Conversion Technologies for Advanced Biofuels

Preliminary Roadmap & Workshop Report

December, 6–8 2011

Arlington, VA

Conversion Technologies for Advanced Biofuels Workshop Executive Summary

IntroductionThe appeal of developing renewable energy sources within the United States is largely coupled with the promise of attaining an elevated level of domestic energy security in the future. This idea is widely espoused throughout the entire renewable energy field, but it is specifically pronounced within the realm of biofuels development, where every day, engineers, researchers, and industry leaders are confronted with the significant task of displacing over 4.3 billion barrels of crude oil and petrochemical imports annually1—all the while maintaining price parity with foreign fossil imports. In a recent speech, the President affirmed:

“Biofuels are an important part of reducing America’s dependence on foreign oil and creating jobs here at home. But supporting biofuels cannot be the role of government alone… partnering with the private sector to speed development of next-generation biofuels will help us continue to take steps towards energy independence and strengthen communities across our country.”

–President Barack Obama, August 16, 2011

It was in this spirit that the Department of Energy’s (DOE) Office of the Biomass Program in the Office of Energy Efficiency and Renewable Energy (EERE) hosted the Conversion Technologies for Advanced Biofuels Workshop (CTAB) from December 6–8, 2011. The purpose of the conference was to engage industry, academia, and the national laboratories in defining the most important technical challenges and research activities that must be addressed to hasten the expansion of a domestic advanced biofuels industry. The primary focus was on developing hydrocarbon biofuels (renewable gasoline, diesel, and jet fuel) from lignocellulosic biomass-derived intermediates.

DOE’s Biomass Program was established to focus on the development and transformation of domestic, renewable, and abundant biomass resources into cost-competitive, high-performance biofuels, biopower, and bioproducts through targeted planning, research, development, and demonstration leveraging public and private partnerships. Focused originally on cellulosic ethanol, the Program is transferring knowledge gained through its cellulosic ethanol research, development, deployment, and demonstration (RDD&D) experience to accelerate advances in other advanced biofuel pathways.

1 http://www.eia.gov/dnav/pet/pet_move_imp_dc_NUS-Z00_mbblpd_a.htm

BackgroundThere are three types of challenges associated with pioneering a successful biofuels industry— technical, economic, and policy. Though the CTAB workshop primarily focused on technical challenges, it is impossible to refrain from mentioning the other two categories. The Biomass Program’s targets are framed by a host of federal laws and economic policy incentives, most notably the Energy Independence and Security Act of 2007 (EISA) which established the Renewable Fuel Standard (RFS2) and requires blending 36 billion gallons of renewable fuel by 2022 (21 billion gallons of which cannot be ethanol or corn-starch derived). Currently, the fledgling U.S. biofuels industry is just beginning to grapple with the reality of producing commercial-scale quantities of cellulosic ethanol (on the 20–25 million gallon per year plant capacity scale), which can only be used to displace light-duty vehicle fleet gasoline consumption at a 15% maximum blend wall. CTAB’s ultimate goal was to generate enough information necessary to help formulate technical targets for the Biomass Program looking ahead to 2022 to commercialize hydrocarbon biofuels technology and to update the Program’s existing technology roadmaps, which were published in conjunction with the DOE Office of Science in 2007.

Nearly 150 stakeholders with diverse subject matter expertise and backgrounds convened at CTAB to provide input to 10 technical breakout tracks over two days. The breakout tracks were organized into two groups dedicated to the following topics:

Production of carbohydrate derivatives from biomass and their subsequent upgrading to hydrocarbon biofuels.

Production of bio-oils from biomass and their subsequent upgrading to hydrocarbon biofuels.

Each breakout session was led by two co-chairs, one representing a national laboratory and the other from industry, academia, or government. Scribes were available in each session to capture notes and track group discussion. The aforementioned topic groups reflect existing Critical Technology Goals (CTGs) within the Biomass Program that revolve around producing and upgrading carbohydrates and bio-oils to “drop-in” fuels. Participants were encouraged to suggest, discuss, and prioritize technical barriers and research and development (R&D) activities. Crosscutting themes in barriers that emerged throughout all breakout sessions were:

Feedstock supply, logistics, and pre-processing considerations Techno-economic and life-cycle analyses Catalysis issues Separation science needs Process integration.

The following is a summary of the findings from the Workshop. Results are presented in terms of overarching themes and recommendations that emerged from the sessions. Preliminary findings from the Workshop indicate that additional years of basic and applied research are needed to fully realize a commercially successful advanced biofuels industry.

Bio-OilsProduction of bio-oil increases the energy density of raw biomass and converts it into a product that is amenable to additional processing en route to producing a liquid hydrocarbon fuel. Although there is no single composition for bio-oil and its chemical makeup is largely dependent upon the starting feedstock and process variables during production, bio-oils contain a variety of destabilizing components. The destabilizing components may be both inorganic and organic species, in either the vapor or liquid phase, which affect the stability of either the oil or the overall process and may:

Cause the condensed bio-oil intermediate to change physically and chemically over time and under various processing conditions

Cause operational problems in processing Reduce catalyst performance during intermediate upgrading to biofuels Impact existing fuel distribution infrastructure.

Bio-oil is an emulsion with suspended lignin solids, so physical instability may arise from agglomeration of lignin to form larger particles. If bio-oil is allowed to age, a phase separation can occur between the aqueous and organic fractions and large, agglomerated clumps will settle into a lignin-rich sludge at the bottom of the vessel. Chemical instability can arise from polymerization reactions. Species within the bio-oil that contain unsaturated, carbon-carbon and carbon-oxygen double bonds are especially susceptible to participating in polymerization reactions (e.g., aldehydes, aromatics, olefins, and organic acids). In addition to potential instability of the condensed bio-oil, there are components that can lead to instability in the chemical processes, which include degradation of materials and equipment, as well as deactivation of upgrading catalysts through chemical poisoning, fouling, or physical changes via mechanisms such as leaching.

To facilitate the use of bio-oil for production of hydrocarbon fuels, the removal of destabilizing components from the bio-oil is an essential activity. The removal of these components may occur by chemical/catalytic conversion of the unwanted species or by utilizing separation techniques. These removal processes can be implemented on either the vapor phase (e.g., in-situ or ex-situ vapor phase upgrading, hot gas filtration, cyclones) the whole condensed phase (filtration, membranes, liquid-phase catalysis), or either the aqueous or organic phases alone.

Fundamentally understanding how lignin, hemicellulose, and cellulose thermally depolymerize during biomass fast pyrolysis and how inorganic contents vary in different biomass materials (especially in terms of how they impact bio-oil production and upgrading) is crucial to engineering systems to produce bio-oils with desirable qualities. Also, attaining a better fundamental understanding of high-temperature solid-vapor separation was repeatedly noted as a research barrier, particularly as it applies to scale-up of bio-oil technologies. The key research needs that were identified for vapor-phase upgrading included; increased understanding of the catalyst interaction with oxygen functional groups (catalytic deoxygenation) and exploring the use of H2-donor molecules for in-situ hydrogenation and deoxygenation. Other large themes identified during the discussion on bio-oil production and upgrading were as follows:

Hydrogen cost and supply: The clear leading candidate for oil upgrading is catalytic hydrogenation (hydrotreating). Bio-oil contains oxygen that must be removed on the way to finished fuel. Hydrogen is the natural choice and is already used in refinery operations for upgrading, albeit with different heteroatom targets. The cost and logistical difficulties of a distributed hydrogen supply make hydrogen supply to a distributed biomass-based system a challenge. Production of hydrogen from in-field waste streams or improved processes for hydrogen reforming from biomass were both advocated. Internal generation of hydrogen (potentially from a hydrogen donor species) was also discussed. Donors that are in the existing fuel infrastructure that can be left in the process effluent were envisioned, but not defined.

Catalytic processing limitations: Heterogeneous catalysts were assumed to be the only practical solution for processes that have to be inexpensive and robust. Known systems are susceptible to fouling and deactivation. Development of fouling resistant catalysts and those with sufficient lifetime are required. Studies of fouling and deactivation fundamentals are proposed, as are new catalysts or new regeneration regimes.

Process intensification: The upgrading process has to be cost effective, implying that it must be simple. Effective integration with pyrolysis and refining must be examined to be successful. The ambiguity surrounding the integrated process must be clarified in order to develop specific R&D targets.

Oxygen removal without hydrogen addition: Radical new approaches to oxygen removal should be considered, even though well-defined options are lacking. Movement away from hydrogenation could result in process simplification and economic improvement.

Fundamental studies related to upgrading: Publicly available information on the catalyst performance and failure modes is lacking. The potential exists for improvement if better understanding of the mechanisms can be gained.

Carbohydrate DerivativesTechnical barriers to the generation of lignocellulosic sugars or saccharide-derived species

include both feedstock properties and processing techniques. Processes for converting renewable resources into biofuels may be classified in terms of bioprocessing or thermocatalytic/ thermomechanical conversion techniques. Barriers are reflective of the feedstock, such as the carbohydrate content, type and amount of inhibitors, and structural integrity. These may be further classified as being ideal, acceptable, or unacceptable and are anticipated to be different for chemical/catalytic processes compared to biochemical or bio-based processes. Furthermore, barriers will be defined by the properties and robustness of catalysts (either biochemical or chemical) that are used.

Pretreatment and enzymatic saccharification: Pretreatment is needed to enhance the accessibility of lignocellulosic biomass to catalytic enzymes, microorganisms, and other types of catalysts during bioprocessing. Bioprocessing is defined as an enzyme or biological based technology for transforming pretreated lignocellulosic biomass to sugars (oligosaccharides and monosaccharides), followed by either biocatalytic or chemical catalytic transformation of oligosaccharides to monosaccharides, and monosaccharides to biofuel or biofuel precursors other than ethanol. A combined pretreatment and biobased (enzymatic) approach was identified as a key technology in need of focused research to develop an optimized process. Alternatively, if oligosaccharides are obtained, these may be processed to monosaccharides using chemical (catalytic) methods. The ideas feedstock for biofuels production would have reduced recalcitrance (through alterations of lignin or polysaccharides) to pretreatment methods and low inhibitor content such as acetyl groups, aldehydes and phenolics. Current research should be targeted at optimizing hydrolyzate quality while minimizing energy input.

Non-enzymatic routes to carbohydrates: Non-enzymatic sugar production from lignocellulose typically employs a mechanical system to deconstruct or fractionate an aqueous or solvent modified biomass slurry in the presence of acid, base or other reagents under varying temperature and pressure conditions. Biomass can be pre-processed to minimize recalcitrance beforehand or fed directly into a system, although unit operation intensification is typically preferred. One advantage to using non-enzymatic systems is the potential for rapid hydrolysis of biomass-based sugars, but a fundamental issue inherent to such processes involves economically recycling reagents and the technical challenges inherent to developing closed loop systems. Poor separation of biomass and solvent was identified as a major issue and area of active R&D. Difficulties in solubilizing high (greater than 85%) of the bulk sugar content from the biomass was also noted as a significant issue that can hinder process economics. Research on development of extraction techniques for targeting clean separation of organic and water layers, improving solids separations, development of acid inhibitor tolerant materials (e.g. membranes and mesopourous materials) and mechanical separation systems (e.g. screw extruders, supercritical fluid systems and shrinking bed reactors) were identified as crucial research targets in advancing the state of the art.

Microbial conversion of carbohydrates to biofuels: Desirable fuel precursors, including fatty acids, alcohols, esters, aldehydes, ketones, isoprenoids, polyketides, neutral lipids, and others, can be synthesized by specialized microbes from sugars released during pretreatment and hydrolysis. Microbes that can produce such compounds in sufficient quantities can be created through metabolic engineering or strain evolution. Central tasks to designing effective organisms are identification and overexpression of genes that encode for enzymes that synthesize precursors to fatty acids, other molecules containing fatty functional groups (??) and straight and branched alkanes. These precursors can then be extracted from either the host organism or the extracellular environment of the host (if excreted), and upgraded to produce hydrocarbon biofuel blends. Customization of a biofuels’ properties is based upon the functionalities catabolized by the production host. Key barrier issues are efficient carbon utilization during bioconversion (especially with regard to C5 sugar use), redox balance, lack of energy- and cost-efficient hydrocarbon product separation systems, identification and elucidation of biological conversion inhibitors and mechanisms, and prioritization of hydrocarbon molecules targeted for production.

Catalytic processes for converting carbohydrates to biofuels: Chemical conversions of carbohydrate derivatives represent new routes to hydrocarbon fuels that can use wide ranges of sugars and sugar-derived intermediates, including carbohydrate dehydration products and organic acids. The primary barriers to demonstrating technical and economic feasibility of these materials can be grouped by issues related to feedstocks, catalysts, carbohydrate processing, and fuel production. The co-design of upstream processes for biomass deconstruction with the downstream catalytic processes to convert biomass-derived intermediates to fuels is important. In particular, upstream processes determine the product slate of biomass-derived intermediates (including intermediates derived from lignin) and the potential introduction of contaminants, catalyst poisons, and fouling agents. The composition of the intermediate streams will significantly impact the final product slates, catalyst lifetimes, separations, and the operation of the downstream unit operations, all of which impact the economics of the processes.

Table of ContentsCrosscutting Issues in the Processing of Biomass to Transportation Fuels..................................................9

Feedstock Handling.................................................................................................................................9

Catalysis and Biocatalysis........................................................................................................................9

Separation and Purification.....................................................................................................................9

Techno-economics and Process Data....................................................................................................10

Production of Cellulosic Sugars and Carbohydrate Derivatives from Biomass and their Upgrading to Hydrocarbon Biofuels and Oxygenate Blends............................................................................................11

Pretreatment and Enzymatic Saccharification of Lignocellulosic Biomass.............................................11

Nonenzymatic Routes to Sugars and Carbohydrate Derivatives from Lignocellulosic Biomass.............36

Chemical Conversion of Sugars and Carbohydrate Derivatives to Hydrocarbon Fuels..........................38

Biological Conversion of Sugars and Carbohydrate Derivatives: Isoprenoid, Polyketide, Fatty Acid, and Oleaginous Pathways.............................................................................................................................51

Production of Bio-Oils via Direct Liquefaction of Biomass and Upgrading to Hydrocarbon Biofuels and Oxygenate Blends......................................................................................................................................67

Fast Pyrolysis.........................................................................................................................................68

In-situ vapor phase initial catalytic upgrading of the pyrolysis vapors..................................................68

Ex-situ vapor phase initial catalytic upgrading of the pyrolysis vapors..................................................68

Hydropyrolysis.......................................................................................................................................69

Hydrothermal Liquefaction....................................................................................................................69

Special Topics Areas..................................................................................................................................79

Hybrid Biochemical/Thermochemical Processes...................................................................................79

Direct Microbial Conversion to Fuels from Unconventional Sources.....................................................81

Conversion Systems for Genetically Modified/Optimized Feedstocks...................................................85

Lignin Utilization....................................................................................................................................88

Separation Systems...............................................................................................................................96

Solvent Systems...................................................................................................................................101

Crosscutting Issues in the Processing of Biomass to Transportation Fuels

Feedstock HandlingBiomass feedstocks are the keystone to the biofuels industry. As stated in the introduction, the focus of The Department of Energy’s (DOE) Biomass Program is the development and transformation of biomass resources into cost-competitive, high-performance biofuels, biopower, and bio-products. If the singular goal of biomass logistics and handling is to reduce the per ton supply costs of biomass, systems may very well develop with ultimate unintended consequences of highly variable, and reduced quality biomass feedstocks that directly impact the efficiency of conversion processes, perturb system designs, and product specifications. Furthermore, feedstock diversity varies markedly from region to region, from crop type to crop type, and will vary from year to year based on weather conditions, harvesting, transportation, storage, and preprocessing operations. Additionally, preprocessing operations can significantly impact or modify the physical and chemical attributes and uniformity of the biomass feedstock, and ultimately, the viability of the biomass delivered to conversion refineries. Crosscutting feedstock logistics and handling issues affect all aspects of bioenergy production, including:

Feedstock sustainability: The design and implementation of sustainable feedstock production systems typically favor diversity. Maximizing the environmental performance and total productive capacity of a production system is achieved by careful placement of biomass feedstocks on the landscape. This can present both challenges and opportunities when building efficient, cost-effective biofuel conversion processes. Utilizing a diverse set of biomass feedstocks can help reduce the impact of inherent variability in individual feedstock resources. However, utilizing a diverse and potentially dynamic resource base can limit the ability to optimize a conversion process around the characteristics of an individual feedstock. The interface between sustainable production system and process design needs to be well characterized to achieve cost, quality, and efficiency targets.

