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DOE OSTI2019

Microbial communities of the Laurentian Great Lakes reflect connectivity and local biogeochemistry

The Laurentian Great Lakes are a vast, interconnected freshwater system spanning strong physicochemical gradients, thus constituting a powerful natural laboratory for addressing fundamental questions about microbial ecology and evolution. We present a comparative analysis of pelagic microbial communities across all five Laurentian Great Lakes, focusing on Bacterial and Archaeal picoplankton characterized via 16S rRNA amplicon sequencing. We collected samples throughout the water column from the major basins of each lake in spring and summer over 2 years. Two oligotypes, classified as LD12 (Alphaproteobacteria) and acI-B1 (Actinobacteria), were among the most abundant in every sample. At the same time, microbial communities showed distinct patterns with depth during summer stratification. Deep hypolimnion samples were frequently dominated by a Chloroflexi oligotype that reached up to 19% relative abundance. Stratified surface communities differed between the colder, less productive upper lakes (Superior, Michigan, Huron) and warmer, more productive lower lakes (Erie, Ontario), in part due to an Actinobacteria oligotype (acI-C2) that averaged 7.7% of sequences in the lower lakes but <0.2% in the upper lakes. Together, our findings suggest that both hydrologic connectivity and local selective pressures shape microbial communities in the Great Lakes and establish a framework for future investigations.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Metabolism and Evolution of a Biofuel-Producing Microbial Coculture

Specialized features of individual microbial species can be combined in coculture to produce useful fuels and chemicals. Cocultures can also be used to address how microbes cooperate in nature through metabolic interactions but with a level of experimental control that is not possible in natural systems. However, the challenge of maintaining stable microbial relationships and the limited assays available for studying single strains in consortia has impeded progress in characterizing and implementing synthetic communities. In this project, my lab developed a stable and highly reproducible H 2 biofuel-producing coculture between fermentative Escherichia coli and phototrophic Rhodopseudomonas palustris . In this coculture each species provides or 'cross-feeds' a nutrient that the other requires for growth. My lab used this coculture along with mathematical modeling to uncover basic principles governing mutualistic interactions between microbes, including (i) the potential for cross-fed nutrients to alternatively act as toxins depending on the level to which they are produced, (ii) how mutualistic cross-feeding can establish and persist in nutrient-deprived environments where the highest biofuel yields were observed, and (iii) the potential for competition between mutualistic partners for a cross-fed nutrient when that nutrient holds value for both species involved. In using ethanol-producing Zymomonas mobilis in place of E. coli in the coculture, my lab also discovered that Z. mobilis can use N 2 gas as a nitrogen source, with potential cost-savings for the industrial production of cellulosic ethanol.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRSDate not supplied

Studying Microbial Adaptation in the Laboratory: Sensor & Control Upgrades for an Experimental Evolution Biofluidics System

Experimental evolution (EE) involves iteratively exposing a microbial community to specific stressors to study its response to changes in environment over time. EE work is commonly done manually in the laboratory, but, when there are many environmental variables to measure and adjust, it is highly labor intensive, prone to human error, and challenging to scale. Single-purpose automated continuous culturing chambers exist, but implement only limited stressor types. A more general-purpose design is desirable. The BeING Lab at Ames Research Center created the prototype Automated Adaptive Directed Evolution Chamber (AADEC) to address these problems, beginning with Escherichia coli tolerance of short-wave ultraviolet (UV-C) radiation and of temperature. In newer versions, AADEC monitors microbial activity and can adjust the UV-C and temperature levels automatically. An optical density measurement is used to determine how many cells are present in the growth medium—over time, this corresponds to how many survive and reproduce. Oxidation-reduction potential provides information on consumed metabolic energy, and pH and electrical conductivity on metabolic products. Dissolved oxygen content is used to determine aerobic vs anaerobic growth. A Raspberry Pi computer processes all this data to set the UV-C stressor level. AADEC’s auxiliary systems include peristaltic pumps to change media and agitation to counteract cell settling. These actuators can also act as additional stressors. With the Raspberry Pi monitoring sensors and adjusting actuators in real time, AADEC takes measurements and controls the environment much more accurately than can be done with a manual EE implementation. The third and latest AADEC iteration is the first to simplify design and usage with circuits on PCBs and the ability to pre-program experimental protocols. Still planned is expansion to a multi-well design for the study of varying cell cultures in parallel, which will enable researchers to retain and re-inoculate cultures exhibiting the desired trait most strongly while flushing out others. AADEC’s special capabilities make it a valuable tool for studying life under multiple stressors, enabling scientists to replicate changes in climate on microbes for study in a lab setting.

