Early Career Awards

The Early Career Research Program is managed by DOE’s Office of Science and awards research grants to young scientists and engineers at U.S. universities and national laboratories. The grants are designed to bolster the nation’s scientific workforce by providing support to exceptional researchers during the crucial early years of their careers.

Opportunities exist in the following program areas: Advanced Scientific Computing Research, Biological and Environmental Research (BER), Basic Energy Sciences, Fusion Energy Sciences, High Energy Physics, and Nuclear Physics. For more information, see the DOE Office of Science Early Career Research Program website.

GSP Early Career Award Winners














Fiscal Year 2022

Synthetic Membrane Biology in Microbial Cell Factories
Itay Budin
, University of California San Diego

Yeasts and bacteria can be engineered to function as cell factories for the production of next-generation biofuels and biochemicals. Like all organisms, microbial cell factories are compartmentalized by cell membranes composed of lipids and proteins. These structures are only a few nanometers thick yet play critical functions during bioproduction. Membranes serve as the molecular microenvironment for the Electron Transport Chain (ETC), which is composed of a number of protein complexes and other molecules that carry out cellular respiration to provide energy to the cell. Therefore, membranes play a key role in controlling the rate of energy-producing reactions in central metabolism. They also act as the primary chemical interfaces with the extracellular environment, regulating the transport and efflux of internally produced molecules. The overall goal of this project is to engineer the structure and properties of cell membranes to improve the performance of industrially relevant microbes. The project’s first objective is to enhance the rate and efficiency of the respiratory metabolism by engineering the organization of the ETC. Engineering efforts will define the limits of respiratory metabolism and seek to increase the production of energy-intensive next generation biofuels. The second objective is to apply the emerging biochemistry of intracellular lipid trafficking pathways to develop new transporters for the capture of valuable biochemicals produced by the engineered yeasts. Soluble transporters will be developed for terpene-based substrates and be applied to develop new efflux pathways for this class of bioproducts. The planned research activities will thus harness fundamental membrane biology to develop novel biotechnologies to advance towards the development of renewable energy production systems.

Investigation of Encapsulin Nanocompartment Systems as a Scaffold for Biomaterials Synthesis in
Mimi C. Yung, Lawrence Livermore National Laboratory

With recent innovations in synthetic biology, genetically engineered microorganisms can now produce a wide variety of bioproducts, including fuels, commodity chemicals, plastics, and inorganic materials, to support a robust bioeconomy. However, challenges such as poor reaction efficiencies and toxicity often limit yields and prevent the production of desired chemicals. One strategy to overcome these challenges is to isolate biosynthetic reactions into protein compartments or cages within engineered microorganisms. Confining biochemical reactions into such cages may help increase reaction yields by bringing the reaction components closer together. At the same time, keeping the desired bioproducts inside these cages can prevent any potential toxic effects of the products, or the conversion of those products into undesired molecules. Some bacteria, such as the emerging model bioproduction bacterium Rhodococcus jostii, naturally produce protein cages, called encapsulin nanocompartments. The goal of this research is to identify mechanisms for engineering compartmentalized biosynthesis in this bacterial strain. This project will develop approaches to control encapsulin production by investigating the native regulation, biosynthesis, and maintenance of the encapsulin system. Gene-editing methods will be used to engineer encapsulins with novel structural properties for expanded bioproduction capabilities. As a case study, the encapsulin system will be used to biosynthesize cadmium sulfate (CdS) nanoparticles, which are semi-conducting materials used in many important optical and electronic applications. Ultimately, this work will establish encapsulin compartmentalization systems as a means of improving yields and enabling new biosynthetic routes toward next-generation bioproducts and biomaterials in support of DOE’s mission to build a strong bioeconomy and thus enhance our energy security.

Fiscal Year 2021

Defining the Influence of Environmental Stress on Bioenergy Feedstocks at Single-Cell Resolution
Benjamin J. Cole
, Lawrence Berkeley National Laboratory

Plant biomass from bioenergy crops is an important resource that enhances energy independence and promotes good environmental stewardship. Environmental stresses such as drought or nutrient deficiency hinder optimal performance of these crops. Therefore, the development of new strategies to improve plant biomass production will require a better understanding of how plants tolerate and respond to environmental stress. Plant responses to drought are complex and involve the coordinated action of many different types of cells with specialized functions. For example, cells that compose stomata (pores in the leaf that open and close to exchange carbon dioxide, oxygen, and water vapor) may respond very differently to drought than cells of the plant vasculature. The objective of this project is to use innovative technologies to measure how individual cells respond to drought and nutrient limitation in two prominent bioenergy crops, sorghum, and switchgrass. This will require the construction of large, curated datasets detailing the regulation of genes in hundreds of thousands of individual plant cells. In addition, the planned research will analyze gene expression under drought and nutrient stress using sophisticated plant growth chambers that closely mimic agricultural field conditions. Lastly, this project will investigate the impact of beneficial soil microorganisms on plant growth under stress. The results of this research will significantly advance our foundational knowledge of how plants coordinate their responses to environmental stresses and will ultimately enable us to target genes in specific cells for crop improvement.

Understanding the Effects of Populus-Mycorrhizal Associations on Plant Productivity and Resistance to Abiotic Stress
Melissa A. Cregger
, Oak Ridge National Laboratory

Harnessing plant–microbial interactions that occur in bioenergy crop plantations provides an opportunity to create sustainable, multipurpose bioeconomies. In these plantations, globally important biofuel feedstocks can be produced while simultaneously maximizing soil health and mitigating adverse impacts on climate. Over the past two decades, it has become increasingly clear that interactions between plants and microorganisms alter the way in which plants grow and respond to environmental stress. These interactions have been coined “plant holobionts,” which are biological units consisting of the plant host plus all of the symbiotic microorganisms associated with the plant. To increase sustainability within biofeedstock plantations, this research is focused on building optimal plant holobionts between biofeedstock trees within the genus Populus and fungi that form symbiotic associations (mycorrhizae) with the trees’ roots. This will be accomplished by identifying high-performing varieties of Populus species and hybrids that are resistant to drought and pairing them with diverse mycorrhizal consortia. This work will examine the ecosystem-level consequences of these assembled Populus holobionts to understand how manipulating those interactions influences nutrient cycling and carbon storage in a Populus tree plantation. Furthermore, this project will establish a unique collection of plant, microbial, and common garden resources that can be leveraged to engineer the next generation of bioenergy crops.

Improving Candidate Gene Discovery by Combining Multiple Genetic Mapping Datasets
Rubén Rellán-Álvarez
, North Carolina State University
Identifying the genes involved in the adaptation of plants to their local habitat and to environmental stresses are essential goals of plant scientists across a range of research fields. Knowledge of such genes helps plant breeders to introduce beneficial traits from wild relatives into high-yielding modern crop varieties. Plant biologists can also use that information to understand the role of genetic variation in plant development, evolution, and stress response. However, it is complicated to validate the function of genes at the molecular and physiological levels and determine the importance of the different variants of a gene responsible for a particular trait. Identifying gene function is further complicated when several genes are responsible for a trait of interest. Reducing the number of candidate genes and making an informed decision on which ones should be validated is particularly challenging. This project’s goal is to understand the impact of phosphorus deficiency and cold stress on sorghum lipid metabolism and develop mathematical approaches that will integrate results from different genome-wide association studies and population genetics indexes of selection. With these approaches, metabolic profiling data from a large number of sorghum lines under phosphorous and cold stress will be combined with analyses of geolocated natural populations adapted to those stresses and measures of genetic differentiation. Combining multiple independent datasets will enable the identification and ranking of candidate genes and metabolic pathways involved in these stress responses. The methods developed as part of this project will be applicable to other potential bioenergy crops and different environmental stresses.

Fiscal Year 2020

Infective Viruses and Inert Virions: Illuminating Abundant Unknowns in Terrestrial Biogeochemical Cycles
Joanne B. Emerson
, University of California, Davis
Soil viruses have been recognized as highly abundant but virtually unknown members of the soil microbiome. By infecting soil microbes, viruses likely have substantial, as-yet unknown impacts on terrestrial biogeochemical processes under their hosts’ control. Viral particles (virions) may also play more direct roles in soil biogeochemical cycling as packets of carbon, nitrogen, and phosphorous, but the time scales and environmental conditions that determine virion infectivity, transport, and/or sorption to soil particles are unknown. This project will use a combination of laboratory and computational approaches to distinguish between infective and degraded virions and assess their respective contributions to soil biogeochemical cycling. Using a multi-omics approach, this research will establish spatiotemporal patterns in soil viral community composition and virus-host dynamics in forests, chaparrals, grasslands, and wetlands. To identify feedbacks between soil viruses and carbon dynamics in additional changing environments, planned research includes field experiments after a high-temperature prescribed forest fire as well as in a boreal forest ecosystem, leveraging the U.S. Department of Energy’s Spruce and Peatland Responses Under Changing Environments (SPRUCE) experiment. By integrating the knowledge gained in the analysis of these different environmental conditions over time, this project will expand the understanding of the global soil virosphere and its influence on global biogeochemical cycles.