Feedstock quality attributes: Inherent feedstock attributes such as energy, ash, and carbohydrate content require explicit system designs. For robust or insensitive conversion processes feedstock quality may have little impact, conversely feedstock quality may be critical for certain pathways; off-spec feedstocks can impair catalysts, contribute to slag formation, lead to subsequent instability of product intermediates, and require intensive chemical fractionation and chromatography, significantly impacting conversion economics.

Feedstock variability: Feedstock variability (e.g., variability of carbohydrate, ash, and moisture content and composition) is one of the foremost crosscutting challenges facing the biofuels industry, forcing the over design of conversion systems, increasing design and operational costs.

As pioneer biorefineries move from technology development to production and their focus changes to process optimization, variability in feedstock quality will become a central parameter.

Feedstock characterization: The need to accurately and effectively assess field-run feedstock quality and variability in a rapid manner is significantly underestimated, but is vital to ensuring that feedstocks meet refinery operational specifications. The sheer size and logistical issues associated with the feedstock supply system compound the issue. A lack of understanding of the quality of feedstock entering the throat of the conversion reactor can have significant effects on process operation and efficiency.

Transportation: Transportation costs are largely a function of the distance travelled, bulk density of the biomass type, and form of transportation. Reducing transportation costs by transporting more biomass per unit volume will help in ensuring economical production of biofuels and bioproducts. Regardless of the transportation system used to move the biomass, the only limitation is that it must fit within the existing transportation infrastructure.

Feedstock handling and flowability: The comprehensive particle attributes of particle size, size distribution, shape, friction, and cohesiveness influence flowability and overall engineering design and conversion performance. Feedstock particle characteristics ultimately need to be optimized based on the conversion process requirements and material handling/flowability constraints. Feedstocks must efficiently deconstruct into a uniformly flowable form to support consistency in handling systems and eliminate process obstructions.

Feedstock stability: Stable feedstock resources are critical for the long-term viability of a U.S. energy source. The inherent perishable nature of raw biomass is unacceptable for downstream converters and energy users, not only from the economic impacts caused by uncertain yields, but also from the risks associated with yearly variability in feedstock quantities and the inability to maintain a strategic supply in times of supply crisis like drought, fire, floods, pests, and eventual global demand.

Conversion performance: Biomass recalcitrance is one of the major hurdles for the biorefinery industry. Recalcitrance in the feedstocks limits the conversion performance by reducing the feedstock product yields. The inherent mechanisms of plant biomass to resist natural assault directly correlates to then need for strong acids usage, slow reactions, increased operational costs and capital expenditures.

Downstream intermediate insertion: Insertion of feedstock intermediates early into conventional petroleum processes may introduce rogue chemical species that could perturb the refining process or act as a toxin to other biofuels processes. This crosscutting issue transcends both feedstocks and separations challenges (specifically product purity requirements), and can directly impact intermediate and finished energy product purity.

Commoditization: Stable feedstock pricing and supply is directly dependent on a logistical system that can store and deliver feedstocks when and where they are demanded. A commodity supply system would, by definition, allow for long-term storage and long-distance transport of biomass feedstocks. Commodity feedstocks would also adhere to national/international standards, ensuring quality feedstocks for the conversion process and finished energy product. Feedstock consistency also allows for flexibility and interchangeability within multiple conversion technology pathways.

Waste management: Initial feedstock composition has a direct effect on waste issues as well. In a 60 million gallon per year biorefinery for example, soil contamination at a level of 5% ash (in addition to physiological levels) can substantially increase yearly variable operational costs (estimated at > $1 million). This increase is for the variable operational costs only and does not include the fixed costs of additional infrastructure required for soil handling, accumulation prior to shipment off-site, and increased equipment wear.

Catalysis and BiocatalysisCatalysis is the study of catalysts, both inorganic and organic, which interact with reactants and reduce the activation energy barrier for chemical transformations of the reactants, hence increasing the rate of reaction. Furthermore the catalyst can increase the partition of products towards the desired product. With the need for more cost and energy effective processes, the cross cutting role of catalysts both for robust productivity and selectivity of desired products is critical. Catalysts, when applied to biomass deconstruction and subsequent conversion to fuels, are typically in two classes—those used in biological systems, mostly enzymes, and those typically used at temperatures significantly above ambient conditions, mostly inorganic catalysts and heterogeneous catalysts, in order to aid with process separations. Catalysts play a major role in the industrial sector and significant amounts of the Gross Domestic Product (GDP) leverage catalysts. As such, the biomass conversion to fuels and chemicals sector is anticipated to be extremely dependent on catalysts. Investment in enabling catalysis and biocatalysis research and development (R&D) will support the Biomass Program mission of enabling commercial production advanced biofuels for the nation. Critical crosscutting catalysis challenges include:

Poor selectivity toward desired reactions: Biogenic carbon is a limited and often costly molecular building block for biofuels with increased catalyst selectivity carbon efficiencies will render processes economically more optimal. Additionally, reduced production of side products that can act as inhibitors and deactivation agents for downstream catalytic processes would lead to increased productivity and time on stream.

Insufficient understanding of reaction fundamentals: Detailed understanding of reactions mechanisms and kinetics enable the development of improved catalyst productivity and the de-emphasis of competing reactions at surfaces and interfaces. With the development of

the kinetics of the reaction(s) the process can be modeled at different length scales in order to ultimately refine techno-economic analyses and scaling of technologies. With additional understanding of mechanisms and correlated competitive reactions in realistic biomass derived streams, the work in Office of Science developing the cleavage of specific atomic linkages and critical molecular transformations could be readily leveraged to accelerate development of conversion technologies.

Limited catalyst regeneration and lifetime data: Deactivation and inhibition rates and subsequent replacement and/or regeneration schemes have not been developed in order to reduce the catalyst replacement costs and the risks to process scaling, control, sustainability, and economics. With detailed understanding of how to maintain optimal catalyst performance over significant lengths of time and in the event of typical process excursion events, technology could be more readily transferred into integrated bio-refineries.

References

Basic Research Needs: Catalysis for Energy Report from the U.S. Department of Energy, Basic Energy Sciences Workshop August 6–8, 2007 Bethesda, MD.

Separation and PurificationSeparations play a crucial role in conversion technologies and can be the largest contributor to process economics. In general, conversions technologies involve multiple steps that require different separations techniques. Separations technologies tend to be specific to the feedstocks, products, process streams, and conversion technologies. Separations are classified based on the types and concentrations of species, solvents, and reaction conditions. There are significant opportunities to improve crosscutting separations technology that will enable specific or proprietary industrial deployment in integrated biorefineries. Investment in enabling separations R&D will support the Biomass Program’s mission of commercial production advanced biofuels. Critical crosscutting separations challenges include:

Feedstock variability: Feedstocks are produced from a range of agricultural materials. Even with a specific feedstock, biomass composition, as well as water and ash content, are dependent on the conditions for growth, harvesting, processing, transport, and storage. Choice of an appropriate separations technology is driven by composition and variability of the feedstock.

Product purity requirements: Intermediate and products may have significant different purity requirements depending on subsequent processing requirements. As a general rule of thumb, separations costs exhibit logarithmic type behavior. Therefore targeted purification requirements must be well defined.

Product heterogeneity: Most conversion systems target producing single products. To meet fuel specifications, advanced biofuels will require a distribution of intermediates and products. Separations systems must be designed that retain the targeted distributions.

Unknown contaminants: With a limited understanding of reaction mechanisms during development phases, byproduct, contaminant, and inhibitor concentrations or even their existence are uncertain. During scale-up, general separations schemes will need to adapted to specific feedstocks, biorefinery operations, and product portfolios.

Distinct conversion processes: Advanced biofuels production will use mixtures of biochemical, thermal, and catalytic processes. Each conversion platform has distinct separations demands and limitations including operations conditions, inhibitors, and product concentrations.

Low concentration of targets: Biomass feedstocks typically require large water volumes during pre-processing and conversion. Intermediates, byproducts, and products can be present at dilute concentrations. Depending on the nature of the species, sometimes it is more efficient to remove solutes from the solvent and sometimes the opposite. Effective separations at low concentrations are essential. Low concentrations increase energy consumption, system footprint, and capital equipment costs.

Water management: A significant fraction of both the energy demand and waste discharge associated with biorefinery operations can be attributed to water management. For example, distillation of an 85%–95 % water fraction is a significant energy consumer. Biorefinery facilities may have significant restrictions on release of wastewater. Separations technologies that improve water treatment and reuse can reduce both water input and wastewater discharge.

Conversion route divergence: Conversions frequently involve transformations through different physical forms of matter (i.e., solid, liquid, and gas) of the components for separation. Change in physical form (e.g., precipitation or evaporation) can facilitate or complicate separations and must be considered in process design.

Compatibility at operating conditions: Conversion routes typically consist of multiple process steps with different operating parameters (temperature, pressure, etc.). Therefore, intermediate and product stability, as well as materials compatibility, must be considered at all potential operating conditions. Separations must be designed to avoid incompatibility, instability, and undesired reactivity.

Coordination of multiple separations steps: Design of a conversion/separations train must consider the difficulty of separating species or classes of species in the process stream. Separations systems are typically designed based on an increasing degree of difficulty or complexity, i.e., species that are very similar chemically or physically are separated last.

General separations platforms: Crosscutting R&D investment in general separations platforms will enable more rapid deployment of specific integrated biorefineries and facilitate commercialization of advanced biofuels.

Techno-Economics and Process DataTechno-economic analysis (TEA) is a powerful tool that can be utilized to develop a cost-driven research and development program. TEA couples process design and cost analysis with experimental and pilot-scale research results to evaluate the current economic state of technology of a given process. TEA can

also serve to develop an understanding of how process economics relate to experimental and research developments and process improvements. As such, TEAs offer perspectives into which process areas are most costly, hence, potentially bring opportunity for the greatest reductions in cost. Early scoping studies that include economics can help to identify unknowns and uncertainties in processes that need additional experimental investigation and quantification. The Biomass Program has adopted this TEA approach to develop and track research targets for the production of cost-competitive cellulosic and advanced biofuels.

The initial success of TEA in driving research on economically viable technologies and conversion strategies for the production of advanced biofuels has motivated the identification of further research needs in TEA. Several key research needs in understanding processing costs and providing full techno-economic analyses were identified for the bio-oil conversions strategy, including:

1. High-level studies: Performing high-level economic studies of innovative conversion strategies identified early in the R&D pipeline can be used to develop promising processing strategies and identify key uncertainties that must be addressed through further R&D.

2. Utilization of biomass-derived intermediates: One way to reduce the cost of producing biofuels is to leverage existing capital assets, such as petroleum refineries. Investigation of the utilization of biomass-derived intermediates into existing refinery infrastructure can help address process requirements and highlight value chain opportunities.

3. Tracking research progress: The tracking of R&D progress toward specified targets, especially when coupled to the development of detailed TEA models that provide greater optimization opportunities.

4. Publically available experimental data: Robust mass and energy balances require understanding of a given system and process.

Production of Cellulosic Sugars and Carbohydrate Derivatives from Biomass and their Upgrading to Hydrocarbon Biofuels and Oxygenate Blends

Pretreatment and Enzymatic Saccharification of Biomass

BackgroundThe notion of biomass recalcitrance is based on the ability of plants to evade attack from the animal, insect, and microbial worlds using a multi-length scale defense strategy. Macroscopically, plants use bark and rinds as the primary defense to invasion. Microscopically, complex systems of cells consisting of thin-walled primary, thick-walled secondary and heavily lignified vascular cell walls provide a secondary defensive layer. Going deeper, the polymer matrices within the cell walls provide chemical and physical barriers to deconstruction on the ultra-structural level. Finally, the insoluble nature of cellulose itself provides resistance to facile conversion (in contrast to the relatively more rapid enzymatic digestion of starch). It is the

Biomass Program’s objective to develop more efficient technologies for the conversion of hemicelluloses and cellulose polymers in energy plant cell walls to fermentable sugars, thus providing a critical biological intermediate for eventual conversion to a variety of biofuels, including first generation alcohols as well as second generation and beyond direct drop-in hydrocarbons.

New directions include the use of cutting edge tools for understanding the relevant structure of plant cell walls, the effects of thermal chemical pretreatment at the micro- to-nano scale, critical enzyme structure/function relationships, the uses of protein engineering to improve cellulases and hemicellulases, and the development and utilization of new high throughput techniques for screening biomass types and improved enzymes. Recent advances in metabolic engineering and synthetic and systems biology have allowed the engineering of microbes to produce hydrocarbon-based advanced biofuels or its precursors that can “drop in” to the existing transportation infrastructure. Engineering of microorganisms for producing hydrocarbon-based biofuels in yields, titers, and rates high enough to be useful for commercialization requires significant effort in not only engineering of microbial metabolism for advanced biofuel synthesis at high yields, but also engineering the microorganisms’ capability for utilization of the lignocellulosic substrates (preferably the use of pretreated biomass directly). Using tractable heterologous hosts, a number of hydrocarbon-based fuel substitute or precursors have been produced which significantly advanced our knowledge about producing these advanced biofuels. Future research innovations will rely not only on developing more genetically tractable platform microorganisms, but also using alternative microorganisms with attractive cellulolytic capabilities and ability to produce advanced fuels at high yields, titers, and rates. Development of more efficient and controllable synthetic biology tools in both genetically tractable and industrial microorganisms will enable us to reach our goals.

Cell Wall Structure of Energy PlantsPlant cell walls are composed primarily of cellulose, hemicellulose, lignin, and pectin. These polymers give structural rigidity and strength to the plant, deter pathogens, and retain extracellular water. Cellulose, a highly crystalline, insoluble polymer of beta-(1, 4)-cellobiose, comprises about 50% of the plant biomass. Although cellulose does not degrade easily, it can be hydrolyzed to glucose by the synergistic action of three distinct classes of enzymes: endoglucanases, exoglucanases, and cellobiases (1, 2). In contrast with the insoluble linear cellulose homopolymer, hemicelluloses are water- or base-soluble heteropolymers, comprised of a variety of branched and substituted polysaccharides. In addition to providing water retention and structural reinforcement, hemicelluloses act as cross-linking agents. The complex structure of hemicelluloses has dictated an accordingly diverse array of hemicellulases. Generally, each structural feature in hemicellulose has an associated enzyme that can hydrolyze or modify this feature (in theory, at least). Pectins are acidic polysaccharides that retain large amounts of water and act as an “adhesive” between adjacent plant cells, and, along with lignin, comprise much of the middle lamella. Lignin, by contrast, is a heterogeneous polymer of phenyl propanoid units containing various phenolic derivatives. Lignin is often thought of as the binder that cements the cell wall components together. The possibility of enzymatic degradation of

lignin is still somewhat controversial, with various hydrolytic and oxidative mechanisms proposed. In recent years, a considerable body of work has been published on the mechanisms of microbial, especially white and brown rot fungal degradation of lignin; however, the Program is unaware of a unified molecular mechanism for native lignin depolymerizaton by purified enzymes. Many system-wide studies have been published recently, including those of lignin degradation by mycorrhizal fungi, dye decolorization by white rot fungi, lignin biodegradation in compost, applications in pulp and paper and ruminant feed, and the emerging molecular genetics of ligninolytic fungi (3–8).