Microbial Adaptation↗

DOE OSTI2021

Deeplasmid: deep learning accurately separates plasmids from bacterial chromosomes

Plasmids are mobile genetic elements that play a key role in microbial ecology and evolution by mediating horizontal transfer of important genes, such as antimicrobial resistance genes. Many microbial genomes have been sequenced by short read sequencers and have resulted in a mix of contigs that derive from plasmids or chromosomes. New tools that accurately identify plasmids are needed to elucidate new plasmid-borne genes of high biological importance. We have developed Deeplasmid, a deep learning tool for distinguishing plasmids from bacterial chromosomes based on the DNA sequence and its encoded biological data. It requires as input only assembled sequences generated by any sequencing platform and assembly algorithm and its runtime scales linearly with the number of assembled sequences. Deeplasmid achieves an AUC–ROC of over 89%, and it was more accurate than five other plasmid classification methods. Finally, as a proof of concept, we used Deeplasmid to predict new plasmids in the fish pathogen Yersinia ruckeri ATCC 29473 that has no annotated plasmids. Deeplasmid predicted with high reliability that a long assembled contig is part of a plasmid. Using long read sequencing we indeed validated the existence of a 102 kb long plasmid, demonstrating Deeplasmid's ability to detect novel plasmids.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2023

Biogeochemical evolution of ponded meltwater in a High Arctic subglacial tunnel

Subglacial environments comprise ∼10 % of Earth's land surface, host active microbial ecosystems, and are important components of global biogeochemical cycles. However, the broadly inaccessible nature of subglacial systems has left them vastly understudied, and research to date has been limited to laboratory experiments or field measurements using basal ice or subglacial water accessed through boreholes or from the glacier margin. In this study, we extend our understanding of subglacial biogeochemistry and microbiology to include observations of a slushy pond of water that occupied a remnant meltwater channel beneath a polythermal glacier in the Canadian High Arctic over winter. The hydraulics and geochemistry of the system suggest that the pond water originated as late-season, ice-marginal runoff with less than ∼15 % solute contribution from subglacial sources. Over the 8 months of persistent sub-zero regional temperatures, the pond gradually froze, cryo-concentrating solutes in the residual water by up to 7 times. Despite cryo-concentration and the likely influx of some subglacial solute, the pond was depleted in only the most labile and biogeochemically relevant compounds, including ammonium, phosphate, and dissolved organic matter, including a potentially labile tyrosine-like component. DNA amplicon sequencing revealed decreasing microbial diversity with distance into the meltwater channel. The pond at the terminus of the channel hosted a microbial community inherited from late-season meltwater, which was dominated by only six taxa related to known psychrophilic and psychrotolerant heterotrophs that have high metabolic diversity and broad habitat ranges. Collectively, our findings suggest that generalist microbes from the extraglacial or supraglacial environments can become established in subglacial aquatic systems and deplete reservoirs of nutrients and dissolved organic carbon over a period of months. These findings extend our understanding of the microbial and biogeochemical evolution of subglacial aquatic ecosystems and the extent of their habitability.

Ashley J Dubnick↗

DOE OSTI2019

Assessing microbial community patterns under incipient soil formation from basalt

Microbial dynamics drive the biotic machinery of early soil evolution. However, integrated knowledge of microbial community establishment, functional associations, and community assembly processes in incipient soil is lacking. This study presents a novel approach of combining microbial phylogenetic profiling, functional predictions, and community assembly processes to analyze drivers of microbial community establishment in an emerging soil system. Rigorous sub-meter sampling of a basalt-soil lysimeter after two years of irrigation revealed the contrasting depth-dependent distribution patterns of soil parameters and community diversity. Phylogenetic analysis of 16S rRNA gene indicated the presence of diverse bacterial and archaeal phyla, with high relative abundance of Actinobacteria on the surface and a consistently high abundance of Proteobacteria at all depths. Predicted functional gene analysis suggested dominant autotrophy signatures, evidence of an incomplete nitrogen cycle, and predicted enzymes of iron (III) to iron (II) conversion followed by intracellular uptake, transport, and regulation. Null modeling revealed microbial community assembly predominantly governed by variable selection. The relative influence of the variable selection decreased with depth, indicating unique and relatively harsh environmental conditions near the surface and more benign conditions with depth. Additionally, high levels of dispersal influenced community composition near the center of the domain, suggesting that spatial processes interact with deterministic selection imposed by the environment. Characterizing the complexity in incipient soils can advance research insights about microbial community diversity and assembly in other incipient systems (e.g., transformation of marginal soils, or soils recovering from disturbances such as fire, erosion, intensive agricultural practices, or contamination).