Crosstalk: Interkingdom Interactions in the Mycorrhizal Hyphosphere and Ramifications for Soil Carbon Cycling
Erin E. Nuccio
, Lawrence Livermore National Laboratory
Arbuscular mycorrhizal fungi (AMF) are ancient symbionts that form root associations with 80% of the world’s plants. AMF play an important role in global nutrient and carbon cycles, and understanding their biology is crucial to predict how carbon is stored and released from soil. The region of soil surrounding the fungal body (or hyphae) is called the hyphosphere, which is the zone where much of this nutrient and carbon cycling occurs. However, the hyphosphere is one of the least understood components of the plant-mycorrhizal-soil system. It is hypothesized that AMF rely on microbes living in the hyphosphere to access key nutrients from soil organic matter. In fact, synergistic interactions between AMF and soil microbes have been estimated to contribute 70,000 tons of assimilated plant nitrogen annually. This research will investigate the basic mechanisms that underpin these interactions and drive nitrogen and carbon cycling in the hyphosphere, addressing DOE’s mission to understand and predict the roles of microbes in Earth’s nutrient cycles. By coupling isotope-enabled technologies with next-generation DNA sequencing techniques, this project will investigate soil microbial communities in their notoriously heterogeneous natural environments. This work will provide a greater mechanistic understanding needed to determine how mycorrhizal fungi influence organic matter decomposition and will shed light on large-scale nutrient cycling processes in terrestrial ecosystems.

Characterizing Virus-Driven Alterations of Microbial Metabolism in Model Soil Ecosystems
Simon Roux
, Lawrence Berkeley National Laboratory
Soil microbes are globally important, as they shape major nutrient cycles, drive energy conversion processes, and strongly impact terrestrial ecosystems relevant to the U.S. Department of Energy’s mission in energy and the environment. In aquatic ecosystems, viruses are important regulators of microbial physiology. In soils, even though a large diversity of viruses has been discovered, their role and impact on microbial activity remain poorly understood. This research will use a combination of experimental approaches and large-scale data analysis to better characterize how viruses infect soil microbes and affect ecosystem function. The project will characterize the networks connecting soil viruses to their microbial hosts and their associated metabolisms in a comprehensive way. A comparison of arid and humid ecosystems over time will uncover how virus-host interactions are transformed by environmental conditions. The mechanisms by which soil viruses control their host cells and alter their metabolism will be studied in further detail using simplified soil communities in culture, as well as microfluidic devices. Such high-resolution measurements will enable the integration of viral biology into predictive models covering multiple scales, from the single cell to the ecosystem level.

Fiscal Year 2019

Spatiotemporal Mapping of Lignocellulose Decomposition by a Naturally Evolved Fungal Garden Microbial Consortium
Kristin Burnum-Johnson
, Pacific Northwest National Laboratory
Some microbial communities composed of bacteria and fungi can readily breakdown plant matter into its component sugars. Leaf-cutter ants take advantage of such microbial communities by maintaining fungal gardens that release energy-rich carbohydrates from plant biomass. As these decomposition products are released, they are consumed by bacteria that also live in the garden and in turn, further transform those products into nutrients that promote fungal growth. This symbiotic system has great potential for biological production of biofuels and bioproduct precursors from plant biomass. However, the identity of most of the microbial species and their precise role within the fungal gardens is not known. This project will carry out a multi-omics approach to uncover the mechanisms that drive cooperative fungal-bacterial interactions that result in the degradation of lignocellulosic plant material extracted from the fungal garden ecosystem. To understand how the fungal garden is able to degrade plant matter with such efficiency, it is necessary to study the metabolic interactions and biochemical pathways utilized by its microorganisms in each microscopic region of the fungal garden. This research will accomplish that with a novel microscale proteomics approach that can analyze very small samples, providing detailed information on the location and function of fungal and bacterial proteins. This approach will provide the knowledge needed for a predictive systems-level understanding of the fungal-bacterial metabolic and signaling interactions that occur during cellulose deconstruction in an efficient, natural ecosystem and should provide new strategies for generating precursors of advanced biofuels. This knowledge will provide the foundation for developing efficient consortia composed by well-defined and optimized microbial strains to efficiently produce valuable compounds from lignocellulosic feedstocks, advancing DOE’s goal of developing sustainable bioenergy resources.

Systems metabolic Engineering of Novosphingobium aromaticivorans for Lignin Valorization
Josh Michener
, Oak Ridge National Laboratory
In a typical biorefinery, sugars derived from plant material (or biomass) are fermented to fuels by microorganisms. However, a substantial fraction of the plant biomass that contains a polymer called lignin cannot be easily degraded and is instead burned for heat. Lignin could be converted into value-added bioproducts, offering a potential source of additional revenue to improve the economics of biofuel production. Chemical conversion of lignin is challenging, but specialized bacteria with the necessary biochemical capabilities could potentially produce desired compounds from different mixtures of lignin-rich mixtures. Although bacteria that are suited for lignin conversion are known, they have not been extensively studied or manipulated. This project will characterize the biochemical pathways for assimilation of lignin-derived compounds in a bacterium that can metabolize a wide range of such compounds. New pathways will then be engineered into this bacterium to convert depolymerized lignin into valuable bioproducts. To achieve this goal, a novel genetic method will be used to build a predictive systems biology model and identify additional genetic targets for further metabolic optimization. These efforts will result in new methods to predictively model and engineer a promising microbe for lignin valorization that can ultimately be applied to a wide range of emerging microorganisms relevant for BER’s mission in sustainable bioenergy.

Elucidating Aromatic Catabolic Pathways in White-Rot Fungi during Lignin Decay
Davinia Salvachúa Rodríguez
, National Renewable Energy Laboratory
Lignin is a heterogeneous polymer found in the cell walls of terrestrial plants and accounts for 30% of the organic carbon in the biosphere. White-rot fungi are undoubtedly the most efficient lignin-degrading organisms in nature and are thus responsible for a substantial amount of carbon turnover on Earth. Lignin conversion to carbon dioxide and water by these organisms has been studied for decades and is very well accepted. However, the biochemical pathways that allow white-rot fungi to deconstruct and further metabolize lignin remain largely unknown. Indeed, it is still a matter of controversy whether or not these organisms utilize lignin degradation products as a carbon and/or energy source. Furthermore, the chemical units that constitute lignin could be used as precursors of valuable compounds. However, due to its complex nature and the difficulty to break it down into smaller components, lignin is an undervalued substrate for biorefineries that use plant biomass to produce biofuels. This research will apply systems biology and computational modeling approaches to elucidate the metabolic pathways for lignin conversion in white-rot fungi and understand the biological roles of lignin degradation. The knowledge gained through this work will serve as a foundation to employ white-rot fungi in lignin bioconversion into value added bioproducts, advancing towards a sustainable plant-based bioeconomy.

Genetic Tools to Optimize Lignocellulose Conversion in Anaerobic Fungi and Interrogate Their Genomes
Kevin Solomon
, Purdue University
Specialized microbes can convert renewable plant material into biofuels and other bioproducts. However, the microbes currently used for industrial biofuel production cannot break down the plant cell wall material (or plant biomass) into its component sugars unless it is previously partially broken apart with heat and chemicals. Anaerobic fungi that live in the guts of animals that eat plants have the capacity to degrade plant biomass more efficiently than industrial microorganisms and without the need for prior treatment. In spite of the natural advantages of these fungi, they are not used industrially because no genetic tools are available to identify and manipulate the enzymes that constitute the biochemical machinery responsible for their biomass degradation capabilities. This project will develop and leverage high resolution -omics resources to better understand the physiology of anaerobic fungi using systems and synthetic biology approaches. Novel genetic and epigenetic tools will be designed to engineer new strains of anaerobic fungi with improved biomass degradation capacity. In parallel, cell-free strategies will be applied to rapidly characterize fungal proteins and identify improved enzyme functions. With these approaches it will be possible to identify the genetic machinery involved in the breakdown of lignocellulosic plant biomass in these fungi and provide systems-level insight into the genomic basis of biomass deconstruction. Ultimately, this project will help establish design principles to engineer new fungal strains for lignocellulose deconstruction, while creating enabling genetic tools for metabolic engineering of anaerobic fungi. Planned experiments will leverage DOE genomics, molecular, and computational analysis resources at the Joint Genome Institute, the Environmental Molecular Sciences Laboratory, and the Systems Biology Knowledgebase. The knowledge gained from this project will enable predictive biology and genome engineering in anaerobic fungi, advancing BER’s mission in the development of sustainable bioenergy resources.

