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EBI Research Scope

An overview of the research goals envisioned for the EBI can be obtained by reading the proposal that was submitted to BP, which is available online at www.ebiweb.org. It is important to bear in mind that the proposal was intended to be illustrative of the intellectual and material resources of the host institutions rather than a prescription for the research program. The research program will emerge from proposals submitted by PIs at the participating institutions. Additionally, in the context of the biofuels aspect of the research program, a document resulting from a workshop on biofuels sponsored by the DOE provides a useful overview of many of the technical issues and is available at http://genomicsgtl.energy.gov/biofuels/b2bworkshop.shtml

Biological Sequestration

Terrestrial sequestration involves the removal of CO 2 from the atmosphere by photosynthesis and the storage of CO 2 in biomass, soils, and sediments. We are particularly interested in proposals that combine bioenergy feedstock cropping and sequestration into soils. The EBI seeks to understand whether it is feasible to significantly enhance biological sequestration by management practices that increase the net fixation of CO 2 by photosynthetic organisms, enhance the accumulation of soil organic matter, reduce the emission of CO 2 from soils, or increase the capacity of lands to sequester carbon. These should be accompanied by methods of full cycle validation at the field scale, and should consider utilizing the Feedstock Research, Development and Demonstration core facility described in the full EBI proposal at http://www.ebiweb.org/proposal.htm. Additionally, the EBI would consider proposals for employing microbial species to facilitate geochemical reactions that enhance CO 2 storage, by promoting microbial processes that facilitate mineral trapping of CO 2 or through the development of precipitates and biomass that can seal natural fractures, which could otherwise serve as conduits for CO 2 seepage. Additional areas of interest associated with both geological and terrestrial sequestration include the development of an improved understanding of plant-soil-microbe-mineral-pore fluid processes involved in sequestration; prediction, monitoring, and validation of sequestration processes; and life cycle and risk assessment.

Biofuel Feedstocks

The large-scale production of liquid biofuels for transportation will depend upon a stable supply of large amounts of uniform but inexpensive cellulosic biomass obtained sustainably in relatively close proximity to conversion facilities. For this reason maximizing yield and yield stability for minimized inputs will be critical. The EBI welcomes proposals that are designed to address basic research questions associated with the identification of suitable feedstock species, the development of improved feedstocks, and the applied management practices associated with growing, harvesting, and storing the feedstocks. The EBI has a global perspective so both tropical and temperate feedstocks are of interest. Areas of interest include the following:

Feedstock production

In the US, corn stover and other crop residues represent a possible feedstock that is available in large amounts. However, there is concern that removal of residues may harm soil productivity by mechanism such as depleting soil organic carbon. Studies that define the amounts that may be sustainably removed coupled with mechanistic insights into the relationship of crop residues to soil fertility and erosion would be welcome.

Dedicated low-input perennial energy crops represent a much larger and more sustainable potential source and are expected to be major sources of feedstocks. Dedicated annuals such as sorghums, which may form a stop-gap supply while perennial plantations are being established, may also be considered. Relatively little is known about the agronomy, genetics and breeding of the species that are frequently mentioned as possible perennial energy crops. The limited work conducted has often been limited to only a narrow portion of the germplasm.

Areas that need significant research include integrated basic and applied studies of efficient methods for sustainably producing high yields of biomass and of ecotypic variation to better understand potential ranges of these crops. Agronomic questions include issues such as how to establish the crops; when to rotate; fertilizer requirements; recycling of minerals from fermentation sludge; weed, pathogen, and pest control; fire control; and many related factors. In some cases, it will also be important to assess the potential invasiveness of potential energy crops. Research on methods of harvesting and storage are also of interest. Development of models able to use this new and extant trial information to predict yield and yield stability for these crops for different climates and soils, both at the global and local scale will be critical to development of a biofuel industry around residues and dedicated biofuel crops. Ideally these models will be mechanistically based and will interact with the above practical research to identify critical needs to improve prediction.

