the proportion of mCeMbio bacteria upon surface-sterilization, implying distinct skin- and gut-microbiomes. We find that some strains of bacteria, including Enterobacter sp. JUb101 , are primarily found on the nematode skin, while others like Stenotrophomonas indicatrix JUb19 and Ochrobactrum vermis MYb71 are predominantly found in the animal’s gut. Finally, we show that this skin microbiome promotes host cuticle integrity in harsh environments. Together, we identify a skin microbiome for the well-studied nematode model and propose its value in conferring host fitness advantages in naturalized contexts. IMPORTANCE The genetic model organism C. elegans has recently emerged as a tool for understanding host–microbiome interactions. Nearly all of these studies either focus on pathogenic or gut-resident microbes. Little is known about the existence of native, nonpathogenic skin microbes or their function. We demonstrate that members of a modified C. elegans model microbiome, mCeMbio, can adhere to the animal's cuticle and confer protection from noxious environments. We combine a novel micro-swab tool, the first 16S microbial sequencing data from relatively unperturbed C. elegans , and physiological assays to demonstrate microbially mediated protection of the skin. This work serves as a foundation to explore wild C. elegans skin microbiomes and use C. elegans as a model for skin research.

16S RNA↗

NASA NTRSDate not supplied

Cleanroom Microbes Survive Drying, Vacuum, and Proton Irradiation

Introduction : The goal of planetary protection at NASA is to mitigate the risk of contaminating sensitive target bodies with biological life. While many cleaning procedures have been put in place to reduce bioburden on spacecraft, microbes are experts at evolving to survive harsh conditions. Specifically, the dry, low-nutrient environment of a cleanroom (commonly used for assembly of spacecraft) can represent an environment where extremophiles can survive. Methods : Scientists at NASA MSFC wished to gather a snapshot of the microbial population within a variety of cleanrooms on site. A study was undertaken to collect air, surface, and floor samples from clean-rooms and isolate unique morphologies. From this study, 95 isolates were collected and saved in a microbial library. About 86% of these were identified at least to a genus level. Following identification, 24 microbes were selected, based on a literature review, as potential extremophiles. These were grown in liquid cultures, diluted to a set optical density, washed with water, and then applied to a sterilized Kapton coupon. Droplets were allowed to dry overnight in a biosafety cabinet. Coupons were then installed in a pelletron and pumped down to high vacuum (~1E-6 Torr). Samples were then subjected 100 keV protons at a fluence of 2x10 15 p+/cm 2 up to 4x10 15 p+/cm 2 . Following exposure, samples were returned to the microbiology lab where they were pro-cessed by submerging in water, vortexing, and then plating either droplets or spread plates. Recovery data collected was qualitative with a ranking or +, minor, or – for growth. Some selected radiotolerant strains were sequenced using the Illumina sequencing platform. The resulting genomes were annotated with the Rapid Annotations using Subsystems Technology (RAST) server and analyzed for conserved and unique stress response relevant genomic signatures to identify clues related to specific tolerances. Results and Discussion : After five rounds of proton radiation, we narrowed our isolates to five, non-spore forming bacteria that demonstrated survival: Arthrobacter koreensis, Paenarthrobacter nitroguajacolicus, Mycetocola manganoxydans , and an Erwinia sp. Furthermore, we exposed these four microbes to 254 nm wavelength light at an intensity of 80 W/m 2 at a distance of ~18 cm for 10 minutes. Only A. koreensis demonstrated survival following UV exposure. Finally, we performed whole genome sequencing on the four strains to look for genetic markers of stress resistance. When we compared the genomes of the four strains, we found that genes coding for GGDEF and EAL domains with PAS/PAC sensors were only found in A. koreensis . These domains, modulated by PAS/PAC sensors, are hypothesized to facilitate survival under drying, desiccation, and proton irradiation. Drying and Desiccation : PAS domains sense hydration changes and modulate GGDEF and EAL domain activity to adjust c-di-GMP levels, enhancing resistance to desiccation. For instance, in Pseudomonas aeruginosa , the PAS domain of RbdA modulates activity under varying hydration conditions, affecting stress responses [1]. Proton Irradiation : Proton irradiation causes oxidative stress, leading to ROS generation. PAS domains detect this stress and modulate GGDEF and EAL domains to manage oxidative stress responses. In Shewanella , EAL domain proteins modulated by PAS sensors help bacteria adapt to extreme conditions [2]. These genes upregulate other stress response genes, protecting membrane function, protein stability, DNA repair, and antioxidant defenses. The modulation of c-di-GMP by PAS domains is crucial for bacterial adaptation to stress conditions, enabling dynamic physio-logical adjustments [3]. Understanding these mechanisms provides insights into bacterial stress responses and strategies for controlling bacterial growth [4]. Conclusions : These findings indicate that clean-rooms harbor extremophile microbes that may be able to survive conditions in deep space. Furthermore, while we identified certain stress-response genes that may be at least partly responsible for the phenotypes observed in this study, there are likely unidentified genes or characteristics about A. koreensis , and other bacteria, that may allow them to survive in harsh environments. Future studies will focus on identifying these unknown genes and characteristics, further elucidating the mechanisms of extremophile survival and potentially informing the development of new biotechnologies for space exploration and other extreme environments.