Plant cell walls can be divided into two sections, the primary and the secondary cell walls (9). The primary cell wall, which provides structure for cell expansion, is composed of the major polysaccharides and a group of basic glycoproteins, primarily extensins (10). The predominant polysaccharide in the primary cell wall is cellulose, the second most abundant is hemicellulose, and the third is pectin. Because cellulose is made up only of beta-(1, 4)-linkages, it has a highly linear structure that encourages the formation of strong hydrogen bonds between chains of cellulose. The high level of hydrogen bonding among the chains makes it much more difficult to attack or depolymerize, either chemically or biologically. Hemicelluloses are biopolymers of six- and five-carbon sugars that are branched in grasses and trees with a wide spectrum of substituents, including acetyl and 4-O-methyl glucuronyl esters, along the backbone polysaccharide. The more branched and amorphous nature of hemicellulose makes it more vulnerable to conversion than cellulose, but organisms in nature do not as readily utilize some of its various sugars due to the complex nature of the chemical linkages. Hemicelluloses are thought to hydrogen bond to cellulose, as well as to other hemicelluloses, which helps stabilize the cell wall matrix and render the cell wall insoluble in water. The secondary cell wall, produced after the cell has completed growing, also contains polysaccharides and is lignified (9). Lignin is a high-energy content biopolymer rich in phenolic components. The combination of hemicellulose and lignin provide a protective sheath around the cellulose and this sheath must be modified or removed before efficient hydrolysis of cellulose can occur.

The primary carbohydrate components of lignocellulosic biomass consist of D-glucose, D-xylose, L-arabinose, D-galactose, and D-mannose. Glucose (from cellulose) and xylose (from hemicellulose) are the two principal carbohydrates present in most biomass feedstocks. The levels of the minor carbohydrates L-arabinose, D-galactose, and D-mannose (also derived from hemicellulose) vary considerably with biomass type. Softwoods typically contain more galactose and mannose than hardwoods, whereas hardwoods, herbaceous plants, and agricultural residues generally contain higher levels of arabinose and xylose. In some herbaceous crops and agricultural residues, arabinose levels are high enough that conversion of arabinose (in addition to glucose and xylose) is required to achieve overall economic viability.

Pretreatment of Feedstocks Lignocellulosic biomass can be converted into mixed-sugar solutions plus lignin-rich solid residues by the sequential use of thermochemical pretreatment and enzymatic saccharification. Sugars from hemicellulose and cellulose can then be fermented to ethanol and other products for fuel production. There is a long and rich history of using acid and base catalysts to release

the sugars found in cellulose and hemicellulose dating back to the discovery of wood sugars in the 19th century. The technology was commercialized during World War I in the United States, during World War II in Germany, and later in the 20th century in the Soviet Union (11–28). More advanced schemes for biological processing are under development today; however, they rely on this chemical hydrolysis step only as a pretreatment for removal of hemicelluloses and some lignin. Biologically mediated hydrolysis of cellulose is now viewed as the most selective and efficient means of hydrolyzing or depolymerizing the cellulose biopolymer to release its glucose sugar monomers. Many workers in the field agree that cellulose decrystallization and depolymerization are indeed the rate-limiting steps in the enzymatic conversion of lignocellulosic biomass. Removal of hemicellulose by dilute-acid pretreatment has been the classic means of rendering biomass more amenable to cellulase action (29). In a hallmark study, Soltes and coworkers (30) showed that biomass with reduced acetylation responded significantly more favorably to cellulase action than did native biomass. Although still controversial, there is some indication that biomass with reduced lignin content is also more readily hydrolyzed by cellulase action (31, 32). One key to understanding cellulase action on biomass is the fact that the structural and reactive chemical components of the substrate—primarily defined as acetyl and lignin contents—strongly affect enzyme access to cellulose. Another is that once cellulase component enzymes are available in sufficient ratio and concentration at the site of hydrolysis, the degree of cellulose crystallinity controls the hydrolytic rate (31, 33). The types of pretreatment most commonly studied, and often recommended for site specific application, are listed below.

Steam ExplosionSteam explosion processes date back to the development of the MasoniteTM process on wood chips in the 1920s (34). In steam explosion, chipped or coarsely shredded biomass is contacted with high-pressure saturated steam at high solids loadings in a pressure vessel for a residence time that is generally 20 minutes or less (35-38). Depending on the feedstock used and the objective of the pretreatment, steam explosion pretreatment temperatures are generally in the range of 140 to 260C. At the end of the pretreatment time, the pressure vessel contents are rapidly decompressed into an atmospheric pressure flash tank, which causes significant disruption and defibration of the biomass. Even without the addition of any chemical catalysts, hydrolysis reactions in steam explosion are catalyzed by the release of organic acids that are liberated from acetyl functional groups associated with hemicellulose. This results in some lignin solubilization and hemicellulose hydrolysis, although yields of xylose from the hemicellulose fraction of most biomass types is typically no higher than 65% of theoretical, primarily due to extensive sugar degradation reactions that occur under typical uncatalyzed steam explosion reaction conditions (35, 39, 40).

Ammonia Fiber Explosion The Ammonia Fiber Explosion (AFEX) process is essentially the alkaline equivalent of sulfur dioxide-catalyzed steam explosion pretreatment (36). In the AFEX process, biomass is treated with liquid anhydrous ammonia at temperatures between 60C to 100C at pressures of 250 to 300 psig, and residence times of about 5 minutes (41). The pressure is then rapidly released

resulting in an explosive decompression, the combined chemical and physical effects of which lead to physical disruption of biomass fibers and partial decrystallization of cellulose. Partial lignin solubilization and conversion of hemicellulose to oligodextrins are also observed (42, 43). AFEX is typically conducted at high solids loadings (about 40% solids) and high ammonia loadings (about 1.0 g NH3/g dry feedstock). The associated complexity and costs of ammonia recovery processes may be significant and must be better understood in order to assess the commercial potential of the AFEX process (44). AFEX has been shown to deacetylate and increase the digestibility of biomass (45-47), although it does require that both cellulose and hemicellulose be enzymatically hydrolyzed due to limited hemicellulose hydrolysis during AFEX pretreatment. The AFEX pretreatment is more effective on agricultural residues and herbaceous crops, with limited effectiveness demonstrated on woody biomass and other high-lignin feedstocks (43).

Liquid Hot Water PretreatmentsIn addition to uncatalyzed steam explosion pretreatments, other uncatalyzed pretreatment processes using pressurized liquid hot water without rapid decompression have been investigated in both batch and percolation modes. Process conditions have been developed for cellulose hydrolysis at very high temperatures of about 260C (42, 48). High yields of soluble sugars from the hemicellulose fraction of some biomass types (primarily herbaceous crops and agricultural residues) can be achieved, but liquid hot water processes generally liberate the sugars in an oligomeric form and thus require a secondary acidic or enzymatic hydrolysis step to produce fermentable monomeric sugars. A typical approach for liquid hot water pretreatment is to use chemicals as agents to control the pH in the range of pH 4 to 7 (42, 49). With some feedstocks, such as corn stover, there may be enough inherent buffering capacity from the feedstock that the target pH range is achieved without any requirement of pH-controlling chemicals. In general, liquid hot water pretreatments are attractive from a process cost-savings potential (no pretreatment catalyst usage, low-cost pretreatment reactor construction due to low corrosion potential). Pressurized liquid hot water that is percolated or otherwise forced through a packed bed of biomass particles has also been shown to result in high removal of both hemicellulose and lignin, with high recovery of hemicellulose-derived sugars (primarily in oligomeric form) and high digestibility of the resulting pretreated solids.

Dilute Acid Batch/Co-Current Pretreatment Dilute acid pretreatments are probably the most thoroughly investigated biomass pretreatment technique. A variety of acidic catalysts have been investigated in numerous batch/co-current dilute acid pretreatment reactor designs on a wide range of woody, herbaceous, and agricultural residue feedstocks. For cost reasons, most dilute acid pretreatment studies have utilized sulfuric acid or gaseous sulfur dioxide (in steam explosion applications), although several processes that utilize nitric, phosphoric, hydrochloric, or carbonic acid have also been investigated. Dilute acid batch and co-current pretreatments are generally aimed at achieving near-complete solubilization of the hemicellulose fraction of biomass, while also achieving high yields of hemicellulose-derived sugars. Many processes seek to directly achieve monomeric

sugar formation, although care must be taken to prevent excessive sugar degradation product formation from monomeric sugars. If performed properly, dilute acid pretreatment can be effective at achieving both reasonable monomer sugar yields via hemicellulose hydrolysis and high resulting enzymatic digestibility of the cellulose in the pretreated solids across a range of biomass feedstock types (50). For reasons similar to liquid hot water percolation processes, dilute acid processes that employ a percolation mode of operation have also been investigated. Very high yields of monomeric and oligomeric xylose have been obtained in a two-stage percolation process on hardwoods, with resulting high enzymatic hydrolysis yields of the cellulose in the pretreated solids (51). The high digestibility achieved in this approach has been attributed to significant lignin solubilization and removal from the pretreated solids in the continuously-flowing percolation process.

Sodium Hydroxide PretreatmentAlkali pretreatment processes generally do not hydrolyze hemicellulose as extensively as acidic pretreatments, but can be effective at removing lignin, which can lead to an increase in the enzymatic digestibility of alkali pretreated solids. This pretreatment approach causes swelling of fibers, leading to an increase in internal surface area, reduction in the degree of polymerization, a decrease in crystallinity, separation of the structural linkages (primarily esters) between lignin and carbohydrates, and disruption of lignin structure (52). The effectiveness of sodium hydroxide pretreatment has been correlated to feedstock lignin content, with high lignin feedstocks, especially softwoods, showing poor performance using this approach (43). Dilute sodium hydroxide pretreatment has been shown to be quite effective on low lignin (10%–18% lignin content) straw feedstocks (53).

Ammonia PretreatmentIn addition to the rapid decompression AFEX pretreatment process, which utilizes ammonia to achieve both chemical and physical changes to biomass, there are a number of additional ammonia pretreatment processes. The simplest ammonia pretreatment process involves a relatively low-temperature soaking (ambient temperature up to 90C) using aqueous ammonia (various strengths up to 29 weight percent [wt %] NH4OH) at solids loadings of 10% to 50% and residence times from a few hours to up to one day (54-56). In these processes, up to 80% delignification has been reported on feedstocks such as wheat straw and corn stover, with much lower extents of hemicellulose solubilization. However, good enzymatic digestibility of the remaining cellulose and some of the remaining hemicellulose can be achieved using commercial cellulase preparations (56).

Lime PretreatmentPretreatment using lime has been studied as a low-cost process that primarily achieves acetyl and lignin solubilization (42, 54, 57-59). Lime pretreatment has been practiced at a wide range of temperatures, from 25C to about 130C, with lime loadings of about 10 wt % (on a dry feedstock basis) and solids loadings of 20% or less. At the higher temperatures, the pretreatment times are reasonably short (minutes to hours), but can extend to several weeks at

lower temperatures. Despite the lengthy residence time at low temperatures, lime pretreatment can be conducted in a pile arrangement without expensive pressure reactors and can be performed as part of the feedstock storage system (57). Near-complete deacetylation generally occurs upon lime pretreatment of low-lignin herbaceous feedstocks and agricultural residues, with about 30% lignin removal.

Organic Solvents (Organosolv)Numerous organic or organic-aqueous solvent mixtures utilizing methanol, ethanol, acetone, ethylene glycol, triethylene glycol, and tetrahydrofurfuryl alcohol have been used as biomass pretreatment processes to solubilize lignin (42, 52, 36, 60, 61). In some studies, inorganic acid catalysts, such a sulfuric or hydrochloric acid, are added to achieve significant levels of hemicelluloses hydrolysis and even cellulose hydrolysis (62) along with lignin solubilization. In some cases, the main components of biomass (cellulose, hemicellulose, and lignin) can be effectively fractionated, with each component potentially used for separate value-added products (63). Solvents must be effectively recovered and recycled using appropriate extraction and separation techniques without leaving behind any inhibitory levels of residual solvents in process streams that undergo subsequent biological processing. While residual cellulose-rich pretreated solids from such processes may be highly digestible using cellulase enzymes, the cost of such processes and the potential value of the relatively pure fractions may make them better suited to higher-value applications.

Cellulose-Dissolving SolventsCellulose and cell wall dissolving solvents, such as cadoxen, concentrated mineral acids, dimethylsulfoxide (DMSO), zinc chloride, and ionic liquids are also used to prepare biomass for conversion (42, 52). While these agents can be effective at directly releasing sugars from the carbohydrate fractions of biomass and/or producing a solid residue containing cellulose that is highly digestible by enzymes, the use of such solvents in pretreatment processes for the production of fuels and commodity chemicals from biomass will be challenging due to the expense of such catalysts, catalyst recycle requirements, and the requirement for clean process streams for subsequent biological conversions.

Oxidative ProcessesOxidative processes for biomass pretreatment applications are often referred to as wet oxidation processes. This approach was born out of efforts in the pulp and paper industry to develop oxygen delignification processes to reduce chlorine use in pulping. The most common approach for wet oxidation as a biomass pretreatment involves the injection of pressurized O2

into a pretreatment reactor at temperatures up to 200C and pressures up to about 1.5 MPa (64). Much of this work has included the use of alkaline buffers (usually sodium carbonate) to maintain reaction pH in the neutral to alkaline range. Wet oxidation extensively delignifies biomass with production of monomeric and oligomeric phenols, followed by oxidative cleavage to a variety of carboxylic acids. When the reaction is not buffered and pH is allowed to drift naturally down, extensive formation of furfurals occurs, which can also be cleaved to form

carboxylic acids in the oxidative environment. Hemicellulose is typically solubilized to about 70% conversion, primarily as oligomers. The combination of extensive delignification and at least 50% hemicellulose removal can result in highly digestible pretreated solids (65).

Enzymatic Hydrolysis of Plant Cell WallsFollowing pretreatment and conditioning (usually pH adjustment and cool down), enzyme formulations are added either before fermentation or concurrently with fermentation (SSF or SSCF). New process options also suggest that modest loadings of thermal tolerant enzymes can be added to the hot, neutralized slurry following pretreatment and held during cool down. The outcome may be reduced loading of the primary enzyme formulation during SSF.

Free Enzyme Systems In cellulolytic bacteria and fungi, the cellulases all hydrolyze the same type of bond of the cellulose chain, i.e., the beta-(1, 4)-glucosidic bond. They do so, however, using different modes of action. The definitive enzymatic degradation of cellulose to glucose is generally accomplished by the synergistic action of three distinct classes of enzymes: (i) The "endo-beta-(1,4)-glucanases" or beta-(1,4)-D-glucan-4-glucanohydrolases (EC 3.2.1.4), which act randomly on soluble and insoluble beta-(1,4)-glucan substrates and are commonly measured by detecting the reducing groups released from carboxymethylcellulose, (ii) the "exo-beta-(1,4)-D-glucanases," including both the beta-(1,4)-D-glucan glucohydrolases (EC 3.2.1.74), which liberate D-glucose from beta-(1,4)-D-glucans and hydrolyze D-cellobiose slowly, and beta-(1,4)-D-glucan cellobiohydrolase (EC 3.2.1.91), which liberates D-cellobiose in a “processive” manner (successive cleavage of product) from berta-(1,4)-glucans, and (iii) the "beta-D-glucosidases" or beta-D-glucoside glucohydrolases (EC 3.2.1.21), which act to release D-glucose units from cellobiose and soluble cellodextrins, as well as an array of glycosides. The above classification scheme is not entirely rigid, and a few enzymes have properties that do not fit one of the above definitions.