Feedstock genetics

Studies that facilitate breeding and selection of improved varieties of energy crops are of interest to the EBI. This may include associating genetic variation in species such as sugarcane, switchgrass, Miscanthus, and certain woody species, with traits relevant to the productivity and conversion qualities of these crops. These may include photosynthesis, cell wall composition, winter survival (for perennials), nutrient remobilization, disease resistance, and abiotic stress tolerance. Defining the full range of available germplasm and its ecological range will be important to this goal for the more poorly known species, and adapting these crops to diverse production environments. Innovations that allow wide crosses (e.g. between Sorghum, Miscanthus and Saccharum, or within these perennial grasses) may also facilitate this goal. Because the perennial grasses may exhibit sterility or self-incompatibility, it will also be necessary to investigate the genetic mechanisms controlling flowering, self-incompatibility, and other issues related to propagation and breeding. Studies may involve characterizations of genome structure and genetic diversity and the construction of high-density genetic maps for marker-assisted breeding. The overall goal should be to expand knowledge that would facilitate breakthroughs in the production of new breeding lines, rather than to produce cultivars per se.

Biomass composition

Plant biomass is largely composed of cell wall polysaccharides and lignin. In order to facilitate the development of efficient technologies for converting biomass to liquid fuels, deeper knowledge about the structure of plant cell walls may be useful. Additionally, improved understanding of cell wall structure and function may allow breeding or directed genetic modification of feedstocks for enhanced conversion efficiency.

Biotic stress

Large-scale cultivation of a species is associated with amplification of pest and pathogen populations. Studies that expand knowledge of the characteristics of pests and pathogens of prospective and existing energy crops, and the genetic diversity within these crops for resistance or tolerance to pests and pathogens are of interest.

Ecosystem Services and Sustainability

The EBI seeks to understand the environmental effects of large-scale conversion of land from current use to feedstock cropping to support biofuel processing plants in regions with high biomass production capability around the world. Key issues are how change will affect biogeochemical cycles (C & N), water use and quality, soil quality, soil erosion, biodiversity, landscape resilience and appearance, net greenhouse gas emissions, particulate emissions, and the potential invasiveness of biomass crops. Equally important is the sustainability of bioenergy feedstock cropping, particularly with respect to impacts on soil quality. Trials so far suggest that perennial energy crops increase soil organic carbon when they replace annual row crops. However, it is unclear how they affect soil microbial and detritivore diversity, and what long-term impacts these may produce.

It is equally unclear whether management can increase free-living nitrogen fixers in the perennial grass crops. Low fertilizer requirements, extensive perennial root systems and winter harvest may improve water quality, decrease soil erosion, increase soil organic matter, and provide better habitat for nesting birds and insects, and cover for other animals. But these changes might also provide habitat for unwanted organisms. Other uncertainties include the possible effects of adding the biofuel fermentation residues back to the cropping system. Increased soil organic matter (SOM) deposition will result in increased sequestration at least in the short term. Establishing the turnover time of this carbon will be necessary to project longer-term sequestration and management that will slow turnover. Increasing SOM and altered quality associated with a new crop will affect microbial and soil insect diversity, including potential pathogens and pests. These changes may also in turn affect emissions of other greenhouse gases, notably methane and nitrous oxide. It will be important to understand altered land-use effects on greenhouse warming potential, and carbon credits that may be earned with biofuel cropping. Risk of genes and plants spreading into native communities must also be assessed and then used as a basis for any management recommendations to minimize such risks.

Planting, Harvesting, Transport and Storage

Farm machinery operations with biofuel feedstocks from planting through to delivery to the point of use can represent significant energy, carbon and economic costs. Present systems utilize farm equipment designed for food crops, which are sub-optimal for these new crops and new uses of existing crops (e.g. corn stover). This should be addressed in a systems analysis context to identify where the greatest gains may be achieved for the least cost. While companies are best placed to produce farm equipment, experiments that facilitate equipment design and use and associated logistics related to biofuel feedstock production and delivery are areas of interest to EBI. Prototypes and modifications of existing equipment should be considered where particularly large gains may be achieved, e.g. in planting technologies. Technologies such as compaction and wrapping that may help to minimize losses during storage are also an area of high priority. For efficiency of operation, proposals for herbaceous crops should plan to use the Feedstock RD&D core facility described in the full EBI proposal at http://www.ebiweb.org/proposal.htm.