Sengupta, Aditi↗

DOE OSTI2023

Investigating the eco‐evolutionary response of microbiomes to environmental change

Abstract Microorganisms are the primary engines of biogeochemical processes and foundational to the provisioning of ecosystem services to human society. Free‐living microbial communities (microbiomes) and their functioning are now known to be highly sensitive to environmental change. Given microorganisms' capacity for rapid evolution, evolutionary processes could play a role in this response. Currently, however, few models of biogeochemical processes explicitly consider how microbial evolution will affect biogeochemical responses to environmental change. Here, we propose a conceptual framework for explicitly integrating evolution into microbiome–functioning relationships. We consider how microbiomes respond simultaneously to environmental change via four interrelated processes that affect overall microbiome functioning (physiological acclimation, demography, dispersal and evolution). Recent evidence in both the laboratory and the field suggests that ecological and evolutionary dynamics occur simultaneously within microbiomes; however, the implications for biogeochemistry under environmental change will depend on the timescales over which these processes contribute to a microbiome's response. Over the long term, evolution may play an increasingly important role for microbially driven biogeochemical responses to environmental change, particularly to conditions without recent historical precedent.

54 ENVIRONMENTAL SCIENCES↗

NASA NTRS2018

Enabling Experimental Evolution: Multi-Parameter Sensor System Integration into a Culture/Stressor Biofluidics System

Experimental evolution (EE) exposes microbial communities to ecological stressors, simulating dynamics up to near-extinction events. Combined with comparative sequencing and other molecular tools, such data can inform the genetic and other biological mechanisms underlying extremophile adaptation, and other observed effects. Automating this type of experiment using biofluidics can mitigate many traditional obstacles, including delays in assay results and environment adjustment and the need for many replicates. A first-generation device for automating EE procedures, the Automated Adaptive Directed Evolution Chamber (AADEC), was developed at NASA Ames. UV-C radiation was the stressor, an LED-photodiode array measured optical density, magnetic agitation and peristaltic pump systems ensured nutrient availability, and Arduino microcontrollers provided control. Escherichia coli in LB kanamycin media was used for testing and performance verification. A manual laboratory procedure with timed exposure to UV-C was performed to typify tolerance acquisition. Approximately a 106 factor increase in survival ratio was recorded over multiple iterations. Currently, a second-generation device is being developed integrating more real-time sensors: redox potential (ORP), indicating available/consumed metabolic energy; dissolved oxygen (DO), indicating aerobic/anaerobic growth; pH, indicating metabolic products; and electrical conductivity (EC), another indicator of metabolic products. The EC sensor system was constructed and calibrated in-house and matched commercial sensors in the required range. A Raspberry Pi computer automated the electrical system, allowing real-time data acquisition. The fluidics card was made of CNC-milled polycarbonate for biocompatibility. Each sensor parameter can also be used as a selection pressure alone or in combination with others to create extreme microbial environments. As a proof of concept, this work demonstrated sensor operation in one pair of growth-sensor chambers. It can be expanded to a multi-chamber system to enable inter-culture comparisons and multi-population studies. The prior Arduino system will be ported to the RPi system. Future stressors to be added include thermal, reactive oxygen species, and varying nutrient availability.