Free cellulases frequently bear a cellulose-binding carbohydrate-binding module (CBM) that delivers the catalytic module to the surface of its crystalline cellulosic substrate (66). In aerobic fungi, the CBM is invariably from family 1, which is very small (~30 to 35 amino acid residues). The ancillary CBMs of bacterial cellulases are often from family 2 or 3, which are much larger than their fungal analogues, comprising approximately 100 and 150 residues, respectively. Despite the differences in size, these types of cellulose-binding CBMs all exhibit a planar array of aromatic residues located on a relatively flat surface of the CBM molecule. These planar-strip residues are generally highly conserved and are believed to align against the hydrophobic face of the glucose along the length of a single cellulose chain of the cellulose surface, thus providing the structural rationale for substrate binding of the CBM and the parent enzyme.

Structurally, the topology of the active sites differs between the endoglucanases and exoglucanases. The active sites of endoglucanases typically attain a cleft-like topology. Thus, a cellulose chain can be accessed in random fashion by an endoglucanase, and bond cleavage can occur anywhere along the chain of the substrate. In contrast, the active site of the exoglucanases resemble a tunnel, formed by long loops of the protein molecule that fold over

the active site residues (67). Consequently, a single glycan chain is fed into one end of the tunnel-like active site, followed by subsequent bond cleavage in the center of the tunnel and release of cellobiose product from the other end (68, 69). Because the chain is fixed within the active site tunnel, successive cleavage events can continue in procession in a unidirectional manner along the glucan chain (70, 71). However, some differences in this mechanism occur among the different types of exoglucanases (72), perhaps reflecting the length of the tunnel, the directionality of action (from non-reducing to reducing end or vice versa), and flexibility of the loops that form the tunnel.

Cellulosomes In general, the multi-enzyme cellulosome complex is composed of two major types of sub-unit: the non-catalytic scaffoldin(s) and the enzymes (73-75). The assembly of the enzymatic sub-units into the cellulosome complex is facilitated by the high-affinity recognition between cohesin modules of the scaffoldin subunit and enzyme-born dockerin modules. Scaffoldins usually contain multiple cohesin modules, thereby enabling numerous different enzymes to be assembled into the cellulosome complex. In addition, a multiplicity of scaffoldins has been found in some species, which lends a higher level of complexity to cellulosome assembly. Theoretically, over 70 different dockerin-containing components can be assembled into the cellulosome of C. thermocellum (76,77). Since the scaffoldin subunit in this bacterium contains only 9 cohesin modules, the varied collection of individual cellulosomes is immensely heterogeneous. Another important scaffoldin-born component is the cellulose-specific CBM, which functions as the major binding factor for specific recognition of cellulosic substrates. The CBM of the scaffoldin serves to deliver the entire complement of cellulosome enzymes collectively to the lignocellulosic substrate, thus fulfilling another important requirement for efficient degradation.

In many aspects, cellulosomal enzymes are very similar to their free counterparts, except their catalytic modules are attached to a dockerin rather than a CBM. The scaffoldin-based CBM serves as a single cellulose-targeting agent for all cellulosomal components. Members of the same families of cellulases and hemicellulases that are involved in the free enzyme systems also serve as cellulosomal enzymes, with some exceptions. In this context, the GH7 and GH45 cellulases that occur exclusively in fungi never appear in the cellulosomal context. Intriguingly, however, GH6 enzymes that occur both in fungi and some bacteria have not been found in native cellulosome systems. Compared to free enzyme systems, the cellulosome brings the catalytic modules into close physical association with each other, and collectively, to the cellulose surface, thereby promoting their essential synergistic action by concentrating the enzymes with complementary functions at defined sites on the lignocellulosic substrate.

Oxidative Cellulose Fragmentation In the last couple of years, a new enzymatic mechanism of cellulose hydrolysis has emerged (78-79). Polysaccharide monooxygenases (PMOs) are produced primarily by white-rot and other saprophytic fungi and are believed to function through the oxidative cleavage of cellulose. The striking difference in mechanism between these two modes of cellulose fragmentation (hydrolytic versus oxidative) has led to numerous studies and theories regarding

probable synergy between these systems and suggested benefits from incorporation of PMOs into new commercial cellulase formulations (80). On the surface, PMOs appear to provide a limited amount of cellulose fragmentation in comparison to classical cellulases and may also require the presence of other enzymes, such as cellobiose hydrogenase (81). In addition, they produce oxidized sugars as products, potentially reducing yields if the fermentative microbes cannot utilize these modified sugars, or if these products are inhibitory to cellulases; however, the actual number of catalytic events is low compared to classical cellulases and the demonstrated synergy suggests that this limited action plays a critical role in liberating substrate that is not readily converted by the classic system. New structural studies of PMOs reveal the distinct role of the catalytic copper ion with possible implications for the oxidative fragmentation of hemicelluloses and even lignins (82, 83).

Hemicellulases and Accessory EnzymesThe complex nature and interconnectivity of plant cell wall polymers preclude straightforward enzymatic digestion. There are dozens of enzyme families involved in plant cell wall hydrolysis, including cellulases, hemicellulases, pectinases, and lignin-modifying enzymes. As may be expected for a complex series of biopolymers, synergism has been demonstrated between beta-xylanases and acetylxylan esterase (84), alpha-L-arabinofuranosidase (85), and beta-glucuronidase (86). Synergy is a major factor in degradation efficiency, making measurement of these activities for single enzymes difficult. Studies show correlations between the enzymatic digestibility of cellulose and the removal of hemicellulosic sugars and lignin, supporting the notion of close spatial relationships (87, 88). Of further complication is that the actions of glycosyl hydrolases often change the chemical environment of the partially degraded substrate, which in turn affects the actions of other glycosyl hydrolases. For example, partly because of the substituents attached to the main chain, most hemicelluloses are quite water soluble in their native state. These side chains disrupt the water structure and help to solubilize the hemicellulose. Debranching enzymes that remove these substituents generally decrease substrate solubility, and in turn lower the polysaccharide’s susceptibility to endo-acting hydrolases [89]. Thus, a xylan that has been subjected to acetyl xylan esterase is less susceptible to enzymatic degradation than a xylan subjected to a mixture of branching and debranching enzymes [90]. As the substituents are removed, xylan can become less soluble, forming aggregates that sterically hinder and finally block further degradation (91). The endoxylanases, for example, cleave the main chain linkages and are often quite specific about the type of linkage, type of sugar, and presence or absence of nearby substituents (92).

As noted for cellulases, hemicellulose depolymerizing enzymes are divided into three classes; endo-acting enzymes, exo-acting enzymes, and oligomer-hydrolyzing enzymes. Although mechanisms of hemicellulose hydrolysis have been steadily studied over the years, they have not received the attention given to cellulose hydrolysis. Despite this, a general pattern of degradation is beginning to emerge. Although there are specific examples of endo-acting enzymes requiring side chains for maximal activity (93), the majority of the endo-acting hemicellulases tend to be more active on de-branched hemicellulose, especially in the case of xylanases. However, these modified polysaccharides tend to become more insoluble as the de-

branching process continues. Concomitant reduction in chain length from the activity of endo-hemicellulases tends to compensate for this effect, allowing the shorter, less substituted fragments to remain soluble. Overall, a balance must be met between removing the branching side chains from the polysaccharide backbone, decreasing the average chain length, and hydrolyzing the oligomers into free monomers, all while maintaining enough solubility of the fragments to allow enzyme access. The concerted action of the various hemicellulase enzyme classes probably accounts for the high synergy observed when the enzymes are mixed (94).

Pretreatment Considerations in Fermentation of Biomass Derived Sugars

Bioethanol FermentationConversion efficiency and robust fermentation of mixed-sugar lignocellulose-derived hydrolysates are critical for producing fuels at a low cost to realize a commercially viable biorefinery. Biomass sugars are typically released by thermochemical pretreatment followed by enzymatic hydrolysis of chopped or milled biomass. In diluted acid pretreatment, most of the hemicellulosic sugars (xylose, arabinose, galactose, and mannose) are solubilized; however, a fraction of the hemicellulose remains insoluble and associated with the cellulose, preventing ready enzyme access to at least a portion of the cellulose. The glucose component remains in the solid form as cellulose, where it is depolymerized by cellulases. This step is often combined with microbial fermentation of the sugars to relieve the product inhibition of cellulases, the so-called simultaneous saccharification and fermentation (SSF) process. A process based on the fermentation of pentose sugars (derived from the hydrolysate) combined with the saccharification of cellulose and fermentation of glucose (derived from simultaneous enzymatic saccharification) is referred to as a simultaneous saccharification and co-fermentation (SSCF). To be successful, this scheme requires that the microorganisms are capable of fermenting hexose and pentose sugars equally well. Alternatively, a hybrid process with partial enzymatic hydrolysis (to obtain high cellulose hydrolysis rate by operating at high temperature) and co-fermentation may be used to achieve high overall conversion rates of biomass sugars to ethanol. Additionally, microorganisms are often susceptible to inhibitors, such as acetic acid, furfural, and phenolic compounds librated from lignocellulose during chemical pretreatment (95, 96). Because of this, a detoxification step, such as the “over-lime process” is generally applied to reduce the toxicity of the hydrolysate. Alternatively, adapted and engineered fermentative strains can be created which are resistant to the various inhibitory compounds. Although a number of microorganisms can efficiently ferment glucose to ethanol, only recently has conversion of the pentose sugars in the hemicellulosic fraction become feasible (97). The few organisms that were known to utilize either D-xylose or L-arabinose typically grow slowly on pentoses and achieve relatively low ethanol yields and productivities (98). Because of this, the identification and development of microorganisms capable of selectively converting D-glucose, D-xylose, and L-arabinose to ethanol at high yield has been the focus of extensive research during the past 10 to 15 years. In the past decade, the sophistication of molecular biology has grown tremendously and numerous attempts have been made to use recombinant DNA technologies to engineer superior microorganisms for bioethanol production.

Consolidated Bioprocessing More recent process scenarios have been proposed that combine key process steps, thus reducing overall process complexity and cost. One notable example is the consolidated biomass processing (CBP) technology proposed by Zhang and Lynd (99) for the Clostridium thermocellum (C. thermocellum) case. Their work reminds us that C. thermocellum hydrolyzes cellulose by a different mode of action compared to the classical mechanism associated with fungal-derived cellulases, the “cellulosome.” Furthermore, for C. thermocellum, the bioenergetic benefits specific to growth on cellulose are result from the efficiency of oligosaccharide uptake combined with intracellular phosphorolytic cleavage of beta-glucosidic bonds, another pathway not known in fungi. Zhang and Lynd believe that these benefits exceed the bioenergetic cost of cellulase synthesis, supporting the feasibility of anaerobic processing of biomass. Another option for CBP is to enable yeast, already ethanologenic, to produce cellulases (100). In this case, expression of active and effective cellulases in yeast has proven challenging (101); however, endoglucanases and beta-glucosidases appear more amenable to yeast processing (102). A third route has been proposed for use in advanced hydrocarbon fuel production. Engineering of cellulolytic filamentous fungi, to produce hydrocarbon fuels or precursors directly through metabolic pathway engineering holds promise for effective conversion of biomass to these new fuels. Numerous minor metabolic pathways towards these products exist in these fungi and new fungal engineering techniques are advancing the tweaking of these pathways to redirect carbon and energy towards these products. Several small seed projects at NREL have demonstrated the potential of this method and NREL is rapidly building the genetic tools needed to expand this work.

Future DirectionsFor the engineer seeking to improve and employ the microbial production of the advanced biofuels process, many of the key challenges encountered in the production of bioethanol remain. Indeed, new challenges also arise. In other cases, lessons learned from bioethanol production R&D also benefit the related advanced biofuels production schemes and act to leverage these new processes. Firstly, biomass depolymerization must be made a more rapid and less costly process; this means the development of enzymes with improved characteristics. The recent discovery of the new class of cellulose depolymerizing enzymes, the PMOs, supports the idea that traditional fungal cellulases and hemicellulases can be further enhanced by the addition of enzymes that function via new mechanisms. Furthermore, the application of new tools, such as informational tools (systems biology) (112-114), biophysical tools (advanced imaging) (115), and computation tools (molecular simulations using molecular dynamics and quantum mechanics) (116) has already brought new insights to the problem of improving enzyme performance. Because biomass pretreatment and enzyme use are closely linked, pretreatment science remains a critical research area. Most researchers in the field today agree that this objective will be met by the “tuning” of pretreatment chemistry and severity to plant type and enzyme cocktail intended for use. The objective is to optimize the reduction in pretreatment severity with respect to process schemes and enzyme components. Reduction in pretreatment severity benefits the process by reducing the cost of the pretreatment unit operation, especially materials of construction. New combinations of targeted chemical

treatments applied prior to traditional pretreatment, such as the NaOH deacetylation, and mechanical processing, such as disc refining, should be closely examined to enhance enzyme cost and reduce slurry toxicity. New unit operations, such as high temperature hold steps, appear to be an effective way to introduce highly active new enzymes which operate at temperatures greater than the fermentative strains can tolerate.

As was the case for bioethanol fermentation, strains producing advanced biofuels must also be required to process the full spectrum of five- and six-carbon sugars released from cellulose and hemicelluloses to product. For the case of bioethanol, the advent of efficient genetically engineered organisms equipped with metabolic pathways to handle all biomass sugars is a key improvement in the process that has occurred just in the past decade or so (117-122). Despite initial success in demonstrating microorganisms capable of producing some advanced fuels, there is currently a dearth of candidates for industrial scale production of advanced fuels. These processes demand robust performance at low pH and high temperature, as well as a high tolerance to product. As stated above, unlike the starch-based glucose streams, hydrolysates derived from lignocellulosic feedstocks can contain many toxic compounds that inhibit microbial growth and fermentation (123). In addition, toxicities of the diverse advanced biofuels can also present challenges in microorganism’s ability to tolerate inhibitors due to the microbial toxicities of some of the compounds. Improving our understanding of inhibition mechanisms and microbial physiology during hydrolysate fermentations for advanced biofuels production will require full use of the advanced analytical and “omics” metabolic engineering and modeling tools recently made available. This approach will greatly enhance our capability to develop a new class of robust industrial microorganisms capable of efficiently and productively converting all biomass sugars to advanced biofuels under actual industrial processing conditions.