Socio-Economic Issues Associated with Biofuels or Sequestration

A major challenge in making the transition to sustainable energy is the integration of individual agricultural, silvicultural, and technological solutions into an energy system that is consistent with natural cycles, the economy, society, transportation networks, the power grid, and urban infrastructures. Some of the key areas of research that are important in providing an integrated and systems-oriented approach to the transition towards sustainable biofuels production and use globally are outlined below to illustrate but not restrict the scope of proposals. Proposals concerning the socioeconomic aspects of biological sequestration would also be welcome.

Global Socioeconomic Impacts

The prospects of the development of regional and a global trade in biofuels will impact nations in many different ways. The EBI is interested in understanding the possible effects of various scenarios on socioeconomic questions of food availability, social equity and trade from a global perspective. The appetite of the large economies for energy, if coupled with technical advances in production of biofuels, could result in undesirable transfer of resources from less developed nations to the large economies. The EBI would welcome research into the government policies, local actions, and social networks needed to avert undesirable socioeconomic effects from diffusion of new technologies and expanding markets for biofuels.

Next-Generation Assessment

The introduction of a large-scale biofuels industry will have a significant impact on energy, agricultural and food systems, and the environment. To understand these challenges, a new framework for assessing the social and environmental implications of biofuels is needed, one that uses the best available tools and methods from life-cycle assessment (LCA), fuel-cycle analysis, computer-based systems analysis, cost estimation, multicriteria decision-making, sustainability science, and environmental impact assessment. The EBI is interested in proposals to integrate LCA with economic models and land management and land-use assessment methods. Additional areas of interest include the potential for low-carbon, low-water and other input biofuels to support community and national economic and sustainability objectives.

Biofuels Evaluation and Adoption

If biofuels are to make a substantial contribution to the world’s energy needs, new crops, new cropping practices, and new fuel production technologies will have to be adopted by a wide range of economic actors. The EBI seeks to understand information needs, policies, and incentives that may affect the adoption and acceptance of new biofuel technologies, including intellectual property rights and fiscal policy that are likely to affect innovation and production around the world. EBI is interested in understanding the energy, agricultural, and environmental impacts of current and potential biofuels, including potential costs and environmental implications of different production pathways and barriers that could prevent deployment of each pathway. Additionally, analysis of regulatory issues that may affect establishment of a biofuels industry is needed (eg., aphis regulations, herbicide re-validation, CRP limitations). Because of the complexity of the issues, a useful analytical modality may be to consider a specific location and then address the issues of planning, crop conversion, transport, micro-economics, farmer attitudes towards adoption, community attitudes and current legislative barriers/opportunities. One possible workshop approach here would be to consider the issues surrounding a specific hypothetical or actual case; e.g. a new cellulosic fuel plant fed by crop residues and perennials in the Midwest or a new ethanol plant in a sugarcane producing region of Brazil.

Biofuels Markets and Networks

The productivity, cost effectiveness, land use, environmental impacts, and transportation requirements of bio-energy crops needs to be integrated and modeled in a regional context, linking local, national, and global dimensions of supply and demand. This should include analysis of the allocation of land and other resources among competing alternatives to meet various levels of demand for biofuels, including the potential for food-fuel tradeoffs among producers. A possible workshop might focus on inclusion of disadvantaged socioeconomic groups, lessons from past experiences (e.g. the ‘Green Revolution’), and proactive approaches to avoid many of the pitfalls of past efforts, as well as opportunities to make biofuel markets benefit, not harm, the global poor.

Social Interactions and Risks

Development of a large-scale international biofuels industry will create changes at many levels in producer nations and may, therefore, create social concerns about biofuels. What impacts, and opportunities, exist along gender lines, for and on minority communities, and in particular agro-ecological and socio-cultural regions. Citizens need relevant information at an early stage, must have the chance to participate in the planning and decision-making process, and must have compensatory mechanisms for costs and risks. EBI seeks insights into the design of processes and policies concerning the public understanding of biofuel technologies and the modeling of social adoption in different political contexts on a global scale.