Govinda Raj, Chinmayee↗

DOE OSTI2018

Dynamic root exudate chemistry and microbial substrate preferences drive patterns in rhizosphere microbial community assembly

Like all higher organisms, plants have evolved in the context of a microbial world, shaping both their evolution and their contemporary ecology. Interactions between plant roots and soil microorganisms are critical for plant fitness in natural environments. Given this co-evolution and the pivotal importance of plant–microbial interactions, it has been hypothesized, and a growing body of literature suggests, that plants may regulate the composition of their rhizosphere to promote the growth of microorganisms that improve plant fitness in a given ecosystem. In this paper, using a combination of comparative genomics and exometabolomics, we show that pre-programmed developmental processes in plants (Avena barbata) result in consistent patterns in the chemical composition of root exudates. This chemical succession in the rhizosphere interacts with microbial metabolite substrate preferences that are predictable from genome sequences. Specifically, we observed a preference by rhizosphere bacteria for consumption of aromatic organic acids exuded by plants (nicotinic, shikimic, salicylic, cinnamic and indole-3-acetic). The combination of these plant exudation traits and microbial substrate uptake traits interact to yield the patterns of microbial community assembly observed in the rhizosphere of an annual grass. Finally, this discovery provides a mechanistic underpinning for the process of rhizosphere microbial community assembly and provides an attractive direction for the manipulation of the rhizosphere microbiome for beneficial outcomes.

54 ENVIRONMENTAL SCIENCES↗

NASA NTRS2000

Modern Microbial Ecosystems are a Key to Understanding Our Biosphere's Early Evolution and its Contributions To The Atmosphere and Rock Record

The survival of our early biosphere depended upon efficient coordination anion- diverse microbial populations. Microbial mats exhibit a 3.46-billion-year fossil record, thus they are the oldest known ecosystems. Photosynthetic microbial mats were key because, today, sunlight powers more than 99 percent of global primary productivity. Thus photosynthetic ecosystems have affected the atmosphere profoundly and have created the most pervasive, easily-detected fossils. Photosynthetic biospheres elsewhere will be most detectible via telescopes or spacecraft. As a part of the Astrobiology Institute, our Ames Microbial Ecosystems group examines the roles played by ecological processes in the early evolution of our biosphere, as recorded in geologic fossils and in the macromolecules of living cells: (1) We are defining the microbial mat microenvironment, which was an important milieu for early evolution. (2) We are comparing mats in contrasting environments to discern strategies of adaptation and diversification, traits that were key for long-term survival. (3) We have selected sites that mimic key environmental attributes of early Earth and thereby focus upon evolutionary adaptations to long-term changes in the global environment. (4) Our studies of gas exchange contribute to better estimates of biogenic gases in Earth's early atmosphere. This group therefore directly addresses the question: How have the Earth and its biosphere influenced each other over time Our studies strengthen the systematics for interpreting the microbial fossil record and thereby enhance astrobiological studies of martian samples. Our models of biogenic gas emissions will enhance models of atmospheres that might be detected on inhabited extrasolar planets. This work therefore also addresses the question: How can other biospheres be recogniZed" Our choice of field sites helps us explore Earth's evolving early environment. For example, modern mats that occupy thermal springs and certain freshwater environments experience conditions such as low O2 and sulfate and high inorganic carbon and sulfide levels that resemble those of ancient marine environments. Later in history, both biologically-induced carbonate precipitation and the trapping and binding of suspended grains of carbonate became a dominant mechanism for carbonate deposition. Modern marine carbonate platforms and alkaline offer good examples of microbiologically-induced calcification. Both marine platforms and solar salterns illustrate microbially-driven trapping and binding. We are also exploring the effects of water composition upon the exchange of biogenic gases with the atmosphere.

DesMarais, David J.↗

NASA NTRS2002

Solar Radiation as Driving Force In Early Evolution

Ultraviolet radiation (UVR) has provided an evolutionary challenge to life on Earth in that it is both an agent of mutation and as well as a selective force. Today surface fluxes of UVR vary diurnally, seasonally, etc. Still, the UVR flux was probably substantially higher during the early phases of evolution, suggesting that its role in evolution was even more prominent during this time. In this presentation, the creative role of UVR in evolution is discussed, specifically in connection with the role that UVR may have played in the evolution of early microbial ecosystems. The presentation will include discussions of the direct influence of UVR on such processes as photosynthesis and genetic damage, as well as the indirect influence of UVR as mediated through the production of reactive oxygen species. These biological effects of UVR will be viewed against the backdrop of the physical nature of the early Earth, surely a very different place then than now.