Barrier Area 1: Feedstocks Research Activities Ideal feedstock qualities for

producing hydrocarbons are not well known

Few characterization studies on feedstock impacts during various conversion/upgrading processes have been performed

Feedstock variability (moisture, ash content, etc.) changes the severity required during pretreatment

Investigate how multiple pretreatment/enzymatic saccharification regimes are impacted by the use of uniform feedstock formats

Assess the best way to rehydrate dried and densified feedstocks for biological processing

Investigate the microbial deconstruction of biomass prior to pretreatment

Develop harvesting, collection, and storage methods to minimize soil pickup and material losses

Investigate the use of genetically modified feedstocks for enhanced sugar yields and reduced pretreatment severity

Barrier Area 2: Pretreatment Research Activities Fundamental aspects of

pretreatment chemistry are still largely unstudied and poorly characterized

Fractionation technologies are still relatively immature

No cost/benefit analysis on the advantages to increasing or decreasing pretreatment severity exists

Identify feedstock particle size reduction tradeoffs for digestibility, lower pretreatment severity and enzyme usage

Perform R&D centered on reducing biomass recalcitrance should be specifically focused on reducing the degree of polymerization and crystalinity in the cellulose fraction

Perform applied R&D on the mechanical front-end needs to focus on fractionation (i.e. clean lignin removal) and the production of a highly concentrated C5/C6 sugar stream

Define the effects of increased xylan concentration in C6-specific unit operations and associated cost impacts

Develop new (fundamental) methods for species-selective adsorption during integrated cleanup

Barrier Area 3: Enzyme Science & Biotechnology

Research Activities

The mechanistic basis underlying the action of most hydrolytic enzymes are still largely misunderstood

Poor categorization and understanding of the natural diversity in hydrolytic enzymes

The advantages and disadvantages of using lignolytic enzyme systems have received relatively little R&D focus

The specific activity of hydrolytic enzymes are typically low

Critical need for understanding applied processes (not basic science)

End products (sugars and degradation products) inhibit enzymes, preventing high

Fulfill need for increased substrate structure chemistry R&D (characterize enzymes interaction and changes during digestion)

Investigate integrating thermochemical/thermomechanical pretreatment with enzymatic saccharification

Design new cellulase enzymes with enhanced thermostability and pH tolerance

Perform fundamental studies to ascertain why glycans and C6 oligomers decrease cellulase activity

Design new hydrolytic enzymes to work synergistically in mild pretreatment conditions

Focus on applied R&D for enzyme reuse/recycle in industrial conditions

sugar concentrations Lignin derived species hinder

enzyme efficiencies Cellulase enzyme loading with

high solids (>20% w/w) is cost prohibitive

Barrier Area 4: Separation Issues

Research Activities

Low sugar concentration hydrolyzates require extensive purification and cleanup

Separation processes for sugar concentration are typically energy intensive

Solids from biomass deconstruction complicate product recovery

Separation and cleanup of C5/C6 sugars following pretreatment is cost prohibitive (i.e., too many steps typically required)

R&D on the integration of reactor and separation system design; focus on converting batch processes to continuous processes

Develop economically valid technologies for high solids separations with process-compatible filtration aids such as flocculants and low-cost polymers

Develop ultra-low fouling membrane filtration system Design integrated processes that require minimal washing

of solids R&D on sugar concentration technologies (i.e., membrane

filtration technology, selective adsorption systems and mesoporous materials)

Barrier Area 4: Economics Research Activities Sugar is a commodity

(changes in cost will make some conversions not cost effective)

Production of useless proteins in enzyme preparation hinders economics

Limited understanding of the cost of process stream cleanup unit operations

CAPEX and OPEX in pretreatment and

Perform TEA in industrially relevant environments (high solids, modest enzyme dose) for varying pretreatment conditions and methods

Modify/customize processes to allow for integration and re-tasking of decommissioned and pre-existing infrastructure, such as pulp and paper mills

saccharification excluding enzymes and feedstock accounts for >80% of process costs

Feedstock cost and availability

Thermodynamics may be limiting economics

Cost of pretreatment equipment due to material of construction is a limiting factor

References

1. Baker J.O., Thomas S.R., Adney W.S., Nieves R.A., Himmel M.E. 4664 (1994) The cellulase synergistic effect—binary and ternary-systems. Abstract Paper Am. Chem. Soc. 207:50.

2. Nidetzky B., Steiner W, Hayn M., Claeyssens M. (1994) Cellulose hydrolysis by the cellulases from Trichoderma reesei: a new model for synergistic interaction. Biochem. J. 298:705–710.

3. Burke R., Cairney J. (2002) Laccases and other polyphenol oxidases in ecto- and ericoid mycorrhizal fungi. Mycorrhiza 12(3):105–116.

4. Shah V., Nerud F. (2002) Lignin degrading system of white-rot fungi and its exploitation for dye decolorization. Canada J. Microbiol. 48(10):857–870.

5. Tuomela M., Vikman M., Hatakka A., Itavaara M. (2000) Biodegradation of lignin in a compost environment: a review. Bioresource Technol. 72(2):169–183.

6. Garg S., Modi D. (1999) Decolorization of pulp-paper mill effluents by white-rot fungi. Crit. Rev. Biotechnol. 19(2):85–112.

7. Cullen D. (1997) Recent advances on the molecular genetics of ligninolytic fungi. J. Biotechnol. 53(2–3):273–289.

8. Zadrazil F., Kamra D., Isikhuemhen O, Schuchardt F., Flachowsky G.(1996) Bioconversion of lignocellulose into ruminant feed with white rot fungi—review of work done at the FAL, Braunschweig. J. Appl. Animal Res. 10(2):105–124.

9. Sjostrom E. (1993) Wood chemistry: fundamentals and applications. Academic, San Diego, pp 13–17.

10. Fry S.C., Miller J.G. (1989) Toward a working model of the growing plant cell wall. In: Lewis NG, Paice MG (eds) Plant cell wall polymers. American Chemical Society, Washington, DC, pp 33–46.

11. LaForge FB, Hudson CS (1918) The preparation of several useful substances from corn cobs. J. Ind. Eng. Chem. 10(11):925–927.

12. Sherrard EC (1922) Ethyl alcohol from western larch—larix occidentalis, nuttal. Ind. Eng. Chem 14(10):948–949.

13. Sherrard EC, Gauger WH (1923) Effect of salts upon the acid hydrolysis of wood. Ind. Eng. Chem. 15(1):63–64.

14. Dunning J., Lathrop E. (1945) The saccharification of agricultural residues: a continuous process. Ind. Eng. Chem. 37(1):24–29.

15. Faith W. (1945) Development of the Scholler process in the United States. Ind. Eng. Chem. 37(1):9–11.

16. Saeman J. (1945) Kinetics of wood saccharification: hydrolysis of cellulose and decomposition of sugars in dilute acid at high temperature. Ind. Eng. Chem. 37(1): 43–51.

17. Harris E., Berlinger E. (1946) Madison wood-sugar process, R1617. U.S. Forest Product Laboratory, Madison, WI.

18. Harris E. (1949) Wood saccharification. In: Advances in carbohydrate chemistry. Academic, New York, Vol 4, pp 163–188.

19. Gilbert N., Hobbs I., Levine J. (1952) Hydrolysis of wood using dilute sulfuric acid. Ind. Eng. Chem. 44(7): 1712–1720.

20. Wenzl H. (1970) Chapter IV: the acid hydrolysis of wood. In: The chemical technology of wood. Academic, New York, pp 157–252.

21. Grethlein H. (1978) Comparison of the economics of acid and enzymatic hydrolysis of newsprint. Biotechnol. Bioeng. 20:503–525.

22. Wright J. (1983) High-temperature acid hydrolysis of cellulose for alcohol fuel production, SERI/TR-231-1714. Solar Energy Research Institute, Golden, CO.

23. Grohmann K., Himmel M., Rivard C., Tucker M., Baker J., Torget R., Graboski M. (1984) Chemical–mechanical methods for the enhanced utilization of straw. In: Biotechnology and bioengineering symposium. Wiley, New York, pp 137–157.

24. Church J., Woolbridge D (1981) Continuous high solids acid hydrolysis of biomass in a 1.5 in plug flow reactor. Ind. Eng. Chem. Prod. Res. Devel. 20:371–378.

25. Wright J., d’Agnicourt C. (1984) Evaluation of sulfuric acid hydrolysis for alcohol fuel production. In: Biotechnol. Bioengin. Symposium. Wiley, New York, pp 105–123.

26. Wright J., Power A., Bergeron P. (1985) Evaluation of concentrated halogen acid hydrolysis processes for alcohol fuel production, SERI/TR-232-2386. Solar Energy Research Institute, Golden, CO.

27. Farone W.A., Cuzens J.E. (1996) Methods of producing sugars using strong acid hydrolysis of cellulosic and hemicellulosic materials. U.S. Patent 5,562,777, 8 Oct 1996.

28. Wright J.D., D’Agincourt C.G. (1984) Evaluation of sulfuric acid hydrolysis processes for alcohol fuel production. Biotechnol. Bioeng. Symp 14:105–121.

29. Grohmann K., Torget R., Himmel M. (1985) Optimization of dilute acid pretreatment of biomass. Biotechnol. Bioeng. Symp. 15:59–80.

30. Kong F., Engler C.R., Soltes E.J. (1993) Effects of cell-wall acetate, xylan backbone, and lignin on enzymatic hydrolysis of aspen wood. Appl. Biochem. Biotechnol. 34/35:23–35.

31. Chang V., Holtzapple M. (2000) Fundamental factors affecting biomass enzymatic reactivity. Appl. Biochem. Biotechnol. 84–86:5–37.

32. Vinzant T.B., Ehrman C.I., Himmel M.E. (1997) SSF of pretreated hardwoods: effect of native lignin content. Appl. Biochem. Biotechnol. 62:97–102.

33. Henrissat B. (1994) Cellulases and their interaction with cellulose. Cellulose 1:169–196.

34. Cadoche, L. and Lopez, G.D. (1989) Assessment of size reduction as a preliminary step in the production of ethanol form lignocellulosic wastes. Biol. Wastes, 30: 153-157.

35. Hsu, T.A. (1996) Pretreatment of biomass. In: Handbook on Bioethanol, Production and Utilization (ed, C.E. Wyman), pp. 179-211, Taylor & Francis, Washington, D.C.

36. Duff, S.J.B. and Murray, W.D. (1996) Bioconversion of forest products industry waste cellulosics to fuel ethanol: a review. Bioresource Technol. 55: 1-33.

37. McMillan, J. D. (1994) Pretreatment of lignocellulosic biomass. In: Enzymatic Conversion of Biomass for Fuels Production (eds M.E. Himmel, J.O. Baker & R.P. Overend), pp. 292-324, American Chemical Society Symposium Series 566, Washington D.C.

38. Saddler, J.N., Ramos, L.P. and Breuil, C. (1993) Steam pretreatment of lignocellulosic residues. In: Bioconversion of Forest and Agricultural Plant Wastes (ed J.N. Saddler), pp. 73-92, C.A.B. International, Wallingford, UK.

39. Wright, J.D. (1988) Ethanol from biomass by enzymatic hydrolysis. Chem. Eng. Progress, 84: 62-74.

40. Brownell, H.H. and Saddler, J.N. (1984) Steam explosion pretreatment for enzymatic hydrolysis. Biotechnol. Bioeng. Symposium, 14: 55-68.

41. Teymouri, F., Laureano-Perez, L., Alizadeh, H, & Dale, B.E. (2005) Optimization of ammonia fiber explosion (AFEX) treatment parameters for enzymatic hydrolysis of corn stover. Bioresource Technol. 96, 2014-2018.

42. Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y., Holtzapple, M. & Ladisch, M. (2005) Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresource Technol. 96, 673-686.

43. McMillan, J.D. (1994) Pretreatment of lignocellulosic biomass. In: Enzymatic Conversion of Biomass for Fuels Production (eds, M.E. Himmel, J.O. Baker and R.P. Overend), pp. 292-324, American Chemical Society Symposium Series 566, Washington D.C.

44. Eggeman, T. & Elander, R.T. (2005) Process and economic analysis of pretreatment technologies. Bioresource Technol. 96, 2019-2025.

45. Dale, B.E., Leong, C.K., Pham, T.K., Esquivel, V.M., Rios, I. & Latimer, V.M. (1996) Hydrolysis of lignocellulosics at low enzyme levels: Application of the AFEX process. Bioresource Technol. 56, 111-116.

46. Moniruzzaman, M., Dale, B.E., Hespell, R.B. & Bothast, R.J. (1997) Enzymatic hydrolysis of high moisture content corn fiber pretreated by AFEX and recovery and recycle of the enzyme complex. Applied Biochem. Biotechnol. 67, 113-126.

47. Gollapalli, L.E., Dale, B.E. & Rivers, D.M. (2002) Predicting digestibility of ammonia fiber explosion (AFEX)-treated rice straw. Applied Biochem. Biotechnol. 98-100, 23-35.

48. Bobleter, O. (1994) Hydrothermal degradation of polymers derived from plants. Progress in Polymer Science, 20, 2083-2093.

49. Weil, J.R., Brewer, M., Hendrickson, R., Sarikaya, A. & Ladsich, M.R. (1997) Continuous pH monitoring during pretreatment of yellow poplar sawdust by pressure cooking in water. Appl. Chem. Biotechnol. 68, 21-40.

50. Esteghalian, A., Hashimoto, A.G., Fenske, J.J. & Penner, M.H. (1997) Modeling and optimization of dilute-sulfuric-acid pretreatment of corn stover, poplar, and switchgrass. Bioresource Technol. 59, 129-136.

51. Torget, R. Hatzis, C., Hayward, T.K., Hsu, T.-A. & Philippidis, G.P. (1996) Optimization of reverse-flow, two-temperature, dilute-acid pretreatment to enhance biomass conversion to ethanol. Appl. Biochem. Biotechnol. 57-58, 85-101.

52. Sun, Y. & Cheng, J. (2002) Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technol. 83, 1-11.

53. Bjerre, A.B., Olesen, A.B. and Fernqvist, T. (1996) Pretreatment of wheat straw using combined wet oxidation and alkaline hydrolysis resulting in convertible cellulose and hemicellulose. Biotechnol. Bioeng. 49, 568-577.

54. Wyman, C.E., Dale, B.E., Elander, R.T., Holtzapple, M., Ladisch, M.R. & Lee, Y.Y. (2005) Coordinated development of leading biomass pretreatment technologies. Bioresource Technol. 96, 1959-1966.

55. Detroy, R.W., Lindenfelser, L.A., St. Julian, Jr., G. & Orton, W.L. (1980) Saccharification of wheat-straw cellulose by enzymatic hydrolysis following fermentative and chemical pretreatment. Biotechnol. Bioeng. Symposium, 10, 135-148.

56. Kim, T.H. & Lee, Y.Y. (2005) Pretreatment of corn stover by soaking in aqueous ammonia. Appl. Biochem. Biotechnol. 121, 1119-1131.

57. Kim, S. & Holtzapple, M.T. (2005) Lime pretreatment and enzymatic hydrolysis of corn stover. Bioresource Technol. 96, 1994-2006.

58. Kaar, W.E. & Holtzapple, M.T. (2000) Using lime pretreatment to facilitate the enzymatic hydrolysis of corn stover. Biomass Bioenergy 18, 189-199.

59. Chang, V.S., Nagwani, M., Kim, C.H. & Holtzapple, M.T. (2001) Oxidative lime pretreatment of high-lignin biomass. Appl. Biochem. Biotechnol. 94, 1-28.

60. Chum, H.L., Johnson, D.K., Black, S., Baler, J., Grohmann, K. & Sarkanen, K.V. (1988) Organosolv pretreatment for enzymatic hydrolysis of poplars: I. Enzyme hydrolysis of cellulosic residues. Biotechnol. Bioengin. 31, 643-649.

61. Thring, R.W., Chornet, E. and Overend, R. (1990) Recovery of a solvolytic lignin: effects of spent liquor/acid ratio, acid concentration and temperature. Biomass, 23, 289-305.

62. Paszner, L. and Cho, H.J. (1988) High efficiency of lignocellulosics to sugars for liquid fuel production by the ACOS process. Energy Exploitation Exploration, 6, 39-60.

63. Black, S.K., Hames, B.R. & Myers, M.D. (1998) Method of separating lignocellulosic material into lignin, cellulose, and dissolved sugars. U.S. Patent 5,730,837.

64. Blanchette, R.A. (1991) Delignification by wood-decay fungi. Ann. Rev. Phytopathol. 29-381-398.

65. Akin, D.E., Rigsby, L.L., Sethuraman, A., Morrison, W.H.-III, Gamble, G.R. & Eriksson, K.E.L. (1995) Alterations in structure, chemistry, and biodegradability of grass lignocellulose treated with the white rot fungi Ceriporiopsis subvermispora and Cyanthus stercoreus. Appl. Environ. Microbiol. 61, 1591-1598.

66. Boraston A.B., (2005) The interaction of carbohydrate-binding modules with insoluble non-crystalline cellulose is enthalpically driven. Biochem. J. 385(2): 479–484.

67. Davies G., Henrissat B. (1995) Structures and mechanisms of glycosyl hydrolases. Structure 3, 853-859.

68. Rouvinen J., Bergfors T., Teeri T., Knowles J.K.C., Jones T.A. (1990) Three-dimensional structure of cellobiohydrolase II from Trichoderma reesei. Science 279, 380-386.

69. Divne C., Ståhlberg J., Reinikainen T. et al., (1994) The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei. Science 265, 524-528.

70. Chanzy H., Henrissat B., (1985) Unidirectional degradation of Valonia cellulose microcrystals subjected to cellulase action. FEBS Lett. 184, 285-288.

71. Imai T., Boisset C., Samejima M., Igarashi K., Sugiyama J., (1998) Unidirectional processive action of cellobiohydrolase Cel7A on Valonia cellulose microcrystals. FEBS Lett. 432, 113-116.