Fiscal Year 2018

Genome-Scale In Vivo Determination of Gibbs Free Energies (ΔG) in Metabolic Networks
Daniel Amador-Noguez
, University of Wisconsin-Madison
Recent breakthroughs in genome editing and metabolic engineering are expected to expand the range of microorganisms and synthesis routes that may be used to produce biofuels and valuable bioproducts from renewable biomass resources. Thus, there is an increasing need for new tools to characterize the metabolic capabilities of nascent industrial organisms and improve the efficiency of their production pathways. Thermodynamic analysis can help us understand how energy is transferred and transformed within metabolic networks and has emerged as a powerful tool for pathway design and metabolic engineering. This project will integrate thermodynamic analysis with advanced mass spectrometry, computational modeling, and metabolic engineering to develop an approach for in vivo determination of Gibbs free energies (ΔG) in metabolic networks. This project will also investigate how the thermodynamics of biosynthetic pathways in microbial biofuel producers change dynamically as substrates are depleted or products accumulate. This research will result in the construction of computational models that quantitatively define trade-offs between energy efficiency of biosynthetic pathways and their overall catalytic rates. The approach developed in this project will be useful for identifying thermodynamic bottlenecks in native and synthetic pathways and pinpoint the enzymes whose expression levels will have the largest effect on production rates and final product yields. It will be suitable for high-throughput analysis of a wide range of organisms and aid the design of new and more efficient metabolic routes for advanced biofuel production.

Enabling Predictive Metabolic Modeling of Diurnal Growth Using a Multi-Scale Multi-Paradigm Approach
Nanette Boyle
, Colorado School of Mines
Although photosynthetic microorganisms such as algae and cyanobacteria have great potential as renewable sources of energy and valuable chemicals, a lack of genetic engineering tools has prevented their use as biofactories. Furthermore, most research on these organisms is conducted with cells grown in constant light, which does not replicate the conditions of outdoor ponds with natural day/night cycles. By integrating a variety of mathematical modeling techniques and experimental data, this research will develop an advanced predictive model of growth and productivity in photosynthetic organisms, capturing the changes in metabolism during day/night cycles, interactions between cells, and the availability of nutrients and light in the environment. The project will focus on the emerging model green algae Chromochloris zofingiensis, which accumulates large amounts of lipids that can be used to synthesize biodiesel and other high-value added products. The performance of the model will be tested using data collected from large-scale outdoor algal growth studies. The model generated in this project will not only allow more predictive computer simulations of algal physiology, but it will also enable advanced and safe engineering of photosynthetic microorganisms for optimal growth in outdoor ponds. The model will be easily adaptable for other organisms as well as more complex microbial consortia, supporting BER’s mission in energy and the environment.

Building a Comprehensive Understanding of Ice Nuclei Sources from the Ground Up: Establishing the Impact of Sea Spray and Agricultural Soils
Susannah Burrows
, Pacific Northwest National Laboratory
Prediction of ice crystal concentrations in clouds is an outstanding problem in atmospheric sciences. Among the obstacles to achieving this predictive understanding is the challenge of predicting atmospheric ice-nucleating particles (INPs) across a range of atmospheric conditions. INPs play a critical role in initiating freezing in clouds, impacting cloud radiative properties, and the location and timing of precipitation. Despite their importance, our understanding of the emission, removal, and atmospheric transformation processes controlling the atmospheric abundance of INPs is still in its infancy. Immersion-freezing INPs active at warmer temperatures (> ca. -25°C) are especially critical to mixed-phase clouds, i.e., those that contain both liquid droplets and ice crystals. Their abundance is often controlled by biogenic particles (e.g., organic matter in soils and sea spray), which are challenging to distinguish observationally and to represent in models. This project will transform predictive understanding of the sources of ice-nucleating particles to the atmosphere by advancing understanding of how two less-studied but important particle types contribute to determining real-world INP concentrations, and tackling the grand challenge of predicting INP concentration from the observed characteristics and sources of atmospheric particles. This project will use unique field campaign data from the Atmospheric Radiation Measurement (ARM) user facility and measurement capabilities from DOE’s Environmental and Molecular Sciences Laboratory (EMSL) to take on the grand challenge of predicting INP abundance, with a focus on two less well studied classes of immersion-freezing nuclei active at T > -25°C—sea spray and agricultural soils. The improved process-level understanding developed in this project can ultimately be incorporated into atmospheric models, increasing their process realism and their utility as a tool for understanding complex surface-atmosphere-cloud interactions in the Earth System.

Implications of Aerosol Physicochemical Properties Including Ice Nucleation at ARM Mega Sites for Improved Understanding of Microphysical Atmospheric Cloud Processes
Naruki Hiranuma
, West Texas A&M University
A specific subset of atmospheric particles can act as ice-nucleating particles (INPs) in mixed-phase clouds and, ultimately, influence precipitation and the Earth’s radiative energy balance. Despite the importance of INPs, current ambient INP data derived from field measurements are not well interpreted with detailed aerosol and cloud properties, except for a few short-term field studies. This project will fill this gap by using long-term measurements from DOE’s Atmospheric Radiation Measurement (ARM) sites, complemented with robust and well-characterized INP measurements that will be archived in the ARM database. Detailed ARM observational data of aerosol chemical composition speciation, abundance, cloud condensation nuclei activity, and hygroscopicity are of the utmost importance for better understanding of INP mixing states, as well as their implication in cloud, precipitation, and regional weather patterns. The proposed new INP measurements will experimentally characterize abundance and physicochemical properties of ambient INPs at the ARM Southern Great Plains (SGP), Eastern North Atlantic (ENA), and North Slope of Alaska (NSA) atmospheric observatories. A combination of a new in situ expansion chamber and the offline droplet freezing assay technique for INP measurement, as well as microspectroscopic characterization techniques, will be used to elucidate abundance and physicochemical properties of ambient INPs at the above-mentioned ARM observational sites. Different INP episodes (agricultural, marine biogenic, and Arctic at SGP, ENA, and NSA, respectively) will be assessed and evaluated to help understand convective and mixed-phase cloud systems typically observed in these regions. The proposed research will generate data to understand how particle chemical composition and mixing state influence ambient ice nucleation propensity at the ARM sites. Such datasets have long been a missing piece in the study of cloud microphysics and atmospheric chemistry, and are of importance to improve atmospheric models of cloud feedback and to determine their impact on the global radiative energy budget. Currently, ice formation processes are very poorly represented in weather and earth system models, including DOE’s Energy Exascale Earth System Model (E3SM), and this study will support the DOE mission by providing INP parameterizations representative of the ARM sites. To constrain E3SM, this project will produce a variety of INP parameterizations, such as ice nucleation active surface site density, cumulative number concentration of INPs per volume of air, and water activity-based freezing descriptions.

O-Acetylation and Methylation Engineering of Plant Cell Walls for Enhanced Biofuel Production
Kolby Jardine
, Lawrence Berkeley National Laboratory
Polysaccharides are major components of plant cell walls that can be converted into fuels by microbial fermentation, making plant biomass an important bioenergy resource. However, a substantial fraction of plant cell wall polysaccharides is chemically modified with methyl and acetyl groups that reduce yield of microbial fermentation. Although little is known about the biochemical and physiological functions of those cell wall modifications, it has been shown that their volatile intermediates (methanol and acetic acid) are tightly associated with plant growth, stress, and senescence processes but are not captured by traditional metabolomics analysis, representing an important gap in our knowledge of cell wall metabolism. This project will study the metabolism of those cell wall modifications and volatile intermediates as well as their role in central physiological processes in the emerging biofuel tree species California poplar (Populus trichocarpa) using field settings and controlled environmental conditions. The main goal of this research is to modify the expression of key genes involved in cell wall metabolism in order to reduce the amount of methyl and acetyl groups present on cell walls. These genetic modifications will be evaluated for potential impacts on important plant hydraulic and physiological processes including proper functioning of vascular tissues to support transpiration, leaf water potential and stomatal regulation, net photosynthesis, and high temperature/drought stress responses. Understanding and manipulating the metabolism of cell wall modifications will not only provide important knowledge on the physiology and ecology of plants but will also allow the generation of engineered bioenergy crops such as poplar for sustainable production of biofuels and bioproducts, addressing BER’s goal of developing renewable bioenergy resources.