Rothschild, Lynn J.↗

NASA NTRS2010

Proceedings of the Astrobiology Science Conference 2010. Evolution and Life: Surviving Catastrophes and Extremes on Earth and Beyond

The Program of the 2010 Astrobiology Science Conference: Evolution and Life: Surviving Catastrophes and Extremes on Earth and Beyond, included sessions on: 50 Years of Exobiology and Astrobiology: Greatest Hits; Extraterrestrial Molecular Evolution and Pre-Biological Chemistry: From the Interstellar Medium to the Solar System I; Human Exploration, Astronaut Health; Diversity in Astrobiology Research and Education; Titan: Past, Present, and Future; Energy Flow in Microbial Ecosystems; Extraterrestrial Molecular Evolution and Prebiological Chemistry: From the Interstellar Medium to the Solar System II; Astrobiology in Orbit; Astrobiology and Interdisciplinary Communication; Science from Rio Tinto: An Acidic Environment; Can We Rule Out Spontaneous Generation of RNA as the Key Step in the Origin of Life?; How Hellish Was the Hadean Earth?; Results from ASTEP and Other Astrobiology Field Campaigns I; Prebiotic Evolution: From Chemistry to Life I; Adaptation of Life in Hostile Space Environments; Extrasolar Terrestrial Planets I: Formation and Composition; Collaborative Tools and Technology for Astrobiology; Results from ASTEP and Other Astrobiology Field Campaigns II; Prebiotic Evolution: From Chemistry to Life II; Survival, Growth, and Evolution of Microrganisms in Model Extraterrestrial Environments; Extrasolar Terrestrial Planets II: Habitability and Life; Planetary Science Decadal Survey Update; Astrobiology Research Funding; Bioessential Elements Through Space and Time I; State of the Art in Life Detection; Terrestrial Evolution: Implications for the Past, Present, and Future of Life on Earth; Psychrophiles and Polar Environments; Life in Volcanic Environments: On Earth and Beyond; Geochronology and Astrobiology On and Off the Earth; Bioessential Elements Through Space and Time II; Origins and Evolution of Genetic Systems; Evolution of Advanced Life; Water-rich Asteroids and Moons: Composition and Astrobiological Potential; Impact Events and Evolution; A Warm, Wet Mars?; Titan Versus Europa - Potential for Astrobiology; Habitability Potential of Mars; Biosignatures: Tools and Development I; Origins of Molecular Asymmetry, Homochirality, and Life Detection; Deserts and Evaporite Basins and Associated Microbialite Systems; Ancient Life and Synthetic Biology: Crossroad of the Past and Future; Biosignatures: Tools and Development II; Free Oxygen: Proxies, Causes, and Consequences; Life in Modern Microbialite Systems - Function and Adaptation; Hydrothermal Systems and Organosynthesis Processes: Origin and Evolution of Life; Where Should We Go on Mars to Seek Signs of Life?; Search for Intelligent Life I. Innovative SETI Observing Programs and Future Directions; Integrating Astrobiology Research Across and Beyond the Community; Education in Astrobiology in K-12; Search for Intelligent Life II. Global Engagement and Interstellar Message Construction; Poster sessions included: Extraterrestrial Molecular Evolution and Pre-Biological Chemistry; Prebiotic Evolution: From Chemistry to Life; RNA World; Terrestrial Evolution: Implications for the Past, Present, and Future of Life on Earth; Hydrothermal Systems and Organosynthesis Processes: Origin and Evolution of Life; Virology and Astrobiology; Horizontal Genetic Transfer and Properties of Ancestral Organisms; Life in Volcanic Environments: On Earth and Beyond; Impact Events and Evolution; Evolution of Advanced Life; Evolution of Intelligent Life; Education in Astrobiology in K-12; Origins of Molecular Asymmetry, Homochirality, and Life Detection; Astrobiology and Interdisciplinary Communication; Diversity in Astrobiology Research and Education; Integrating Astrobiology Research Across and Beyond the Community; Policy and Societal Issues: Dealing with Potential Bumps in the Astrobiology Road Ahead; Results from ASTEP and Other Astrobiology Field Campaigns; Energy Flow in Microbial Ecosystems; Psychrophiles and Polar Environments; Deserts and Evaporite Basins and Associated Microbialite stems; Life in Modern Microbialite Systems - Function and Adaptation; Free Oxygen: Proxies, Causes, and Consequences; Bioessential Elements Through Space and Time; Water-rich Asteroids and Moons: Composition and Astrobiological Potential; Biosignatures: Tools and Developments; Robotics and Instrumentation for Astrobiology; State of the Art in Life Detection; Astrobiology in Orbit; Survival, Growth, and Evolution of Microrganisms in Model Extraterrestrial Evolution; Search for Intelligent Life; Habitability Potential of Mars; How and Where Should We Seek Signs of Life on Mars?; Titan: Past, Present, and Future; Extrasolar Terrestrial Planets: Formation, Composition, Diversity, Habitability and Life; Human Exploration, Astronaut Health; Science from Rio Tinto: An Acidic Environment and Adaptation of Life in Hostile Space Environments;