72. Teeri T.T. (1997) Crystalline cellulose degradation: new insight into the function of cellobiohydrolases. Trends Biotechnol. 15, 160-167.

73. Bayer E.A., Belaich J.-P., Shoham Y., Lamed R., (2004) The cellulosomes: Multi-enzyme machines for degradation of plant cell wall polysaccharides. Annu. Rev. Microbiol. 58, 521-554.

74. Demain A.L., Newcomb M., Wu J.H., (2005) Cellulase, clostridia, and ethanol. Microbiol. Mol. Biol. Rev. 69, 124-154.

75. Doi R.H., Kosugi A. (2004) Cellulosomes: plant-cell-wall-degrading enzyme complexes. Nature Rev. Microbiol. 2, 541-551.

76. Schwarz WH, Zverlov VV, Bahl H., (2004) Extracellular glycosyl hydrolases from Clostridia. Adv. Appl. Microbiol. 56, 215-261.

77. Raman B., Pan C., Hurst G.B. et al, (2009) Impact of pretreated Switchgrass and biomass carbohydrates on Clostridium thermocellum ATCC 27405 cellulosome composition: a quantitative proteomic analysis. PLoS ONE 4, e5271.

78. Harris P.V., Welner D., McFarland K.C., Re E., Poulsen J.C.N., Brown K., Salbo R., Ding H.S., Vlasenko E., Merino S., et al, (2010) Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family. Biochemistry 49: 3305–3316.

79. Quinlan R.J., Sweeney M.D., Lo Leggio L., Otten H., Poulsen J.C.N., Johansen K.S., Krogh K., Jorgensen C.I., Tovborg M., Anthonsen A., et al, (2011) Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. Proc. Natl. Acad. Sci. US A 108: 15079 15084.

80. Langston J.A., Shaghasi T., Abbate E., Xu F., Vlasenko E., Sweeney M.D. (2011) Oxidoreductive cellulose depolymerization by the enzymes cellobiose dehydrogenase and glycoside hydrolase 61. Appl. Environ. Microbiol. 77: 7007–7015.

81. Phillips C.M., Beeson W.T., Cate J.H.D., Marletta M.A. (2011) Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa. ACS Chem. Biol. 6:1399–1406.

82. Beeson W.T., Phillips C.M., Cate J.H.D., Marletta M.A. (2012) Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases. J. Am. Chem. Soc. 134: 890–892.

83. Li X., Beeson W.T, Phillips C.M., Marletta M.A., Cate J.H.D. (2012) Structural basis for substrate targeting and catalysis by fungal polysaccharide monooxygenases. Structure 20:1051-1061.

84. Puls J., Tenkanen M., Korte H.E., Poutanen K., (1991) Products of hydrolysis of beechwood acetyl-4-O-methylglucuronoxylan by a xylanase and an acetyl xylan esterase. Enzyme Microb Technol 13, 483-486.

85. Greve L.C., Labavitch J.M., Hungate R.E., (1984) -L-arabinofuranosidase from Ruminococcus albus 8: purification and possible role in hydrolysis of alfalfa cell wall. Appl Environ Microbiol 47, 1135-1140.

86. Johnson K.G., Silva M.C., MacKenzie C.R., Schneider H., Fontana J.D., (1989) Microbial degradation of hemicellulosic materials. Appl. Biochem. Biotechnol. 20/21, 245-258.

87. Grohmann K., Torget R., Himmel M. (1985) Optimization of dilute acid pretreatment of biomass. Biotechnol. Bioeng. Symp. 15:59–80.

88. Kong F., Engler C.R., Soltes E.J. (1992) Effects of cell-wall acetate, xylan backbone, and lignin on enzymatic hydrolysis of aspen wood. Appl. Biochem. Biotechnol. 34/35:23–35.

89. Tenkanen M, Poutanen K (1992) Significance of esterases in the degradation of xylans. In: Visser J, Beldman G, Kusters-van Someren MA, Voragen AGJ (eds) Xylans and xylanases. Elsevier, New York, pp 203–212.

90. Poutanen K, Sundberg M (1988) An acetyl esterase of Trichoderma reesei and its role in the hydrolysis of acetyl xylans. Appl. Microbiol. Biotechnol. 28:419–424.

91. Poutanen K., Sundberg M., Korte H, Puls J. (1990) Deacetylation of xylans by acetyl esterases of Trichoderma reesei. Appl Microbiol. Biotechnol. 33: 506–510.

92. Biely P., Vrsanska M., Kremnicky L., Alfoldi J., Tenkanen M. et al (1994) Family F and G endo-beta-1,4-xylanases—differences in their performance on glucuronoxylan and rhodymenan. Abstr. Paper Am. Chem. Soc. 207: 148-BTEC.

93. Nishitani K., Nevins D.J. (1991) Glucuronoxylan xylanohydrolase. A unique xylanase with the requirement for appendant glucuronosyl units. J. Biol. Chem. 266(10): 6539–6543.

94. de Vries R.P., Kester H.C., Poulsen C.H., Benen J.A., Visser J. (2000) Synergy between enzymes from Aspergillus involved in the degradation of plant cell wall polysaccharides. Carbohydr. Res. 327(4): 401–410.

95. Helle S., Cameron D. et al (2003) Effect of inhibitory compounds found in biomass hydrolysates on growth and xylose fermentation by a genetically engineered strain of Saccharomyces cerevisiae, Enzyme Microb. Technol. 33(6): 786–792 64.

96. Klinke H.B., Thomsen A.B. et al (2004) Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl. Microbiol. Biotechnol. 66 (1):10–26 4419.

97. Dien B.S., Cotta M.A., Jeffries T.W. (2003) Bacteria engineered for fuel ethanol production: current status. Appl. Microbiol. Biotechnol. 63(3):258–266.

98. Dupreez J.C., Bosch M., Prior B.A. (1986) The fermentation of hexose and pentose sugars by Candida shehatae and Pichia stipitis. Appl. Microbiol. Biotechnol. 23(3–4): 228–233.

99. Zhang Y-HP, Lynd L.R. (2005) Cellulose utilization by Clostridium thermocellum: bioenergetics and hydrolysis product assimilation. Proc. Natl. Acad Sci. U S A 102(20):7321–7325.

100. VanRensburg P., Zyl W.H.V., Pretorius I.S. (1998) Engineering yeast for efficient cellulose degradation. Yeast 14(1):67–76 4516.

101. Godbole S., Decker S.R., Nieves R.A., Adney W.S., Vinzant T.B. et al (1999) Cloning and expression of Trichoderma reesei CBH I in Pichia pastoris. Biotechnol. Prog. 15(3): 828–833 4519.

102. VanRooyen R., Hahn-Hagerdal B., Grange D.C.L., VanZyl W.H. (2003) Comparative expression of novel beta-glucosidases in Saccharomyces cerevisiae. Yeast 20: S223

103. Renninger, N.S., Mcphee, D.J., Fuel compositions comprising farnesane and farnesane derivatives and method of making and using same. US patent 7,846, 222, (2008).

104. Zhang, M., (2112) Metabolic engineering of Zymomonas mobilis for hydrocarbon fuels production. SIMB Annual Meeting – 2012.

105. Peralta-Yahya P.P., et al., (2011) Identification and microbial production of a terpene-based advanced biofuel. Nature Communications 2, 483.

106. Bokinsky G., et al., (2011) Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli. Proc. Natl. Acad. Sci. U S A 108, 19949.

107. Steen E.J. et al., (2010) Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 463, 559.

108. Dellomonaco, C., Clomburg, J.M., Miller, E.N., Gonzalez, R., (2011) Engineered reversal of the beta-oxidation cycle for the synthesis of fuels and chemicals. Nature 476, 355.

109. Goh, E.B., Baidoo, E.E., Keasling, J.D., Beller, H.R., (2012) Engineering of bacterial methyl ketone synthesis for biofuels. Appl. Environ. Microbiol. 78, 70.

110. Kalscheuer, R., Stolting, T., Steinbuchel, A., (2006) Microdiesel: Escherichia coli engineered for fuel production. Microbiology 152, 2529.

111. Schirmer, A., Rude, M.A., Li, X., Popova, E., del Cardayre, S.B., (2010) Microbial biosynthesis of alkanes. Science 329, 559.

112. van der Lelie, D., Taghavi, S., McCorkle, S.M., Li, L.L., Malfatti, S.A., Monteleone, D., Donohoe, B.S., Ding, S.Y., Adney, W.S., et al. (2012) The Metagenome of an Anaerobic Microbial Community Decomposing Poplar Wood Chips. PLoS one, 7 (5): Article No. e36740.

113. Li, L.L., McCorkle, S.R., Monchy, S., Taghavi, S., and van der Lelie, D. (2009) Bioprospecting metagenomes: Glycosyl hydrolases for converting biomass. Biotechnol. for Biofuels, 2 : Art. No. 10.

114. Li, L.L., Taghavi, S., McCorkle, S.M., Zhang, Y.B., Blewitt, M.G., Brunecky, R., Adney, W.S., Himmel, M.E., Brumm, P., Drinkwater, C., Mead, D.A., Tringe, S.G., and van der Lelie, D. (2011) Bioprospecting metagenomics of decaying wood: mining for new glycoside hydrolases. Biotechnol. for Biofuels, 4 (Article No. 23).

115. Igarashi, K., Uchitashi, T., Koivula, A., Wada, M., Kimura, S., Okamoto, T., Pentilla, M., Ando, T., Samejima, M., (2011) Surface Traffic Jams Reduce Hydrolytic Efficiency of Cellulase on Cellulose, Science 333, 1279.

116. Beckham, G.T., Bomble, Y.J., Bayer, E.A., Himmel, M.E., Crowley, M.F., (2011) Applications of computational science for understanding enzymatic deconstruction of cellulose, Current Opin. Biotechnol. 22: 231–238.

117. Ingram L., Ohta K., Beall D. (1991) Energy from biomass and wastes. In: Klass D. (ed) Genetic modification of E. coli for ethanol production. Institute of Gas Technology, Chicago, pp 1105–1125

118. Lynd L.R., Weimer P.J., van Zyl W.H., Pretorius I.S. (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. 66(3): 506–577.

119. ISO (1997) ISO 14040: environmental management—life cycle assessment—principles and framework, ISO 14040: 1997(E). International Standardization Organization, Geneva, 15 June 1997.

120. ISO (2000) ISO/TR 14049: environmental management—life cycle assessment—examples of application of ISO 14041 to goal and scope definition and inventory analysis, ISO/TR 14049. International Organization for Standardization, Geneva, 15 March 2000.

121. ISO (1998) ISO 14041: environmental management—life cycle assessment—goal and scope definition and inventory analysis, ISO 14041:1998(E). International Organization for Standardization, Geneva, 1 Oct 1998.

122. ISO (1996) ISO 14004: environmental management systems—general guidelines on principles, systems and supporting techniques, ISO 14004:1996(E). International Organization for Standardization, Geneva, 1 Sept 1996.

123. Himmel M.E., Adney W.S., Baker J.O., Elander R., McMillan J.D. et al (1997) Advanced bioethanol production technologies: a perspective. In: Saha BC, Woodward J (eds) Fuels and chemicals from biomass, Vol 666. American Chemical Society, Washington, DC, pp 2–45

Nonenzymatic Routes to Sugars and Carbohydrate Derivatives from Lignocellulosic Biomass

Background The use of mineral acids in biomass processing has been employed since the early 1900s to yield fermentation sugars, and the Scholler Process developed in Germany in the 1930s resembles a precursor to many dilute acid processing techniques employed today, wherein dilute sulfuric acid was percolated through hardwood at high temperatures to deconstruct the biomass. Since that time, the state of the art has developed considerably and there are some non-enzymatic processing schemes that do not require the use of an acid or base catalyst, though most still do. One inherent advantage non-enzymatic sugar production routes have over enzymatic saccharification is that they can attain higher theoretical production capacities, as the hydrolytic process occurs at a relatively rapid pace in comparison to enzyme assisted biocatalysis. Additionally, separation of the saccharide product stream can be economically feasible and straightforward when compared to purifying the sugar content from a hydrolyzate as produced during enzymatic saccharification. Inasmuch, non-enzymatic routes offer the best opportunity for commoditization of lignocellulosic sugars as stable, transportable biomass intermediates.

Non-enzymatic routes to sugar generally employ a thermomechanical or catalytic processing strategy to fractionate whole biomass into cellulose, hemicellulose and lignin (the separability of the latter two varies by processing regime, whereas it is usually easier to attain clean cellulose). From there, the cellulose and the hemicellulose (which has been made accessible) are hydrolyzed to produce glucose, xylose and other five carbon sugars in high purity. Alternately, whole biomass can be deconstructed and hydrolyzed without fractionation and the resulting monomers and oligosugars can then be purified from the resulting mixture thereafter.

Non-enzymatic routes to sugar from biomass typically suffer from a myriad of problems relating to scale-up. Improved techniques for product and co-product separation are necessary to prove scale-up feasibility. Specifically, the development of extraction techniques for targeting clean

separation of organic and aqueous layers, improve bulk solids separations, development of acid inhibitor tolerant materials (e.g. membranes and other separations) and mechanical separations including (single/twin screw dewatering extruders), improved thermodynamic information, and development of improved computational modeling tools are all warranted. Holistically, research should be focused on the development of closed loop systems. This includes considerations for recycling of reagents, concentrating acids, strategies for dealing with impurity build-up at commercial scale, and concentration of desirable product streams. Such approaches are best suited for the development of one pot reactor process, and rely heavily on process parameters for various product distributions (time, temperature, reagent loading) when considering chemical slates aside from just sugars. In-process separation to recover desired constituents, removal of unwanted constituents early in the process, and removing lignin first from the process stream all impact optimization of processing parameters.

Barrier Area 1: Feedstocks Research Activities Feedstock variability (accepting

diverse feedstocks with varying moisture contents, identifying desired traits for different feedstocks relevant to different conversion processes)

Economics of in-field fractionation (removing only the desired portion of biomass) can be a major cost barrier

Biomass architecture is not ideal for mechanical processing

Focus research and development on overcoming recalcitrance

Improve genetic engineering opportunities to help enable facile biomass deconstruction, focus on designing feedstocks with lignin that easily depolymerizes (i.e. overexpress beta aryl ether linkages)

Scale hydrolysis systems for remote, in-field sugar production facilities (hub and spoke system)

Develop efficient biomechanical methods for processing raw biomass to obtain uniform, non-recalcitrant, easily densified products

Barrier Area 2: Processing Research Activities Product selectivity and control are

largely dependent upon individual process specifications

Strongly hydrogen-bonded biomolecules require severe conditions to digest

Corrosion and material handling issues arise when using strong acids and bases

Scale-up is not straightforward, as the majority of non-enzymatic sugar production routes are unproven at

Focus R&D on non-water-soluble polymeric catalysts that can efficiently interact with sugars from biomass slurries and hydrolyzate streams

Design processes to minimize sugar destabilization/degradation during acid hydrolysis

Study biological conversion of sugars from non-enzymatic routes in terms of toxicity/inhibitor issues

Target high-yielding processes that drive the bulk carbohydrate to a furan or carboxylic acid product instead of a sugar

Design acid inhibitor tolerant materials (e.g. membranes and coatings)

anything above demonstration scale Designing cellulosic wet mills for producing cheap, clean sugars to specifications for industrial use

Barrier Area 3: Separation Issues Research Activities Poor separation capabilities exist for

removing hydrophobic lignin from hydrophillic sugar stream

Poor separation capabilities exist for removing co-solvents from biomass hydrolyzate streams

Economical recycling of reagents (challenges in developing closed loop systems, recovery of salts from acid and base hydrolyzed biomass residue)

Concentrating sugars/products for economical transportation

Solubilizing sugars from the bulk biomass (optimizing biomass to solution surface boundary layer interactions, e.g. shear forces).