Elucidating Processes Controlling Arctic Atmospheric Aerosol Sources, Aging, and Mixing States
Kerri A. Pratt,
University of Michigan
The objective of this project is to determine aerosol chemical composition, sources, mixing states, and aging processes across the entire annual cycle in the high Arctic, and in the Alaskan Arctic during fall – winter, to address the most significant gaps in Arctic aerosol observational data. The proposed project is based on the rapid sea ice loss across the entire Arctic, as well as the major delays in sea ice freeze-up in the Chukchi Sea, off the North Slope of Alaska (NSA). The project will: 1) identify the sources of Arctic aerosols as a function of season, 2) determine the mixing states and aging extents of Arctic aerosols as a function of season, and 3) ascertain the most important factors (e.g., sea ice extent, radiation, meteorology) modulating the sources, chemical composition, mixing states, and aging processes of Arctic aerosols. The proposed study will focus on analysis and interpretation of Atmospheric Radiation Measurement (ARM) field campaign samples and data collected during late fall/early winter at Utqiagvik, AK and during the year-long international Multidisciplinary drifting Observatory for the Study of Arctic Climate (MOSAiC) in the high Arctic. The project will use state of the art measurement techniques including real-time aerosol time of- flight mass spectrometry (ATOFMS), on-line aerosol sizing, and off-line computer-controlled scanning electron microscopy with energy-dispersive X-ray (CCSEM-EDX) spectroscopy. The project will provide an unparalleled opportunity to study seasonal changes in aerosol processes in the high Arctic. Size-resolved number fractions of observed individual particle sources will be determined for each month, with quantitation of locally emitted vs. long-range transported aerosols. Number fractions of aerosols internally mixed with various secondary species will be determined, and aerosol mixing state indices will be calculated. Sea ice extent and fracturing impacts on sea spray aerosol will be examined. For predictions of Arctic atmospheric composition and feedbacks, knowledge of aerosol sources, mixing states, and aging processes is critical and is a significant current gap in our understanding of Arctic aerosols. The proposed project will provide unprecedented and critical knowledge of Arctic aerosol mixing states and processes. The overarching impact of the project will be the generation of Arctic aerosol observational data and improved understanding of Arctic aerosol processes to inform and evaluate future simulations of Arctic atmospheric composition and climate.

Finding Missing Links Associated with Aerosol-Cloud Interactions: Aqueous and Cloud-Phase Secondary Organic Aerosol Formation
ManishKumar Shrivastava
, Pacific Northwest National Laboratory
Interactions between clouds and aerosols play a major role in Earth’s energy budget and are among the largest uncertainties in projections of future Earth-system changes. Cloud-aerosol interactions are manifestations of many nonlinear processes governing the number, size, and chemical composition of aerosols that serve as cloud condensation nuclei. Aqueous- and cloud-chemistry pathways that lead to the formation of secondary organic aerosol (SOA) are some of the least understood of these processes but could be associated with significant regional-scale heterogeneities in Earth’s aerosol, cloud, and radiation fields. To date, representations of aqueous- and cloud-phase organic chemistry in Earth-system models have been overly simplistic, making it difficult to diagnose how variations in the cloud, chemistry, and meteorological regimes in the atmosphere affect aerosol-cloud interactions and radiation. Although recent field campaigns and laboratory experiments provide a wealth of measurements, there is a persistent gap in terms of coherently connecting these measurements to advance our understanding of aqueous- and cloud chemistry pathways of SOA. The vision of this U.S. Department of Energy (DOE) Office of Biological and Environmental Research (BER) Early Career Project is to advance the fundamental understanding of SOA formed within aqueous aerosols and cloud droplets, thus providing a complete description of coupling between cloud-aerosol interactions and chemistry. This will be achieved through a multipronged approach that integrates 1) analyses of laboratory measurements and multiple field measurements of aerosols, clouds, radiation, and the dynamic and thermodynamic structure of the atmosphere and 2) observationally constrained high-resolution modeling approaches, which will be used as analytical tools to assess processes governing the formation of SOA through aqueous- and cloud-chemistry. Many of the measurements used in this research come from BER’s Atmospheric Radiation Measurement (ARM) user facility and complementary data sets from both the Amazon and the Southern Great Plains (SGP) sites. The proposed data analyses will elucidate new aqueous SOA processes, determine the role of aqueous chemistry in the formation and growth of nanoparticles in the atmosphere, and establish parameters needed to improve the predictive ability of models. Aqueous SOA processes determined from analyses of field and laboratory measurements will be incorporated in a high-resolution regional model to investigate how these processes are manifested in the chemical and dynamic evolution of SOA in the atmosphere. Additionally, large eddy simulations will be conducted to investigate how subgrid-scale aqueous SOA chemistry processes and cloud-aerosol cycling affect aerosol-cloud interactions. This work will lead to groundbreaking new insights and discoveries that can be used to improve large-scale, Earth-system models and inform further research on aqueous- and cloud-phase SOA formation. A solid understanding of the coupling between atmospheric aqueous SOA chemistry and clouds is critical to advance our understanding of Earth’s energy budget and to address some of the most critical scientific questions facing the nation and DOE.

Improved Biofuel Production through Discovery and Engineering of Terpene Metabolism in Switchgrass
Philipp Zerbe
, University of California, Davis
Plants employ complex and often species-specific networks of small molecules to cope with environmental challenges as well as to communicate with other organisms. In many major crops, a diverse group of metabolites, called terpenes, function as key biochemical mechanisms to respond to different stresses and are essential to agricultural productivity. As terpenes are also used as precursors for renewable biodiesel and aviation fuels, a better understanding of their biochemical networks can be used to generate stress-tolerant crops with enhanced potential for biofuel production. This project will investigate and harness terpene-mediated stress defense mechanisms in switchgrass (Panicum virgatum), which is a valuable bioenergy crop due to its high net energy efficiency. Genetic, genomic, biochemical, and protein structural analyses will be used to gain detailed knowledge of the biosynthetic machinery controlling the switchgrass terpene network. Plant-microbe interactions will also be studied to better understand the role of terpenes in the plant’s response to stresses such as drought. The knowledge thus obtained on the biological function of terpenes will be applied to carry out advanced genome engineering of switchgrass and develop varieties with improved stress resilience and tailored terpene blends for advanced biofuel production on marginal land, advancing BER’s objective of developing sustainable and renewable bioenergy resources that do not compete with food agriculture.

Fiscal Year 2017

Microbial Environmental Feedbacks and the Evolution of Soil Organic Matter
Nicholas J. Bouskill, Lawrence Berkeley National Laboratory
The vast majority of Earth’s organic matter is stored in soil. The products of microbial metabolism as well as dead microbes (necromass), along with residues from plants and other organisms at different stages of decomposition, constitute a large fraction of that soil organic matter (SOM). The ability of microbes to modify and degrade SOM depends on physicochemical characteristics of the soil, affecting SOM stability and persistence. While the contributions of microbes to the decomposition and loss of SOM have been intensively studied, their role in maintaining the terrestrial SOM is poorly understood. Specifically, how fungi, bacteria, and archaea participate in the production of SOM, the interaction between SOM and minerals, and the formation of soil aggregates remain significant gaps in the understanding of the terrestrial nutrient cycle. The chemical composition of SOM is largely determined by soil bacterial metabolism, which is impacted by changes in rainfall patterns. This research will conduct field and laboratory experiments and computational modeling to understand the role of microbial communities in stabilizing SOM under different water availability conditions in tropical soils. The results of this project will increase understanding of the effects that microbes have on the global geochemical and nutrient cycles, addressing DOE’s mission in energy and the environment.

Awakening the Sleeping Giant: Multi-Omics Enabled Quantification of Microbial Controls on Biogeochemical Cycles in Permafrost Ecosystems
Neslihan Taş Baas, Lawrence Berkeley National Laboratory
Large expanses of permanently frozen soils, called permafrost, are found in the Earth’s polar regions. Arctic soils store large amounts of biomass and water from warmer periods in the history of Earth that became preserved in permafrost during cooling and glaciation events. Permafrost soils contain a broad diversity of cold-adapted microbes, whose metabolic activity depends on environmental factors such as temperature changes that cause cycles of freezing and thawing in the soil. Microbial metabolism leads to decomposition of soil organic matter, substantially impacting the cycling of nutrients and significantly affecting the Arctic landscape. However, the relationship between permafrost microbial properties and biogeochemical cycles is poorly understood. This project will use field experiments, laboratory manipulations, and multi-omics approaches to examine how microbial processes, biogeochemical transformations, and hydrology interact during permafrost thaw in different sites in Alaska to determine how these factors drive biogeochemical cycles in different Arctic soils. This project will lead to an in-depth understanding of the underlying microbial processes governing biogeochemical cycles in an environment relevant to DOE’s mission.

Determining the Genetic and Environmental Factors Underlying Mutualism within a Plant-Microbiome System Driving Nutrient Acquisition and Exchange
David J. Weston, Oak Ridge National Laboratory
The importance of symbiosis is highlighted in plant-microbe interactions where a microbe can acquire nitrogen from the air (nitrogen fixation) and provide it to the plant in exchange for sugars necessary for growth and metabolism. However, such beneficial interactions can shift to commensal (neutral) or even antagonistic, depending on genetic and environmental factors that are poorly understood. This project will provide a fundamental quantitative understanding on the role of plant host and microbial genetics on maintaining beneficial symbiosis during environmental perturbations. With that fundamental understanding, it will be possible to select host and microbes with the appropriate genetic makeup to manipulate symbiotic relationships adapted to different environmental conditions. The study systems will be a community composed of the moss Sphagnum and nitrogen-fixing cyanobacteria because of the genomic resources available for these organisms and their suitability for advanced genomic and imaging technologies. This effort will identify the genes and metabolic functions involved in nutrient exchange between the interacting plants and microbes and determine how symbiotic systems respond to environmental perturbations in laboratory and field settings. Ultimately, fundamental knowledge of the genetic and environmental factors driving plant and microbial nutrient exchange will enhance the understanding of nutrient cycling in natural systems and provide the foundation to improve bioenergy crop productivity in more complex biological communities.