Depolymerization of Biomass

The hydrolysis of lignocellulose into fermentable sugars is hampered by slow reaction rates arising from the crystalline cellulose content of the lignocellulosic feedstock and the inaccessibility of enzyme adsorption sites. This is the result of intricate links between the structure of the lignocellulosic material and the limited availability of enzymatic binding sites. Present methods for deconstructing lignocellulose are costly and energy intensive. A central aim of this component of the EBI program will be to understand the physical and chemical fundamentals of lignocellulose deconstruction and enzymatic conversion into C5 and C6 sugars and lignin byproducts. A long-term goal will be to develop one or more microbes expressing cellulases or cellulosomes that can be used in a consolidated bioprocess with organisms that utilize the simple sugars and lignin products to make a range of biofuels, such as ethanol, butanol, or hydrocarbons. Areas of interest include the following.

Pretreatment Technologies

The first step of biomass depolymerization is typically accomplished with technologies derived from the pulp and paper industry such as dilute acids (HCl, H 2SO 4), explosive decompression (steam), hot water, or treatments with organic solvents. Acid hydrolysis leads to degradation products that are often inhibitory and significantly lower glucose yields. Glucose and xylose degradation products that result from the pretreatment methods include hydroxymethylfurfural (HM F) and furfural, which produce levulinic and formic acids that inhibit the subsequent fermentation of sugars to ethanol. In contrast, cellulase hydrolysis results in almost no by-product formation. Pretreatment processes that increase the surface area accessible to water (largely excluded due to the packing of cellulose fibrils) and provide enzyme binding sites will thus substantially increase glucose yields. These problems underline the importance of developing a pretreatment process that a) converts the initial lignocellulosic material into an easily hydrolysable format and b) must not produce any inhibitory components that will negatively impact the downstream processing of that biomass into ethanol or other biofuels. Promising new approaches such as ionic liquid solvents and ammonia fiber explosion are of interest. The focus should be on understanding mechanisms of deconstruction rather than on incremental improvements in poorly understood empirically derived processes. Quantifying and imaging pretreatment processes may thus be useful in developing predictive models to optimize the pretreatment processes for subsequent enzymatic depolymerization of lignocellulosics.

Enzyme Discovery and Evolution

Lignocellulosic biomass is enzymatically hydrolyzed to sugars and other low molecular weight compounds in natural systems such as soils and rumens at physiological conditions. Many of the enzymes that participate in natural decomposition of lignocellulose are unknown or poorly understood. The EBI will support research focused on expanding knowledge of the properties of enzymes and enzyme systems such as cellulosomes that catalyze depolymerization of lignocellulose components. Additionally, research concerning the use of protein evolution technologies to improve the industrial performance of hydrolytic enzymes is of interest.

Integrated Bioprocessing and Industrial Adaptation

A long-term goal of the biofuels community is the development of an organism that is capable of both secreting enzymes to depolymerize biomass and also of utilizing the hydrolysis products for fuel production. Proposals concerning selected aspects of this long-term goal are of interest to the EBI. For example, understanding how to regulate high-throughput protein secretion may be appropriate. Experiments concerning the regulation of pathways for simultaneous fermentation of various sugars might also be of interest. Understanding the factors that inhibit microbial growth in biomass hydrolyzates is also of interest in this context.

Development of Novel Catalysts

The EBI will support research directed toward development of new chemical catalysts for conversion of biomass to fuels. A priority objective is to explore the development of new catalysts for the cleavage of glycosidic linkages, particularly b-glucans and xylans. The development of such catalysts could obviate concerns about hydrolysis of cellulose under mild conditions and could greatly reduce the formation of toxic byproducts by current pretreatment processes. Another area of interest is the catalytic conversion of glucose to n-alkanes as a potential alternative route to liquid fuels.

Biofuel production

The production of biofuels is hampered by toxicity of lignocellulose depolymerization products and the fuels to the microbial producer, the inability of the microbial host to withstand processing conditions, inefficient conversion of the products of lignocellulose depolymerization into fuels, and a lack of biosynthetic pathways for production of potential next-generation fuels and fuel additives. The Biofuels Production Program of EBI will focus on alleviating these limitations, not only as it relates to ethanol production, but also higher alcohols (e.g., butanol), alkanes, esters and other highly reduced compounds. Topics of interest include studies of the toxicity of various fuels and biomass monomers to potential microbial hosts using the latest experimental technologies in functional genomics and computational models. An overall goal is to acquire the knowledge to facilitate engineering of several platform hosts to be tolerant of the various processing conditions likely to be found in producing biofuels from lignocellulosic biomass. The ability to manipulate pathways as modular elements of metabolism is expected to greatly facilitate pathway optimization and process optimization to create industrial microorganisms capable of efficiently converting biomass to fuels under industrial conditions.