Perform pilot scale R&D on separating water, acids and bases from biomass hydrolyzates at high solids loading (>20% w/w) with and without solvent

Examine extraction techniques to target clean separation of lignin-rich organic and water layers

Design screw extruding systems for co-current/counter-current biomass fractionation and dewatering

Collect thermodynamic data and perform process simulation modeling to understand the mechanistic basis behind non-enzymatic sugar routes

Developing closed loop systems Recycling of reagents Concentrating acids from initial processing stream Perform modeling to assess the impact of impurity build-

up at commercial scale Utilize integrated user scale-up facility from pilot to demo,

commercial Focus lab-scale R&D on designing processes to increase

mass transport of reagents to crystalline, hydrogen-bonded biomass substrate (i.e. overcoming rate limiting steps in hydrolysis)

Chemical Conversion of Sugars and Carbohydrate Derivatives to Hydrocarbon Fuels

The Contrast between Biological Processing of Sugars and Chemical Processing of Sugar and Carbohydrate Derivatives, and Upstream RequirementsBiochemical and thermochemical deconstruction and upgrading processes are the main approaches employed to convert biomass to fuels and products. Figure 1 shows a simplified

flow scheme considering possible uses of these two approaches. Following feedstock selection and handling, processing options include all-biological and all-thermochemical routes, as well as hybrid biochemical/thermochemical processes. Relative characteristics of biochemical and thermochemical processing are compared in Table 1.

The co-design of upstream processes for biomass deconstruction with the downstream processes to convert biomass-derived intermediates to fuels is important. In particular, upstream processes determine the product slate of biomass-derived intermediates (including intermediates derived from lignin) and the potential introduction of contaminants, poisons, and fouling agents. The composition of the intermediate streams will significantly impact the final product slates, catalyst and enzyme lifetimes, separations, and the operation of the downstream unit operations, all of which impact the economics of the processes.

Biochemical deconstruction of complex biomass can result in high selectivities to monomeric C5 and C6 sugars, but at rates slower than thermochemical routes. The thermochemical routes may have higher rates, but selectivity to monomeric sugars is lower. Mixtures of mono- and polysaccharide C5 and C6 carbohydrates are formed at various levels of purity depending on processing conditions. Some thermochemical processes degrade sugars, particularly the more sensitive C5 sugars like xylose.

Figure 1. Process flow diagram showing options for biochemical and thermochemical processing of biomass.

Feedstock

Biochemical Deconstruction

Feedstock Handling

ThermochemicalDeconstruction

Biochemical Upgrading

ThermochemicalUpgrading

Separations

Fuels and Products

Recovered Reagents Wastes

Table 1. Relative Characteristics of Biochemical and Thermochemical Transformations.

Feed Concentration

Product Selectivity

Conversion Rate

Feed Diversity

Biochemical low high low lowThermochemical high low high high

Both deconstruction treatments have the potential to introduce impurities that can affect downstream processing. Such impurities could comprise biomass components (such as lignin degradation products), enzyme degradation products (such as proteins), and acid reagents used in thermochemical deconstruction (such as chloride, nitrate, and sulfate counterions).

Downstream thermochemical upgrading processes will have different feed requirements than biochemical processes. For example, proteins from biochemical pretreatments will likely need to be removed before introduction to the thermochemical fuel producing process, while furans from a thermochemical pretreatment are detrimental to biochemical processes. Dirty hydrolyzate products, therefore, will likely need to be cleaned up before introduction to a catalytic process or fermentation, unless improved catalysts and enzymes are found that are insensitive to these impurities.

Compared to biochemical processes, conversion of intermediates in thermochemical processes is less sensitive to the ring size and degree of oligomerization of the feed sugars. While both monomeric and oligomeric feeds are readily converted under thermochemical conditions, monomeric sugars are needed in most fermentation schemes. C6 sugars are much more readily converted by most organisms in fermentations, while relatively few strains are presently able to convert C5 sugars to products.

Selectivity is also different for the two approaches, with high selectivities to relatively few products in biochemical processes and often low selectivities to wide spread of products in thermochemical conversions. Fuels used today are broad mixtures of diverse chemical species, so product selectivity during fuel production may or may not be a benefit depending on the products formed. All fuels are held to rigorous ASTM standards as a neat fuel or a blend with other hydrocarbon fractions, for boiling point, flash point, freezing point, and many others. Processes with low selectivities often maximize the carbon yield in these favorable fractions with desired properties, minimizing yields in high-end and low-end distillation cuts. Alternatively, any products that can be fed directly into existing refinery processes without refinery process modifications are valuable as feedstocks.

Carbon yield is the biggest driver in process economics and maximizing carbon incorporation into hydrocarbon product fractions with fuel value is often considered the ideal. However, this

may or may not be a reasonable expectation. Conversion of a carbohydrate to a hydrocarbon requires removal of oxygen atoms, regardless of the reaction path taken. Removal as molecular oxygen is infeasible, leaving only a limited number of reasonable routes. Three routes include oxygen removal with hydrogen (H2) to form water, carbon to form carbon monoxide (CO), and CO to form carbon dioxide (CO2). Formation of more highly oxidized organic compounds as co-products might also be possible, but these species will have limited fuel value unless follow-up reactions remove oxygen, for example, as CO2. The first route maximizes carbon incorporation into fuel products, however it requires a H2 source, which may or may not be economically feasible or desirable from a life-cycle standpoint. Oxygen scavenging by carbon or CO, including cases where CO is converted to H2 via water gas shift, reduces the need for external H2 generation, but decreases carbon yield. In either case, finding systems that both maximize carbon yield while minimizing the need for H2 is a major challenge.

Chemical Upgrading to Fuels

SummaryThe co-design of upstream processes for biomass deconstruction with the downstream catalytic processes to convert biomass-derived intermediates to fuels is important. In particular, upstream processes determine the product slate of biomass-derived intermediates (including intermediates derived from lignin) and the potential introduction of contaminants, catalyst poisons, and fouling agents. The composition of the intermediate streams will significantly impact the final product slates, catalyst lifetimes, separations, and the operation of the downstream unit operations, all of which impact the economics of the processes.

Barriers and SolutionsChemical conversions of carbohydrate derivatives represent new routes to hydrocarbon fuels that can use wide ranges of sugars and sugar-derived intermediates, including carbohydrate dehydration products and organic acids. The primary barriers to demonstrating technical and economic feasibility of these materials can be grouped by issues related to feedstock, processing, catalysts, fuel product certification, and economics. These barriers and potential solutions for these issues are discussed below and summarized in Error: Reference source not found.

FeedstockReliable feedstocks provide carbon in a form that can be converted to desired products, but do not introduce contaminants in concentrations incompatible with the process. What constitutes a contaminant depends on the process, but could include both inorganics (ash) and organics, such as nitrogen or sulfur containing compounds or lignin. Feedstock characterization is required to enable biorefineries to manage contaminants, usually by removal prior to processing. Contaminants can lead to corrosion of equipment and reduce feedstock storage stability and catalyst activities and lifetimes.

Variations in carbohydrate profile or even alcohols and organic acids (e.g., produced in the conversion process or by microbial contamination of the carbohydrates) are not a prime concern for feeds to a catalytic conversion process. Catalysts can often utilize a wide range of carbon substrates. Six-carbon and five-carbon sugars as oligomers, monomers, or even in the form of dehydration products, including furfural and HMF, are all viable substrates provided side reactions (such as humin formation) that reduce carbon yield or catalyst performance can be avoided. Many extractive species can also be substrates, such as soluble (non-structural) sugars, sugar alcohols, alcohols, organic acids, fats, fatty acids, and phenols. Some extractives are detrimental to catalytic processing such as compounds that contain heterocyclic nitrogen, sulfur, or compounds that form deposits on a catalyst surface blocking access to active sites or contributing to pressure drop across reactor beds. Lignin is a promising future substrate but lignin utilization is complicated by its typically low solubility and penchant for recondensation to insoluble products.

Ash (inorganic) compounds can have dramatic negative effects on processing. The ash may come from dirt from the feedstock harvesting (extraneous), or may be part of the indigenous ash inherent in the plant cells (vascular and structural). Ash can affect catalytic processes in various ways such as catalyst poisoning. Sulfur, heavy metals, and nitrogen are common catalyst poisons that can reduce activity, selectivity, and lifetime. Some ash compounds can precipitate at high temperatures, forming scale such as calcium carbonate. Ash impurities that do not affect catalytic conversion end up in waste streams and increase disposal costs.

Aside from composition, surface area and particle size are additional feedstock characteristics that are highly variable and can affect processing. Biorefineries would require a minimum particle size and would likely have on-site milling or other size reduction capabilities. Particle size reduction done prior to delivery would reduce biorefinery energy costs and may also increase the bulk density depending on the feedstock formatting. However, a wide distribution of particle sizes complicates conversion (transient mass and heat transfer) and separations (filtrations, for example) and introduces safety hazards (inhalation, dust explosions). Some differences in particle size result from anatomic differences in the feedstock, for instance sugarcane bagasse is composed of 30%–40% pith and 60%–70 wt% fiber. Pith is the cell wall residue (of parenchyma cells) inside the cane and it typically forms substantially smaller particles upon processing than structural fiber cells.

Pre-processing is one means to minimize variability by formatting biomass into a form that is easier and cheaper to transport as well as possibly removing deleterious compounds in raw biomass. High-density, low-moisture feedstocks are needed to reduce transportation costs. Pre-processing might enable the production of valuable co-products (such as animal feed), as well as the removal of deleterious compounds before harsher processing in the biorefinery, or ultimately the return of these materials to the field. Nitrogen, potassium, and phosphorous are essential for maintaining soil fertility and returning these nutrients to the field will reduce fertilizer needs and the life-cycle analysis (LCA) burden that fertilizer brings. New harvesting equipment is required to remove only the desired portion of the biomass from the field. Pre-

processing allows the opportunity to return these components to the field before harsher processing at the biorefinery.

The three main pre-processing categories are chemical, thermal, and physical. Chemical methods include leaching and ensiling. Developing systems that optimize the recycle and recovery of chemical agents used in the pre-processing is of paramount importance. Thermal methods include torrefaction, a mild thermal treatment sometimes conducted in conjunction with densification to produce drier and more stable feeds. Physical methods include grinding and veneering. Some pre-processing can fall into two or all three categories, such as steam explosion and ammonia fiber expansion. Further R&D is necessary in these areas as any improvements in these activities will greatly benefit biorefinery processing. TEA and LCA should also be conducted to estimate impacts on biorefinery economics.

R&D is needed to define ideal feedstock specifications in a way that is useful for the new industry. This will depend on the deconstruction process and catalytic steps, so greater interaction between agronomists and conversion specialists is desirable. Measurement techniques are needed that can be easily distributed to the farm so that producers can target feedstock quality measurements to maximize their profits when they deliver material to the bioprocessing center.

For pretreatment and enzyme conversion approaches there is a need to understand inhibitory effects. Feedstocks that produce more inhibitors (such as greater acetate content or greater content of more friable sugars) or require more enzymes would be subprime and likely discounted on price. Feedstocks should be priced based on a variety of factors (i.e., moisture, format, variability, ash content). As the industry matures, biomass feedstocks may develop into groups designating their relative ease and expense of conversion similar to light sweet crude, Brent crude, and oil sands. Cruder feedstocks may require greater operating expenses or extra unit operations that only a smaller subset of biorefineries will possess. Feedstocks may be processed at a penalty or a premium by different conversion facilities.

R&D should focus on the complete pre-processing, deconstruction, catalytic conversion, and upgrading processes, rather than a single step in isolation. This may reveal opportunities to eliminate deleterious compounds and reduce biomass recalcitrance at the stage in the process that provides the greatest overall reductions in capital and operating costs.

ProcessingThis section considers the conversion of pre-processed sugar-based feedstocks to fuels and chemicals via thermal processes. Potential process routes are numerous and often involve an initial conversion to an intermediate product stream prior to final processing. The generation of desirable compositions of intermediate streams that are compatible with downstream upgrading in the catalytic fuel-forming processes is the key to successful process development. The ultimate gauges of success are carbon efficiency to desired products and favorable process economics as both are required.

Desirable intermediates can be generated from a variety of biomass pretreatment and deconstruction strategies including enzymatic and non-enzymatic hydrolysis (Figure 1). In general, while some processes are being developed that can directly convert raw biomass, capital expenses can be minimized by first overcoming recalcitrance by employing various pretreatment and deconstruction steps. These steps, however, can complicate downstream processing. Recovery of reagents, waste generation, and removal of species that affect catalysts, selectivity, and separations all add unit operations and, therefore, costs.

Biomass deconstruction via acid catalyzed thermal hydrolysis can introduce anions, biochemical methods can introduce proteins, and both can release various organics into the hydrolzate. These components and others can cause catalyst deactivation and changes in selectivity. Process development will necessarily include determinations of the effects of these components and identify needed separations to maintain activity. Processes initially developed with pristine feeds will need to transition to complex biomass hydrolyzates to demonstrate feasibility and identify needed modifications.

The range of potential intermediates and pathways to fuels is broad. Some pathways through sugars, levulinates, and furans have been discussed in the literature, while current work has demonstrated new pathways through these and other oxygenates previously not considered. Catalysts and pathways to remove oxygen from carbohydrates with minimum H2 use are needed for fuel production. Fundamental aspects of these various conversions are not always known. Hybrid approaches combining biocatalysis and thermochemical conversions have capitalized on the ability of biological systems to selectively form Carbon to Carbon (C-C) bonds coupled with the efficiency of thermal processes to convert these intermediates to fuels and products.

Identified pathways invariably require further process development to improve conversion, selectivity, catalyst lifetime, product and reagent recovery, reactor design (especially for exothermic reactions), and other parameters. In order to meet cost targets, fuel-forming processes must have high carbon efficiencies and low processing costs to offset high feedstock costs. Closed loop processes employing reagent recovery and recycle are important to improve economics. Product mixtures are often diverse and separation of value-added individual components can be technically and economically challenging.

In support of process development, analytical tool development is important to quantify species in diverse mixtures. Analytical methods are needed to develop, improve, monitor, and manage processes.

CatalystsCatalytic processes are designed around the performance and behavior of the catalysts they employ. Discovery of new pathways through new intermediates is usually enabled by the discovery or modification of catalysts, so design and testing of catalysts is central to process development. The ultimate goal is to design more robust, enhanced catalysts that can handle a

wide range of biomass-derived inputs and contaminants, including sulfur, nitrogen, and ash, to economically produce fuels and chemicals. In addition, the ideal catalysts will use lignin and its deconstruction products beneficially, without deactivation to accommodate the need for high carbon efficiencies and long catalyst lifetimes. This is a tall order and multiple catalytic steps and separations will likely be needed.

An understanding of catalyst fundamentals is needed to design better catalysts and understand catalyst selectivity. Rational design and development of catalysts could be accomplished using a combination of computational and experimental techniques. Computational tools can give insights into catalyst behavior and suggest alternative improved catalysts. Experimental results provide inputs and feedback to improve computational models and methods. In addition, high-throughput batch and flow testing can be used to quickly screen catalysts and narrow down the most active formulations and process conditions. When conducted correctly, these high-throughput methods give data representative of that obtained in larger systems.

Obtaining high selectivity to fuels is a key challenge. Depending on the target fuel, catalysts and processes that generate hydrocarbons with low oxygen content, in the appropriate carbon-number range, with the appropriate amount of branching, cyclic, and aromatic content, and with other characteristics needed to pass specification and fit-for-purpose testing are needed; meeting all of these requirements while maintaining high carbon yield can be difficult. A better understanding of the generation and conversion of intermediates would help in delineating factors leading to better selectivity. For example, many catalysts are effective at converting oxygenate intermediates to aromatics, but few are able to produce paraffins and isoparaffins. Carbon loss and by-product formation could be minimized by using more selective catalysts.