Genomes to Ecosystem Function: Targeting Critical Knowledge Gaps in Methanogenesis and Translation to Updated Global Biogeochemical Models
Kelly C. Wrighton, The Ohio State University
Natural freshwater temperate wetland systems currently represent the largest natural source of atmospheric methane but are relatively understudied using systems biology tools (e.g., meta-omics) compared to other high-producing methane systems (e.g. peat, tropical, or reconstructed wetlands). Using field investigations at the NOAA-operated sentinel site on Lake Erie, methane-producing activities and responses to geochemical conditions will be determined along seasonal and spatial gradients (Objective 1). Here, a combination of high-throughput activity and gas measurements, combined with high-resolution systems biology and analytical methods, will provide in-depth knowledge of the microbiological, chemical, and physical constraints on methane production in wetlands. Using laboratory microcosms, the formation of anoxic microsites and their capacity to facilitate methane production in wetland soils will be simulated (Objective 2). This objective will validate the findings from the field investigations, offering a more controlled environment for teasing out the role of different, yet interrelated, variables. Lastly, these field and laboratory data will be used for multiscale, process-level evaluation of an ecosystem biogeochemical model that accommodates these newly identified processes and parameterizes representation of these processes along relevant environmental gradients (Objective 3). This research will identify multiple interacting geochemical, ecological, and metabolic constraints that are poorly understood, oversimplified, or missing in global biogeochemical methane models. This proposal targets the role of oxygen limitation on methane processes in soil domains to improve reactive transport models of microbial carbon cycling across terrestrial-aquatic soils and generate data on nutrient cycling activities in Great Lake wetlands. This information could provide new insights into the microbial controllers of Lake Erie eutrophication.

Fiscal Year 2016

Molecular Interactions of the Plant-Soil-Microbe Continuum of Bioenergy Ecosystems
Kirsten S. Hofmockel, Pacific Northwest National Laboratory
The accumulation and stabilization of organic matter in soil are important for the global carbon cycle because organic matter contributes to soil fertility and helps reduce the release of the greenhouse gas carbon dioxide into the atmosphere. A better understanding of the processes related to soil carbon accumulation is critical for designing strategies to increase soil carbon storage. Emerging experimental and theoretical evidence suggests that the residues of dead soil microbes play an important role in increasing the stabilization and long-term storage of carbon in soil. This project will study the deposition of dead microbial cells on different mineral surfaces and its effects on long-term carbon stabilization in soils used for both annual and perennial bioenergy crops. This research will identify the metabolic pathways and chemical components of microbes that contribute to soil carbon accumulation under controlled laboratory conditions. Field experiments will also be conducted to characterize the accumulation of microbial cells in response to crop selection and soil characteristics. The experimental data will be used to develop models of carbon cycling in bioenergy cropping systems under different soil conditions. These models will generate new knowledge on beneficial plant-microbe-soil interactions that increase carbon storage in biofuel agroecosystems. As new marginal lands are cleared and greater quantities of biomass are harvested, this project will provide the basic science needed to develop sustainable biofuel feedstocks to ensure healthy soils and promote a low carbon-economy outcome.

Spatially Resolved Rhizosphere Function: Elucidating Key Controls on Nutrient Interactions
James J. Moran, Pacific Northwest National Laboratory
Microbes play a key role in providing nutrients to plants. A better understanding of plant-microbe interactions is thus important for ensuring sustainable biofuel production from plant feedstocks in the face of a changing climate. Plants acquire their nutrients from the soil around their roots (the rhizosphere) through a process controlled by a dynamic suite of biogeochemical cycles. These cycles facilitate nutrient exchange among the soil, microbes, and plant roots through the rhizosphere interface. There is spatial heterogeneity in both microbial activity and nutrient accessibility throughout this interface. This project will improve the understanding of the spatial controls on rhizosphere nutrient exchange, identify key microbial functions involved in nutrient exchange, and test whether nutrient amendments to the soil can be used to stimulate plant-microbe interactions in spots of high activity (i.e., hotspots) within the rhizosphere to increase plant biomass productivity. Central to this study is the use of a series of spatially resolved techniques to pinpoint specific locations within the rhizosphere where enhanced nutrient exchange between roots and soil organisms occurs. How these nutrient exchange hotspots are generated will be characterized through elemental and functional analyses of the rhizosphere. Fundamental understanding of these crossroads of nutrient exchange at the spatial scale of the rhizosphere will form a knowledge framework for directed manipulations of these complex, yet vitally important, nutrient conduits. Ultimately, effective management of rhizosphere processes will enable enhanced plant nutrient acquisition from marginal lands, thereby contributing to improved biofuel feedstock productivity with lower chemical inputs.

Host-Microbial Genetic Features Mediating Symbiotic Interactions in the Bioenergy Crop Salix
Wellington Muchero, Oak Ridge National Laboratory
Many microbes present in the soil surrounding plant roots (the rhizosphere) can be beneficial for the plant, promoting growth and the incorporation of carbon dioxide into the plant biomass. Yet, only 10% of the microbes in the rhizosphere are able to establish a beneficial interaction with plant hosts due to defense mechanisms that evolved in plants to protect them from microbial infections. Those defense mechanisms pose a fundamental challenge in the utilization of symbiotic microbes to enhance sequestration of carbon dioxide, a potent greenhouse gas, and its fixation into economically valuable plant feedstocks. In compatible plant-microbe interactions, biomass increases of up to 200% have been achieved in perennial feedstocks inoculated with growth-promoting symbiotic microbes. However, compatible interactions are largely host specific, thereby limiting application across diverse plant species. Using Salix (i.e., willow), a widely used biofuel feedstock and pioneer species with increasing presence in the warming arctic region, this project will identify and characterize unique host-derived genetic factors that allow select microbes to successfully evade defense mechanisms and establish a functional presence inside the plant with no adverse effects. The plant cell surface contains proteins called membrane-bound pattern recognition receptors (PRRs) whose function is to recognize microbes with high fidelity through their microbe-associated molecular patterns (MAMPs). Upon recognition of MAMPs, PRRs trigger a signaling cascade that results in the suppression of host defense mechanisms and facilitates plant colonization by the microbe. Understanding these molecular dynamics presents a unique opportunity to couple new growth-promoting microbes with willow to increase carbon sequestration in the vulnerable arctic region. This project will advance DOE’s missions in energy and the environment by increasing plant biomass yields for the sustainable production of cellulosic biofuels.

Does Mycorrhizal Symbiosis Determine the Climate Niche for Populus as a Bioenergy Feedstock?
Kabir G. Peay, Stanford University
Microbes are found in virtually every environment on Earth, and many of them play beneficial roles, maintaining the health of plants and animals. Perhaps the most ubiquitous form of beneficial interaction in terrestrial ecosystems occurs between fungi and plant roots. In these fungus-root (or “mycorrhizal”) symbioses, the plant provides sugars that feed the fungus that in turn supplies the plant with critical nutrients such as nitrogen and phosphorous. Most plants are associated with a diverse variety of mycorrhizal fungi. However, the ecological factors that control the distribution and abundance of mycorrhizal symbioses are still poorly understood. To advance toward understanding the role of climate, soil environment, and mycorrhizal interactions in determining growth and competition in plant communities, this project will focus on Populus, a native North American tree and a potential biofuel feedstock, and the mycorrhizal fungi associated with it. Using a global forest database, the distribution of different Populus-mycorrhizal associations will be mapped and modeled across different regions and climates. Based on those models, laboratory experiments will be conducted to measure the precise ways in which beneficial plant-mycorrhizal interactions determine the distribution of Populus in its natural habitat and how these interactions affect competition with other tree species. Finally, the flow of carbon from Populus to mycorrhizal fungi and other soil microbes will be studied in different environmental conditions using stable isotope labeling. These experiments will not only provide fundamental insights into the way beneficial interactions shape the natural world, they will also allow the prediction of how carbon flow is affected by climate change. The knowledge gained in this project will have a direct impact on predicting the suitability of different environments for bioenergy crops.