Systems biology

A major barrier in the efficient use of biomass-derived sugars is that microbes have a complex network of poorly understood regulatory networks that are designed to achieve different metabolic outcomes from those we desire. Problems include inhibition by deleterious products formed during biomass hydrolysis, yields limited by accumulation of alternative products, unnecessary microbial growth, suboptimal specific productivity resulting from various limitations in the biosynthetic pathways for products (i.e., fuels) and mismatches in conditions with the hydrolysis enzymes, and inhibition by the main fermentation product (e.g., ethanol or higher alcohols) with concomitant low alcohol titer. These and related problems contribute to the cost of lignocellulosic ethanol by increasing capital expenditure, reducing product yields, and increasing water volumes that must be handled as part of relatively dilute product streams. The new tools of systems biology, synthetic biology and evolutionary approaches will facilitate the development of predictable models that can be used to direct metabolic engineering and synthetic biology approaches to development of improved biotransformation processes. A key to systems biology is the development and use of computational models that incorporate whole system types of data (e.g., whole genome transcriptome, proteome, or metabolome datasets) into predictive models. Such models need to be developed to address questions such as what mechanisms control glycolytic flux, what limits photosynthesis, and what are their implications for cellular metabolism. What molecular mechanisms are used by cells to cope with such environmental challenges as high concentrations of sugars and ethanol and the presence of inhibitors from biomass hydrolysis? What genetic and physiological characteristics mediate evolution of wild-type organisms into robust laboratory or industrial strains, and which ones control their functional state in the process environment? Coupled with evolutionary algorithms where selection is for enhancement of the end product, (e.g. maximum sucrose yield per unit light in photosynthetic cells) the in silico cell can guide targets for manipulation in vivo.

Pathway Engineering

No microorganism currently has all of the capabilities that are desirable for production of biofuels from cellulosic biomass. For instance, current industrial strains of yeast are only capable of producing ethanol from glucose, so recombinant ethanologenic organisms (e.g., yeast, E. coli, and Z. mobilis) have been developed to ferment both glucose and xylose by the addition of genes for entire pathways. Additional research is needed on the identification and characterization of biofuels-relevant pathways from a wide variety of organisms and the identification of the enzymes and corresponding genes for relevant pathways at a sufficient level of resolution to permit transfer of pathways to new hosts. A critical goal is to identify pathways for fermentation of all major sugars and also identify pathways for synthesis of various potential fuels such as alkanes, medium-chain alcohols, terpenoids, xylenes, and other hydrophobic molecules.

Most methods of biomass pretreatment to produce hydrolysates also produce side products that are inhibitory to growth of microorganisms and final product titers. The basis for these inhibitory effects needs to be understood and strains developed through rational pathway engineering that exhibit resistance to these inhibitors—preferably by allowing the improved strains to catabolize the inhibitors—thereby improving overall process efficiency.

Biofuel Production Systems

Biofuels may be produced by fermentation of lignocellulose hydrolysis products, photosynthesis, or from metabolism of syngas (CO + H 2) generated by biomass pyrolysis. The EBI invites proposals concerning the integrative aspects of large-scale biofuels production, including design of practical methods of large scale cultivation. For instance, studies of methods for large-scale algal cultivation based on realistic assumptions about production costs and regulatory hurdles may be appropriate. Similarly, studies of fermenter designs that allow continuous cultivation of strains that secrete hydrophobic compounds would be of interest.

Fossil Fuel Bioprocessing

There are large reserves of fossil fuels, such as tar sands, shale, and soft coal that are likely to be extensively used for fuel production in the future. At present, the conversion of such materials to fuels is energy intensive and involves release of large amounts of greenhouse gases. The EBI will consider proposals for research into ways in which biological processing of fossil fuels may facilitate the production of liquid or gaseous fuels (eg. methane) from fossil fuel sources in ways that reduce the environmental impact of processing compared to non-biological methods. Topics may involve either ex situ or in situ methods.

Microbially Enhanced Oil Recovery (MEOR)