Source record↗

DOE OSTI2019

The Hopkins Microbiology Course

For 10 years, (2008-2018), DOE supported the four-week, intensive summer course in Microbiology at the Hopkins Marine Station. This course builds upon the inquiry-driven teaching tradition introduced by C. B. van Niel in his renowned Hopkins Microbiology Course taught in the 1950s and 1960s. The distinguishing aspect of this Hopkins Microbiology Course is the unique integration and application of conceptual ideas and experimentation of microbial physiology, ecology, population genetics and experimental evolution in a cohesive study of the environment. In traditional University curricula, courses in microbial physiology, genetics, ecology and evolution are taught distinctly from each other often competitively and without much cross-referencing. Each field has its own theories, thinking, and vocabulary, but the synthesis and assimilation of these clearly related aspects of microbial life is often lacking. This Hopkins Microbiology Course is not bound to adhere to such a strict curriculum. Therefore, we have assembled an international expert team specializing in physiology, ecology, and evolution to develop a coherent picture of how to understand microbe’s function in nature and the principles of what constitutes Microbial Diversity, how it is generated, and how it is maintained.

54 ENVIRONMENTAL SCIENCES↗

NASA NTRS1980

Early photosynthetic microorganisms and environmental evolution

Microfossils which are preserved as shrivelled kerogenous residues provide little information about cellular organization and almost none about the metabolic properties of the organisms. The distinction between prokaryotic vs eukaryotic, and phototrophic vs chemo- and organotrophic fossil microorganisms rests entirely on morphological comparisons with recent counterparts. The residual nature of the microbial fossil record promotes the conclusion that it must be biased toward (a) most abundant organisms, (b) those most resistant to degradation, and (c) those inhabiting environments with high preservation potential e.g., stromatolites. These criteria support the cyanophyte identity of most Precambrian microbial fossils on the following grounds: (1) as primary producers they dominate prokaryotic communities in modern extreme environments, e.g., intertidal zone; (2) several morphological counterparts of modern cyanophytes and microbial fossils have been established based on structure, cell division patterns and degradation sequences. The impact of anaerobic and oxygenic microbial photosynthesis on the evolution of Precambrian environments is discussed.

Golubic, S.↗

DOE OSTI2021

Using continuous directed evolution to improve enzymes for plant applications

Continuous directed evolution of enzymes and other proteins in microbial hosts is capable of outperforming classical directed evolution by executing hypermutation and selection concurrently in vivo, at scale, with minimal manual input. Provided that a target enzyme’s activity can be coupled to growth of the host cells, the activity can be improved simply by selecting for growth. Like all directed evolution, the continuous version requires no prior mechanistic knowledge of the target. Continuous directed evolution is thus a powerful way to modify plant or non-plant enzymes for use in plant metabolic research and engineering. Here, we first describe the basic features of the yeast (Saccharomyces cerevisiae) OrthoRep system for continuous directed evolution and compare it briefly with other systems. We then give a step-by-step account of three ways in which OrthoRep can be deployed to evolve primary metabolic enzymes, using a THI4 thiazole synthase as an example and illustrating the mutational outcomes obtained. We close by outlining applications of OrthoRep that serve growing demands (i) to change the characteristics of plant enzymes destined for return to plants, and (ii) to adapt (“plantize”) enzymes from prokaryotes—especially exotic prokaryotes—to function well in mild, plant-like conditions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

A Scaling Study of Microbially-Enhanced Methane Production From Coal (Mecbm): Optimizing Nutrient Delivery For Maximized Methane Production