Fiscal Year 2015

Functional Characterization and Regulatory Modeling of Lignocellulose Deconstruction in the Saprophytic Bacterium Cellvibrio japonicus
Jeffrey G. Gardner, University of Maryland-Baltimore County
The degradation of plant biomass (lignocellulose) is a critical component of global carbon cycling and renewable energy production. Every year, microorganisms in the environment degrade 100 billion tons of plant biomass. These microorganisms have found effective ways to completely break down plant biomass and use it for energy. This process is very efficient because the microorganisms in the environment produce large numbers of enzymes that can degrade lignocellulose. While some of the mechanisms of lignocellulose breakdown are understood, less is known about which enzymes are essential to plant biomass degradation and how their production is regulated. There is little understanding of how plant biomass degradation is regulated in bacteria because it is not known how these microorganisms are able to detect plant biomass as a nutrient source. This project will use the plant biomass-degrading bacterium Cellvibrio japonicus to address these questions. Using next-generation DNA sequencing technologies, this research will determine which enzymes are highly produced when C. japonicus is degrading lignocellulose. This information will direct additional experiments to identify the essential enzymes for plant biomass degradation and will compare the enzymes that C. japonicus produces to those currently used for biofuel production. Finally, by understanding how C. japonicus effectively degrades plant biomass, this project will create a model of how microorganisms in the environment are able to detect lignocellulose as a nutrient. The knowledge obtained from this research will help develop biotechnology strategies to enhance the economical production of biofuels.

Defining the Minimal Set of Microbial Genes Required for Valorization of Lignin Biomass
Elizabeth S. Sattely, Stanford University
As the world population surpasses 7 billion, science and engineering are faced with the pressing challenge of creating technology to shift reliance on petroleum resources to renewable feedstocks for the production of liquid fuels and platform chemicals. A primary candidate feedstock is plant biomass, where most current efforts have focused on converting cellulosic sugars to biofuels, replacement petrochemicals, and novel renewable materials. However, the other major fraction of plant biomass is lignin, a hydrocarbon-rich biopolymer left over after cellulose is used to make ethanol and other liquid biofuels. Lignin is the second most abundant biopolymer on Earth and represents a critically underutilized renewable resource that could be a major feedstock for future biorefineries. Unfortunately, without sufficient tools to convert lignin into its simple aromatic components, valuable compounds from this abundant biopolymer cannot be generated; instead, lignin is typically burned for thermal energy. This project will use a novel approach to identify the minimal set of microbial enzymes necessary for the synthesis of valuable chemicals from lignin as a byproduct of biofuel production from biomass. This research will examine two separate stages of lignin breakdown carried out by the microbes that do it best: (1) early breakdown of lignin into soluble fragments by wood-rotting fungi and (2) further conversion of those lignin fragments into useful chemicals performed by specific soil microbes. The initial goal of the project is the discovery and biochemical characterization of the enzymes required for lignin metabolism. The fungal and bacterial genes that code for those enzymes then will be used to engineer a microbial host that will efficiently convert lignin waste streams directly into valuable platform chemicals. This effort will leverage DOE investments in microbial genome analysis and secure a critical channel for lignin biomass utilization that will also help to render lignocellulosic biomass a viable feedstock for the production of renewable liquid biofuels.

Fiscal Year 2014

Understanding Microbial Carbon Cycling in Soils Using Novel Metabolomics Approaches
Trent R. Northen, Lawrence Berkeley National Laboratory
To predict and mitigate the adverse effects of climate change, improved understanding of carbon cycling in soils is urgently needed. Carbon is accumulated in soils as decayed plant matter and chemically transformed by the metabolism of microorganisms that live in the ground. The products (metabolites) of these transformations carried out by microbes make up a large fraction of the soil carbon. While very little is known about the metabolite composition of soils, much is known about the types of microorganisms found in soils. This is a result of significant efforts to study soil microbes using DNA sequencing technologies. Unfortunately lacking, however, are the vital data needed to enable scientists to link this sequence information to the microbial metabolic transformations that govern carbon cycling in soils. The project will help bridge this gap by resolving the current “black box” of soil metabolites and develop approaches to understand how specific microorganisms produce and transform the soil metabolite pools. This will be achieved by pioneering analytical technologies to identify and quantify soil metabolites. These technologies will be used to characterize the cascades of microbial activities that follow wetting of dry soils to correlate soil metabolite composition and microorganisms’ activities. Detailed methods will then be developed to determine the uptake and release of specific soil metabolites by key soil bacteria to make and test predictions of carbon cycling based on DNA sequence data. This program will provide an urgently needed complement to DNA sequencing that will enable the understanding and mathematical modeling of soil carbon cycling, ultimately improving the ability to predict and mitigate the effects of climate change.

Microbial Carbon Transformations in Wet Tropical Soils: The Importance of Redox Fluctuations
Jennifer Pett-Ridge, Lawrence Livermore National Laboratory
Tropical forest soils store more carbon—in the form of plant litter and decomposed organic matter—than any other terrestrial ecosystem and play a critical role in the production of greenhouse gases (e.g., methane, nitrous oxide, carbon dioxide) that affect both atmospheric chemistry and climate. Humid tropical forests also exchange vast amounts of carbon, water, and energy with the atmosphere and can lose large amounts of dissolved carbon via runoff and leaching. The rapid carbon cycling characteristic of wet tropical ecosystems is driven in part by high rainfall and warm temperatures. This combination of environmental conditions causes tropical soils to alternate between oxygenated and anaerobic conditions and affects the behavior of tropical soil microorganisms that regulate many aspects of the belowground carbon cycle. In the coming half century, tropical forests are predicted to see a 2 to 5 degree Celsius temperature increase and substantial differences in the amount and timing of rainfall. Although the importance of tropical soils to the global carbon cycle is clear, the current understanding of how soil carbon cycling in wet tropical forests will respond to climate change is surprisingly poor. This makes predicting future climate impacts extremely difficult. The ability to forecast how new moisture and temperature patterns will shape tropical microbial activity is also a gap in knowledge because so little is known about the fundamental abilities and chemical preferences of tropical soil microorganisms. If wet tropical forests experience shifts in rainfall patterns, becoming generally drier and more aerated, microbially mediated processes that produce greenhouse gases or help store soil carbon will likely be affected. Only a few studies of microbial diversity have been conducted in wet tropical soils, and only a handful of them have evaluated microbial function with modern DNA sequencing technologies. This project will examine the genomic content and potential of tropical soil microorganisms as they experience shifts in soil temperature, moisture, and oxygen availability. By also tracking the degradation and fate of organic carbon compounds, this work will increase the accuracy of predictions about how microbial processes affect whether organic carbon is retained or lost from tropical systems. The mechanistic understanding produced by this research will directly benefit attempts to improve the predictive capacity of mathematical models that forecast future tropical soil carbon balance.

Fiscal Year 2013

Extreme Expression of Cellulases in Poplar
Heather D. Coleman, Syracuse University
Cellulose, the major component of plant cell walls, is composed of long chains of sugars linked together. Plant cellulosic biomass (stalks, trunks, stems, and leaves) provides a vast untapped source of sugars that can be fermented to produce biofuels. Sugars are extracted from biomass using enzymes (cellulases) that break down cellulose. However, a major roadblock to developing an economically viable cellulosic biofuel production process is the cost of those enzymes, typically produced and purified using bacteria or fungi. An alternative and potentially more economic approach is to produce the enzymes within the plant itself. The goal of this research is to implement a new genetic engineering technology to produce large amounts of exogenous cellulases into poplar cells. This technology allows the researcher to control the production of the enzymes within the plant using an inducer substance that triggers the rapid accumulation of the cellulases. The transformation of poplar trees using this approach will increase the efficiency of converting cellulose to fermentable sugars and will increase the understanding of plant cell walls. Furthermore, the cellulases produced by these transformed trees could be purified in a cost-effective way, paving the way for developing a sustainable alternative for the production of biofuels from woody feedstocks.

Developing Synthetic Biology Tools to Engineer Plant Root Systems and Improve Biomass Yield and Carbon Sequestration
Dominique Loque, Lawrence Berkeley National Laboratory
Dedicated crops for bioenergy production must be grown in marginal environments to avoid competition with food crops that are cultivated in high-quality arable land. However, nutrient and water availability is very low in these marginal environments. Therefore, energy crops must be engineered to improve their ability to extract those vital elements from poor soils so that they can reach their full yield potential without the cost and environmental impact of chemical fertilization. The root system not only anchors a plant to the ground but is responsible for acquiring essential mineral nutrients and water and for maintaining interactions with the soil environment, all critical for plant growth. Despite their importance for biomass accumulation, plant roots are relatively understudied and few engineering tools area available to better understand and improve root function. This project will address this need by developing “universal” root expression tools that are functional across a broad range of plant species. These tools will be used to engineer metabolic pathways that will be designed to optimize nutrient acquisition by energy crops such as switchgrass and Camelina. This research will deliver a diversity of building blocks for plant root engineering that will be instrumental in advancing DOE goals for sustainable production of bioenergy.