Chelsi Cassilly↗

DOE OSTI2018

Evaluation of the Effects of Microbes on Autunite Dissolution and the Spectral Induced Polarization Response in Hanford Vadose Zone Sediment - 18440

As a result of the U.S. nuclear weapons program during the second World War and throughout the Cold War, there now exists a significant amount of uranium contamination at the U.S. Department of Energy Hanford site located in Washington State. Much of this contamination is in the form of mobile uranyl carbonates species within the vadose zone, which at Hanford varies up to 100 m thick in the Central Plateau. In-situ immobilization of mobile uranium via injections of a soluble sodium tripolyphosphate amendment may prove effective in the formation of low-solubility uranyl phosphate solid phases, autunite. However, the injected polyphosphate undergoes hydrolysis in aqueous solutions to form orthophosphate, which serves as a readily available nutrient for the various microorganisms in the sediment. Sediment-filled column experiments conducted under saturated oxygen restricted conditions using a geophysical technique known as spectral induced polarization (SIP) have shown the impact of microbes on the dissolution of synthetic autunite. Two sets of experiments were conducted using test columns. Each experiment had a pair of columns inoculated with microbes (with a glucose solution acting as a nutrient source) which were monitored using SIP in conjunction with regular sampling of pore-water. Columns were built with two silver/silver chloride (Ag/AgCl) coiled current electrodes at either end and four Ag potential electrodes along the side, which allowed the measurement of three distinct points along the length of the column. SIP measures phase which is the time delay between an injected current and measured voltage, in this case measured in radians. Three sampling ports were added to correspond with the geophysical measurements. Pore-water samples taken over the course of the experiment were analyzed for dissolved elements including uranium, iron, phosphorous, calcium, and magnesium. SIP results were plotted over time in order to determine correlations between changes in electrical phase and changes in pore-water chemistry. It was determined that changes in the phase and bulk resistivity detected with SIP corresponded to changes in the pore-water chemistry due to the growth of microbes. The first experiment ran for five months and had measurements done once a week, the second only ran one month but had measurements taken five times a week in order to understand initial changes in column chemistry in greater detail. SIP may be an effective way to remotely track changes indicative of bacterial activities on the surrounding environment. This method has the potential for field application at a larger scale for the purpose of tracking microbe growth in response to tripolyphosphate amendment. (authors)

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

DOE OSTI2023

Editorial: Plant-microbe omics

Omics-based studies have evolved over the past two decades to provide rich datasets from which deeper system-level understanding can be unraveled. Omics technologies (e.g., genomics, transcriptomics, proteomics, and metabolomics) can provide specific molecular insights into plant and microbe fitness, disease states, stress, cell signaling/cell-cell communication, and nutrient exchange, and these molecular observations can be correlated with system phenotype and function. The overall health of plant-microbe systems and their surrounding environments can be assessed, and a greater understanding of processes related to nutrient exchange/cycling, plant disease, and ecosystem homeostasis can now be achieved via the latest approaches to plant-microbe omics analysis.

59 BASIC BIOLOGICAL SCIENCES↗