Microbially-enhanced coalbed methane (MECBM) recovery is a novel method to stimulate gas production in CBM reservoirs. We first develop suitable nutrient recipes for native coals from sites each in the Illinois Basin and in the San Juan Basin. The optimal concentrations for statistically significant parameters are identified and the methane yields at laboratory scale are obtained (Part I & II). We then demonstrate the importance of replicating truly in-situ conditions for bio- gasification studies by investigating methane productivity in a core holder simulating in-situ conditions. Based on the experimental results, the resulting gas production correlates positively with the permeability of the samples. We examine the deliverability of engineered microbial solutions to promote coal bioconversion in coals and demonstrate that greater than milli-darcy permeability is required under in-situ stress conditions (Part III). This constraint is supplemented by imaging and a fractal-based characterization approach to evaluate changes in the physical properties of coal due to bio-gasification. High resolution SEM imaging is used to observe swelling of the coal matrix with sub-micron wide continuous pores observed to persist even after microbial treatment (IV). We explore the potential of generating, propping-open and sustaining the functionality of hydraulic fractures in coal using different fracturing fluids (Part V). The alteration of the nano-pore structure due to MECBM treatment is examined by using small angle X-ray scattering (SAXS) to define the in-situ porosity evolution as a proxy to infer permeability evolution (Part VI). Coupled hydro-mechanical and bio-gasification modeling is applied to determine the effectiveness of nutrient delivery by hydraulic fracturing and its field implementation. Compared with both the unstimulated and hydro-shear-dilated cases of the natural fracture network, hydraulic fracturing demonstrates a significant improvement to enhance nutrient delivery. A preliminary analysis of field-scale methane yield is completed with consideration of the combined influence of permeability evolution, solute transport and microbial growth (Part VII). Developing nutrient solutions for Illinois Basin and San Juan Basin (Task 2): To enhance methane release from coal through bio-gasification, suitable nutrient solutions are needed to stimulate microbial activities toward coal depolymerization and conversion to biogas. Specific for bituminous coal in the Illinois basin, a nutrient recipe that can be used to enhance coal bio- gasification in situ is developed. Based on our previous experience working with Illinois coal and the characteristics of the formation water and the indigenous microbial community, four parameters, Fe powder, methanol, ethanol, and trace minerals are optimized through using a Box-Behnken design. The optimal condition predicted by the models is: Fe-powder at 74 mM, methanol at 97.91 mM, ethanol at 100 mM, and trace minerals at 100%. Under these conditions, the predicted methane yield and content is 1,417.35 ft 3 /ton and 80.7%, respectively. These results are then validated by experimental studies. In addition, each added component is evaluated in terms of its contribution to methane generated. Specifically, the role of coal in the bio-gasification process is studied against two other solid materials. Experimental data from different solid matrices prove that coal is indeed degraded by the studied microbial community.

01 COAL, LIGNITE, AND PEAT↗

DOE OSTI2023

Tectonic and geological setting influence hot spring microbiology

Hydrothermal systems form at divergent and convergent boundaries of lithospheric plates and within plates due to weakened crust and mantle plumes, playing host to diverse microbial ecosystems. Little is known of how differences in tectonic setting influence the geochemical and microbial compositions of these hydrothermal ecosystems. Here, for this work, coordinated geochemical and microbial community analyses were conducted on 87 high-temperature (>65°C) water and sediment samples from hot springs in Yellowstone National Park, Wyoming, USA ( n = 41; mantle plume setting), Iceland ( n = 41, divergent boundary), and Japan ( n = 5; convergent boundary). Region-specific variation in geochemistry and sediment-associated 16S rRNA gene amplicon sequence variant (ASV) composition was observed, with 16S rRNA gene assemblages being nearly completely distinguished by region and pH being the most explanatory parameter within regions. Several low abundance ASVs exhibited cosmopolitan distributions across regions, while most high-abundance ASVs were only identified in specific regions. The presence of some cosmopolitan ASVs across regions argues against dispersal limitation primarily shaping the distribution of taxa among regions. Rather, the results point to local tectonic and geologic characteristics shaping the geochemistry of continental hydrothermal systems that then select for distinct microbial assemblages. These results provide new insights into the co-evolution of hydrothermal systems and their microbial communities.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Unlocking nature’s biosynthetic potential by directed genome evolution

Microorganisms have been increasingly explored as microbial cell factories for production of fuels, chemicals, drugs, and materials. Among the various metabolic engineering strategies, directed genome evolution has emerged as one of the most powerful tools to unlock the full biosynthetic potential of microorganisms. Furthermore we summarize the directed genome evolution strategies that have been developed in recent years, including adaptive laboratory evolution and various targeted genome-scale engineering strategies, and discuss their applications in basic and applied biological research.

59 BASIC BIOLOGICAL SCIENCES↗