Engineering Anaerobic Gut Fungi for Lignocellulose Breakdown
Michelle A. O’Malley, University of California, Santa Barbara
Renewable biofuels derived from plant biomass (stems, stocks, and leaves, mainly composed of cellulose and lignin) are attractive alternatives to petroleum-based fuels. To produce biofuels, enzymes are used to break down cellulose into simple sugars, which are then fermented into fuels such as ethanol and butanol. However, because the structure of cellulose is a tightly bound network of crystalline cellulosic fibers and lignin, existing biomass-degrading enzymes are not very efficient. New technologies to break down plant material into sugar can be developed by studying how microbes digest lignocellulose in biomass-rich environments, such as the digestive tract of large herbivores. Anaerobic fungi that live in the absence of oxygen and are native to the gut and rumen of these animals have evolved powerful enzymes to degrade plant biomass. This project will develop new experimental tools to engineer anaerobic fungi for lignocellulose breakdown and biofuel production. To accomplish this goal, a panel of anaerobic fungi will be isolated from different herbivores and screened for their ability to degrade several types of lignin-rich grasses and agricultural waste. Focusing on a model anaerobic fungus, the basic metabolic processes that control enzyme production will be determined. This information will be used to develop new genetic engineering strategies to manipulate gut fungi at the molecular level. Understanding the biology of these anaerobic organisms will result in the development novel platforms for biofuel production.

Application of Next-Generation Sequencing to Engineering mRNA Turnover in Cyanobacteria
Brian F. Pfleger, University of Wisconsin-Madison
The ability to control gene expression in microorganisms is essential for biotechnology applications such as renewable fuel production and carbon dioxide fixation. To carry out their function, genes coded in the DNA must be transcribed into a messenger RNA (mRNA), which is translated into a protein with biological activity. While the processes of transcription and translation are well understood in many organisms, much less is known about the stability of the mRNA and how its lifespan affects gene expression. Thus, genetic engineering tools used to express foreign genes in microbes rarely consider key factors that influence mRNA stability. The goal of this project is to fill this gap in knowledge for a model photosynthetic bacterium, a cyanobacterium. Using the latest DNA sequencing technologies, this research will identify mRNA sequence features that affect the rate of mRNA turnover. Those features will be used to design strategies for altering mRNA stability and to improve oil production in cyanobacteria. The planned experiments will leverage the DOE Joint Genome Institute’s DNA and RNA sequencing capabilities and will contribute data for the computational infrastructure provided by the DOE Knowledgebase. The knowledge gained from this project will help the development of more accurate gene expression models and will facilitate metabolic engineering projects needed to advance toward the sustainable production of biofuels.

Fiscal Year 2012

Repurposing the Saccharomyces Cerevisiae Peroxisome for Compartmentalizing Multi-Enzyme Pathways
John Dueber, University of California-Berkeley
To replace fossil fuels and other chemicals with biofuels and biomaterials from renewable sources, microbes can be engineered to alter their metabolism to maximize production of the desired chemicals. To do this, biosynthetic pathways from other species are added to a host organism, often resulting in the accumulation of new chemical compounds that are detrimental to the engineered microbe. Thus, a major challenge in microbial engineering is to enable high-yielding biofuel production without affecting the microorganism’s health. One solution to this problem is to spatially separate engineered metabolic pathways from the rest of the metabolic machinery within the microbial cell. In fact, many organisms already use subcellular compartments, called organelles, to isolate cellular functions by encapsulating components within impermeable membranes. The goal of this research is to repurpose one of these organelles, specifically the peroxisome, for use in engineered yeasts. The peroxisome is unique in that it is not necessary for healthy cellular growth in most environmental conditions. Therefore, this organelle can serve as a minimal compartment where unnecessary components are replaced by desired ones. This research will determine how the peroxisome can be specialized for encapsulating synthetic metabolic processes that can facilitate the production of biofuels to address DOE’s mission of advancing the development of renewable energy sources.

Metabolism and Evolution of a Biofuel-Producing Microbial Coculture
James McKinlay, Indiana University
Some microbes can convert renewable resources such as carbohydrates and sunlight into biofuels. Therefore, they offer an urgently needed alternative to nonrenewable fuels. Most research efforts in this area have focused on genetically engineering individual microbial species to improve biofuel production. However, a lesson can be taken from nature, where multiple microbial species help each other to thrive on food sources such as plant residues that the individual species cannot use on their own. Furthermore, mixtures of specialized microbes can sometimes outperform a single engineered strain for producing chemicals of value to society. This research will make use of a mixture of two microbial species (i.e., a coculture) that work together using sugar and energy from sunlight to produce more hydrogen gas than either microbe could by itself. A major challenge in using cocultures is ensuring that the different species maintain a long-term cooperative relationship. This research will stabilize such cooperation by forcing each microbe to provide a nutrient that the other requires to survive. This approach enables experiments that will decipher how the metabolisms of the two species interact and thereby how they can be optimized for biofuel production. Studying the evolution of the microbes in the coculture will also lead to the discovery of traits that enhance biofuel production. This information will ultimately lead to the design and engineering of tailor-made microbial mixtures for the economical production of hydrogen gas and other biofuels from renewable resources.

Improved Sensitivity and Utility of Metaproteomics Analyses
Samuel Payne, Pacific Northwest National Laboratory
Microbial communities are found in virtually any environment. Understanding the relationship between microbes and their environment is key to the U.S. Department of Energy’s goal of manipulating microorganisms for biofuel production. Microorganisms use proteins to interact with their natural environments and digest their food for growth and survival. Analyzing proteins from microbial communities thus facilitates understanding the relationship between microbes and their environment. Mass spectrometry is a powerful technique to identify proteins in complex biological samples such as the microbes that constitute an environmental community. The objective of this project is to develop novel computational methods to dramatically improve the ability to detect and identify new proteins in these complex samples. Current protein identification methods require the use of protein databases to infer the function of the proteins present in the sample under study. The methods that will be developed in this project take advantage of the similarity among proteins that perform comparable functions in different organisms, circumventing the need for protein databases. These studies will focus on the cow rumen environment, where the microbial community degrades a variety of renewable resources such as plant residue. A final goal of the project is to improve the ability to identify the species of origin for newly discovered proteins within the cow rumen bacterial community. In a natural environment with thousands of different organisms, this complex process is crucial for understanding the specialized roles that individual microbes play within the community and how they convert plant material into biofuels.

Systems Approach to Engineering Cyanobacteria for Biofuel Production
Jennifer Reed, University of Wisconsin-Madison
Most of the energy consumed in the United States is derived from nonrenewable fuels (e.g., petroleum and natural gas), with a significant fraction of this energy being used for transportation. To reduce the amount of oil used to satisfy U.S. transportation energy needs and to alleviate dependence on foreign sources of oil, renewable sources of transportation fuels are needed. Cyanobacteria offer a promising route for directly converting solar energy and carbon dioxide into biofuels. Certain cyanobacterial strains can be engineered to produce butanol, a biofuel that is compatible with the existing infrastructure for fuel transportation and use. The objective of this research is to integrate computational modeling and experimental approaches to guide the engineering of cyanobacteria with improved butanol production. New computational approaches will be developed to facilitate the design of experiments, predict their outcomes, and evaluate the results. In this way, this project will identify genetic engineering strategies for improving butanol production in cyanobacteria. Experiments will subsequently be performed to construct and evaluate new engineered cyanobacterial strains. The developed approaches will be systematically applied to identify engineering strategies for improving production of a variety of biofuels in five other microorganisms, supporting the U.S. Department of Energy’s mission for developing renewable ways of producing advanced biofuels.

Deciphering the Genetic and Molecular Underpinnings of Carbohydrate-Degrading Systems in Ruminal
Garrett Suen, University of Wisconsin-Madison
Biofuels like ethanol can be obtained from cellulose present in plant cell walls. A challenge in the production of biofuels is the efficient breakdown of cellulose into simple sugars. Current industrial approaches rely on cocktails of cellulose-degrading enzymes. These strategies can be improved by identifying and characterizing more active enzymes. Arguably the most optimized natural cellulose degrading system is found in the rumen of domesticated cows. The rumen contains a diverse group of bacteria with highly active enzymes that digest cellulose in feed and convert this energy source into nutrients usable by the cow. This research will characterize the mechanism through which three bacteria from the rumen degrade cellulose. Each of these bacteria employs different strategies for cellulose degradation and will provide contrasting models that can increase the understanding of this fundamental process. This work will leverage existing genomic sequences for these bacteria to identify the genes and enzymes relevant for cellulose degradation. Importantly, these enzymes will be purified and biochemically tested for their capacity to degrade cellulose. Novel enzymes characterized in this way will not only expand the current set of cellulose-degrading enzymes but will also provide insights into how these specialized microbes accomplish cellulose degradation in natural systems. The results of this research will advance the DOE mission of supporting the development of advanced biofuels.

Enhancing Metabolic Flux to Photosynthetic Biofuels
Jamey Young, Vanderbilt University
Developing liquid transportation fuels that are both renewable and compatible with existing fuel infrastructure is a major research challenge of the next decade. Corn ethanol provides nearly all the renewable fuel currently used in the United States. However, attention is shifting to “advanced” biofuels that more closely resemble gasoline. Several recent studies have demonstrated the feasibility of producing advanced biofuels in engineered strains of photosynthetic cyanobacteria. These organisms could be used to produce liquid fuels directly from sunlight and carbon dioxide (CO2) on land unsuitable for agriculture, thereby minimizing energy-intensive harvesting, transporting, and degrading of plant-derived feedstocks. However, cyanobacterial fuel productivity is currently too low for industrial feasibility. Therefore, this project will test new metabolic engineering approaches for maximizing carbon flux from CO2 to biofuels in cyanobacterial hosts. Tools will be developed for analyzing carbon flux and engineering the metabolic pathways that result in biofuel production. This will be further optimized by reprogramming the “biological clock” that controls daily metabolic rhythms that may affect those metabolic pathways. This work will have an important positive impact on the development of bioprocesses that rely upon photosynthetic microorganisms. In addition, it will provide fundamental insights into the role of biological clock genes in regulating photosynthesis and carbon fixation in engineered cyanobacteria. This research will directly contribute to DOE’s mission by advancing toward production of renewable fuels that do not compete with agriculture.

Fiscal Year 2011

Engineering Robust Hosts for Microbial Biofuel Production
Mary Dunlop, University of Vermont
Microbes contain a vast diversity of metabolic pathways that can be subtly tweaked and redesigned for the conversion of biomass to biofuels compounds. Next-generation biofuels such as short-chain hydrocarbons are particularly attractive target molecules since they would be compatible with existing engines and infrastructure. However, high levels of these compounds are often toxic to the microbes synthesizing them, limiting the potential rate and yield of industrial biofuel production. The objective of this research is to understand hydrocarbon tolerance mechanisms used by microbes inhabiting natural hydrocarbon seeps or oil-contaminated sites, searching genome sequences of these organisms for efflux pumps and other molecular machines that microbes use to separate toxic hydrocarbons from their delicate biological systems. Promising candidates will be introduced into biofuel synthesizing strains of E. coli and tuned for optimal gene expression to determine if it is possible to engineer strains with enhanced tolerance to hydrocarbons and improved efficiency of overall synthesis.

Plant-Microbe Genomic Systems Optimization for Energy
Samuel Hazen, University of Massachusetts-Amherst
To evolve from promise to practice, essential optimization of each step of an advanced biofuel industry based on cellulosic biomass is already underway. In this research, a bioassay will be used to measure rates of ethanol production in various accessions of the energy crop model Brachypodium distachyon and then assess genetic diversity for this trait in this species. In doing so, the research will seek to resolve the mechanisms underlying plant feedstock quality through genetic analysis with a focus on energy crop improvement. As a second phase, to determine the plausibility of specific positive interactions between plant and microbial genotypes, pairwise comparisons will be made, varying both plant and microbial genotypes. Similar to adapting crop varieties to different environments, these experiments will link the need for specific feedstock properties to biomass conversion processes. Importantly, the development and optimization of unified plant-microbe genomic systems will advance the concept of “plant-microbe co-development” within the industry, thus improving the efficiency of cellulosic biofuels production from ecologically and economically sustainable resources without affecting the food supply.

Systems-Level Investigation of Uranium Resistance and Regulation by Caulobacter crescentus
Yongqin Jiao, Lawrence Livermore National Laboratory
Microbes are known to play a major role in influencing the movement of uranium and other environmental contaminants. In addition to simply surviving exposure to radionuclides, some microbes are capable of using these compounds to promote their growth, altering their chemical state to restrict their movement in the environment. However, understanding of the basic mechanisms that microbes use to perform these metabolic reactions is limited, especially in environments exposed to oxygen. This research project will examine the biological systems of the bacterium Caulobacter crescentus that allow it to detect uranium in the environment, accumulate the metal at the cell’s surface, and use it to generate energy via respiratory metabolism (essentially “breathing” uranium). The long-term goal of the project is to develop a conceptual model of uranium cycling that could be used to understand processes occurring at contaminated sites and inform potential bioremediation strategies.

Microbial Communities in Biological Carbon Sequestration
Susannah Tringe, Lawrence Berkeley National Laboratory
Wetland ecosystems are known to cycle and potentially store massive amounts of carbon on an annual basis. Carbon dioxide captured from the atmosphere by plants moves, through the action of roots or the death of biomass, below the water or soil surface where it is subject to the processing by complex communities of microorganisms. This can result in the degradation of organic carbon back to carbon dioxide or methane or to more stable forms that may be stored for long periods of time. Relatively little is known about the organisms performing these processes or what conditions influence the storage or release of carbon. The current research will use cutting-edge genomic techniques to examine microbial community structure and functional properties in a restored wetland habitat in San Francisco bay, with an emphasis on characterizing processes that result in increased biosequestration of organic carbon over time. The study will leverage resources at the U.S. Department of Energy Joint Genome Institute to link activities of dominant environmental microbes to major carbon cycle processes to enhance the understanding of critical biogeochemical cycles and ecosystem sustainability.

Fiscal Year 2010

A Systems Biology, Whole-Genome Association Analysis of the Molecular Regulation of Biomass Growth and Composition in Populus deltoides
Matias Kirst, University of Florida
The goal of this research is to identify the genes that are the basis for the variation in biomass and biofuel productivity-related traits in the genus Populus (the poplar tree). Poplars are the principal woody crop species used for clean, renewable, and sustainable fuels in North America because of their fast, perennial growth habit and wide natural distribution in a broad range of environments. This project will identify genes underlying bioenergy-associated traits in poplar using an innovative approach to map genome-wide changes related to lignocellulosic biomass formation. This novel method—that will use data from previously sequenced poplar cultivars to capture biomass-related genes in a poplar population—promises to reveal a much larger fraction of the poplar genetic diversity than previously possible. The results will help answer the very important question of which genes regulate biomass productivity and its composition.

Integrative Molecular and Microanalytical Studies of Syntrophic Partnerships Linking C, S, and N Cycles in Anoxic Environments
Victoria Orphan, California Institute of Technology
The objective of this research is to improve understanding of anaerobic oxidation of methane by microbes, a globally significant biogeochemical process. Anaerobic methane oxidation results in the consumption of methane, a potent greenhouse gas, in numerous anoxic environments and is thought to have potentially global climate significance as a biogeochemical carbon cycle pathway. This research will focus on a metabolic partnership between sulfate-reducing bacteria and methane-oxidizing archaea that allows metabolism of methane for energy under anoxic conditions. The work emphasizes the optimization of new technologies for visualization of interactions between the partner organisms at the level of single microbial cells and the tracking of joint metabolic processes.

Spatial and Temporal Proteomics for Characterizing Protein Dynamics and Post-Translational Modifications
Wei-Jun Qian, Pacific Northwest National Laboratory
This project aims to develop a suite of quantitative proteomics technologies that enable spatially resolved measurements of subcellular protein abundance changes and the dynamics of post-translational modifications in environmental eukaryotes to gain understanding of the regulation of cellular function. The research will integrate subcellular fractionation, post-translational modifications, and quantitative proteomics technologies to establish a general approach for enabling spatial and temporal proteomics. The effectiveness and utility of these technologies for biological applications will be demonstrated using the filamentous fungus Aspergillus niger, an organism that plays an important role in biofuel production and global carbon cycling, to attain a better understanding of how its morphology is regulated. The unique suite of technologies will have broad application in diverse studies of microbial and plant organisms and in systems biology studies aimed at better understanding of cellular machineries. Such capabilities not only provide core value for current systems biology efforts, but they also add unique datasets for refining gene models, genome annotation, and future predictive modeling.

Consolidating Biomass Pretreatment with Saccharification by Resolving the Spatial Control Mechanisms of Fungi
Jonathan Schilling, University of Minnesota
The aim of this project is to characterize enzymatic mechanisms used by brown rot fungi to degrade woody biomass. The research will examine the enzymatic mechanisms used by Postia placenta, a brown rot fungus, to degrade woody biomass, since this fungus has already “developed” its own solution to the efficient use of lignocellulose for energy production. In particular, the work will address potential spatial partitioning of delignification reactions used by fungi to prepare the wood for digestion from final enzymatic deconstruction of cellulose, a natural analogue to the separate steps used in industrial biomass treatment processes. A combination of physical characterization of partially digested wood samples, microscopic examination of the wood-fungus interface, and analysis of gene expression will be used to address the study questions. The aim of the work is to provide new mechanistic understanding of fungal processes that could be used to develop new approaches to consolidated industrial bioprocessing for biofuels production.