Catalyst lifetime should be improved to increase the economic viability of the process. Dirty hydrolyzates poison catalysts and often the agents and poisoning mechanisms are unknown. Bad actors need to be identified so that either better catalysts or separation methods can be developed. A better fundamental understanding of both chemical conversion and poisoning mechanisms may help to design better catalysts. This understanding might also help to improve pretreatment and deconstruction methods to minimize impacts on the downstream processes. If deactivation cannot be prevented by process improvements, methods to regenerate the catalysts will be needed.

Fuel Product CertificationWhile biological processes typically produce one or a few types of fuel component molecules, catalytic processes produce a wide range of products, similar to petrochemical processing in a refinery. Fuel product certification remains a large barrier for non-petroleum feedstocks. Regulatory agents have a long, expensive process for approving new fuels and blendstocks into the market and there are no overarching agencies with global power. There is a key need to increase interagency activity to help define blending and certification specifications for product materials including, or derived from, oxygenated intermediates. The large volumes required for certification are a significant barrier and usually cannot be provided at lab or pilot scale,

requiring costly capital in the production of a demonstration-scale facility. Any offset of these costs would be a welcome aid.

Guidelines for fuel certification processes are generally defined, but typically iterative in nature. Qualification and approval of new aviation turbine fuels, for example, is defined in ASTM specification D4054. The entire process requires review of data by industry original equipment manufacturers (OEMs) after several stages of testing. At the beginning of the certification process, it cannot be known with certainty exactly how much time and data (and therefore how much fuel volume) will be required to reach final certification. These uncertainties introduce significant risk to biofuel projects launching new technologies.

New analytical techniques are needed for tracking these intermediates. Oxygenated intermediates require upgrading to become advanced biofuels and there is a lack of clarity as to the best place for the upgrading to take place. Domestic refineries have underutilized capacity, but current regulations disincentivize or even prohibit feeding non-petroleum materials to existing refining assets, requiring that biofuels be fully processed in dedicated units that require new and expensive capital. There is a key need to increase interagency activity to help define blending and certification specifications for oxygenated intermediates. ASTM has developed standards that are widely used. Important characteristics are energy density, boiling point, freeze point, toxicology, Reid vapor pressure, octane, and corrosively. If the upgrading of intermediates becomes feasible, standards are needed to ensure efficient processing without negative side effects.

EconomicsBiofuels are in the process of commercial launch and costs will improve as the industry innovates and matures. To speed the penetration of advanced biofuels to market, detailed techno-economic and sustainability modeling is needed. The design reports for cellulosic ethanol provide a good benchmark, and the advanced biofuels reports should strive for analyses at a similar level of detail. These tools can provide visibility of sensitivities unique to advanced biofuels, such as hydrogen usage and refinery integration. Modeling should guide R&D, preventing funds from being allocated to projects with a low likelihood of success or an undesirably long development time. This work should be made available in the open literature to allow for wide dissemination to educate the marketplace and prospective investors. While data used in open literature can’t incorporate confidential intellectual property, models can be made that keep these steps general, or non-confidential analogues are used, such as refinery steps. Modeling can and should guide policy makers to make practical decisions. Sustainability modeling is an important piece of the puzzle; first generation ethanol has been widely criticized for its perceived life cycle footprint and advanced biofuels should mitigate or eliminate steps that reduce sustainability wherever possible.

The economics of advanced biofuels depend on improvements in all areas of the process leading to higher yields and lower capital and operating expenses. R&D should focus on improving the capital utilization, e.g., higher solids concentration at shorter residence time, increasing catalyst yields, and combining steps where feasible. R&D should also focus on

methods to decrease capital costs, including development of processes that require more moderate reaction conditions. There is a need for improved research in materials of construction to determine the corrosivity of candidate materials over the life of a biorefinery and to identify new materials. In the catalyst area, funding should support research in new formulations to lower metal costs.

R&D funding and biorefinery support should strive to be in tune with business realities of the emerging industry to incentivize faster deployment. One opportunity is expanding support for projects that use feedstocks beyond just lignocellulosic biomass. For biological conversion systems, restricting R&D funding to second generation sugars sources is sensible because conversion of first generation commodity sugars is already a commercial process. However, most solid phase catalysts are insensitive to the type of sugar used. At lab and demonstration scale, commodity sugars are easier and cheaper to obtain for proving new catalysts and systems rather than relying on the limited providers of deconstructed biomass sugars. As long as the converter has proven a robust process for sugar production with appropriate sustainability performance, funding agencies for lab and demonstration plant R&D should not overly prescribe the type of feedstock. Catalytic biorefiners can deploy more quickly to commercial scale with commodity sugars because they are widely available at minimal risk, whereas there are significant technological and logistical barriers to the widespread availability of lignocellulosic sugars. Providing government support to advanced biofuels projects that use commodity sugars can speed the deployment of advanced biofuels producers ahead of the supply chain rather than insisting that all areas of the supply chain be developed in lock step with biomass deconstruction and logistics.

Another emerging business model is the appearance of companies that produce biomass sugars as their primary product rather than fuels or chemicals. Decoupling the deconstruction process from the conversion to fuels process (catalytic, fermentative, or otherwise) is a very real possibility and may provide many advantages as summarized in Table 2. In a decoupled “merchant” or distributed model, standalone plants produce the intermediate (such as sugar) separately from the fuel producer. Co-location then becomes optional, which can be a benefit when the optimal location for the biomass deconstructor (close to feedstock) is different than that of the converter (close to a cheap hydrogen source). Deployment is faster due to the reduced complexity. Furthermore, in a merchant model, converters can take advantage of industry innovations in deconstruction and feed production more easily because they are less bound to that portion of the supply chain. On the other hand, a benefit of the captive model is that it is more closely tied to a single suite of products (e.g., biofuels). To date, government funding has typically supported the captive model over the merchant model.

From a macro-economic standpoint, the merchant model enables a distributed business approach in which one company builds and operates deconstruction while another specializes in conversion and upgrading, lowering the risk associated with both steps by allowing more than one party to share in the risk. The intermediate, such as sugars, may have the ability to substitute for commercial cane and corn sugars in some applications providing multiple market outlets and allowing the flexibility to overcome market volatility. A further advantage of the

distributed model is that a conversion facility could more easily benefit from an outside company that develops an improved deconstruction technology lowering the price of biomass sugars. The deconstruction and conversion components of bioprocessing are part of the same enterprise that cannot easily take advantage of external improvements because it is responsible for paying for its own deconstruction capital. In recent years, many companies have emerged with business plans focused on production of biomass sugars as marketable intermediates, without an entire plan for lignocellulosic biomass to products. This is a model that DOE and the U.S. Department of Agriculture (USDA) should consider supporting moving forward.

Table 2. Merchant vs. Captive Economic Models.Merchant Captive

Stand alone Highly integratedCo-location, optional Co-located

Less development required Significant developmentFaster deployment Longer deployment

Higher total cost of production Cheaper costs in the longer term

For these reasons, a solicitation focused on stabilization of biomass intermediates (e.g., sugars) is highly encouraged. Stabilization includes separation of the intermediates from compounds undesirable to the converter. Deconstructed biomass slurries have unique physical properties that provide challenges for commercial separations technologies and because separations account for 50%–70% of capital and operating costs at a biorefinery, R&D should focus on lowering these costs.2 Separations at high temperatures can increase efficiency by lowering viscosities, increasing fluxes, and avoiding formation of precipitants. Purification is a necessary step to remove contaminants, poisons, and fouling agents. As noted before, purity requirements for a catalytic conversion and fermentative conversion are different and in a merchant model, intermediates that satisfy purity requirements for both would have the greatest market opportunity. Cane sugar provides an example of a sugar product that is available at different prices based on quality; world refined sugar sells for a premium (1.8–5.5 ¢/lb) over world raw sugar (USDA data for 2000–2011).

Catalytic systems are more tolerant of a range of carbon inputs than biological systems, and conversion of lignin into products is a real possibility. Lignin has some advantages over carbohydrates as a feedstock for advanced biofuels: it has less oxygen than carbohydrates and it is composed of phenolic moieties that could provide aromatic fuel components. In many biorefinery scenarios lignin is burned to provide thermal energy, the lowest form of energy and the lowest value (in the absence of subsidies). Converting even a portion of the lignin to

2

product should provide greater value. The barriers to lignin usage are difficulty solubilizing it and avoiding side reactions that form more recalcitrant compounds.

Barrier Area 1: Feedstocks & Preprocessing

Research Activities

Increasing feedstock reliability and variability in composition

Mitigating inorganic ash content

Reducing feedstock particle size

Lowering feedstock costs

Study pre-conversion techniques such as leaching, ensiling, torrefaction, steam explosion, and ammonia fiber expansion to assess the potential for removing deleterious compounds in the raw biomass feed and reducing comminution needs

Increase R&D focus at the feedstock logistics and conversion interface to define ideal feedstock specifications for the reactor throat

Develop feedstock and intermediate densification techniques

Study techniques to homogenize feedstocks Optimize systems for recycle and recovery of reagents used

during pretreatment processes. Focus R&D on at reducing biomass recalcitrance at minimal

capital cost as ultimate Identify pretreatments that eliminate contaminants Determine harvesting windows that minimize up take of

nutrients (ash) and maximize carbohydrate yield Develop crops with improved characteristics, greater

productivity, less recalitrant lignin, lower physiological ashBarrier Area: 2. Processing Research Activities Limited upgrading/conversion

chemistries to produce tailored intermediates

Cost-effective production of intermediates

Heat integration Reagent recovery and recycle Moving from mock

hydrolyzates to real biomass hydrolyzates

Dirty hydrolyzates poison catalysts

Product mixture diversity and

Utilize reactive intermediates in processing and focus on:o Increasing fundamental R&D on developing efficient

pathways to producing intermediates which are amenable to upgrading

oAssessing state of fundamental knowledge of reactive intermediates

Assess the potential for integrated production/upgrading non-sugar biomass carbohydrate derivatives (furans, carboxylic acids, etc.)

Design low- and high-temperature separation processes and reactive membranes to generate clean intermediate/product streams

separation issues Develop analytical tool to quantify species in mixtures to improve, monitor, and manage process chemistries

Barrier Area 3: Catalysts Research Activities Understanding catalyst

fundamentals to fine-tune selectivity

Removing oxygen from carbohydrates and minimizing H2 consumption

Understanding generation and conversion of intermediates better

Raising selectivity to fuels Identifying poisoning

mechanisms and Characterization of intermediate streams to define clean-up needs and understanding catalyst poisoning impacts on performance and lifetime

Catalyst life is too short for economical use

Explore design catalysts for conversion in high temperature concentrated sugar solutions, focusing on:oPretreatment at process development scaleo Effects of concentrations on catalysisoOrganic catalysts in addition to inorganic catalystsoDirected evolution, computer-assisted tools to develop

alternative improved catalysts; combined computational/experimental approach

oCheaper catalystsoBasic reaction mechanisms for deoxygenation of

carbohydrates with minimum H2 use Improve selectivity to fuel conversion and decrease carbon

loss/by-products, focus on:oDeveloping selectivity rules and metrics oDesigning catalysts aimed at isoparaffins instead of

aromatics Improve catalyst lifetime and durability, focus onoPoison and intermediate resistanceoPassivation routes, coatingsoPoisoning mechanismsoRegeneration methodologyo Impact of lignin deconstruction products on catalysts as

a function of deconstruction strategy Improve knowledge base on converting intermediate

streams to products, focus on:o Engage in fundamental studies on possible intermediates

and alternative products (e.g., furans to fuel, COOHs to fuel)

oDevelop a “Top 10 Intermediate” list

Barrier Area: 4. Fuel Certification

Research Activities

Lack of performance-based standards for fuel properties

Lack of certification guidance for oxygenates

Refinery integration impacts on refinery operation are

Increase interagency activity to help define blending and certification specifications for oxygenated intermediates

Work with refiners to quantify process impacts and define appropriate insertion points as a function of scale

Examine certification standards and process development to make appropriate fuel mixtures

poorly understood Single-molecule fuels as

blending agents may impact fuels negatively

Barrier Area: 5. Economics and TEA

Research Activities

Insufficient modeling and analysis work

Utilization of lignin Need low-cost, efficient

technologies for intermediate stream clean-up

Feedstocks are expensive Capital costs of conversion

technologies can be high

Develop tools to support individual process development schemes (as oppose to tools to support generalized system dynamics)

Increase focus on sensitivity analysis and tools Apply improved TEA models to guide future research and

development Increase R&D funding to value added products and fuel

from lignin to incentivize technology providers to diversify their product lines and value chain

References

1. Kochergin,, V and Miller, K. “Evaluation of Target Efficiencies for Solid-Liquid Separation Steps in Biofuels Production.” Appl Biochem Biotechnol (2011) 163:90–101

Biological Conversion of Sugars and Carbohydrate Derivatives: Isoprenoid, Polyketide, Fatty Acid, and Oleaginous Pathways

General IntroductionThe biological conversion of sugars to hydrocarbons holds great promise as an ecologically sound route to the production of liquid transportation fuels and chemicals. The diversity of hydrocarbon fuels, fuel precursors, and chemicals found in the spectrum of biological systems presents an opportunity for identifying and manipulating the pathways that produce these molecules in bioprocess platform organisms. This diversity also presents a challenge in regard to the selection of those molecules and pathways on which to focus R&D efforts. Fortunately, a metabolic hierarchy exists from the simplicity of metabolite precursors in common to all of the pathways (pyruvate and acetyl-CoA), and the small number of general pathways (fatty acids, terpenes and polyketides), to the enormous diversity in terms of the array of molecules produced by these three pathways when the terminal production enzymes are added (e.g., terpene synthases and polyketide synthases). Thus, some areas of research will be of benefit to all of the pathways. For example, systems biology studies, including metabolic modeling, of central metabolic processes leading to common intermediates, such as, pyruvate, and acetyl-CoA will be of benefit to all three routes. Other studies may be specific to a particular pathway family, e.g., TAG accumulation is generally triggered by depletion of another macronutrient,

most frequently nitrogen. Conditions that lead to the accumulation of polyketides and terpenes in their natural host are more varied. This may reflect the more varied roles of these compounds, which are generally considered to be secondary metabolites but sometimes primary metabolites (e.g., the terpene ethers of archaea membranes). The promise of synthetic biology is that the pathways can be inserted into well characterized platform organisms and expression of enzymes in the pathway tuned for optimal flux to the desired metabolite.

There are significant technological and economical challenges to implementing bioconversion processes for hydrocarbon production. There is the fundamental stoichiometric limitation of converting carbohydrates as (CH2O)n to hydrocarbons, CnH(2n+2) that means weight yields will be lower than with oxygenated products such as ethanol. Of course the infrastructure compatibility and energy density is higher since much of that loss in weight yield is through elimination of the undesirable oxygen atoms. Percent carbon yields are similar for hydrocarbons and ethanol. In regard to engineering the bioprocess organism, recent advances in systems biology and synthetic biology will accelerate research and development. Systems biology is a holistic approach to the study of biological systems enabled by genomics, functional genomics and computational tools to model these complex systems. This has led to an increase in the understanding of various complex biological systems and will be instrumental to understanding biofuel and biochemical production systems. Similarly great progress has been made in the application of synthetic biology to harness this increased understanding for the synthesis of novel metabolites in well characterized biological hosts (Gibson et al., 2010; Tyo et al., 2007; Stephanopoulos et al., 2004).

This section of the CTAB will be organized around the three principal routes to biological hydrocarbons: triacylglycerides/fatty acids, terpenes and polyketides. Common challenges for all of the routes to hydrocarbons are increasing the flux of carbon from sugars to the precursor metabolites pyruvate and acetyl-CoA, maximizing the fraction of fixed carbon directed towards hydrocarbons with the minimal amount of microbial biomass needed for rapid rate

bioprocesses, understanding the triggers of hydrocarbon accumulation (e.g. nutrient depletion) and how to manipulate these triggers to increase productivity, and understanding responses to lignocellulose hydrolysates with their complex mixture of sugars and potential inhibitors. Some of these challenges are recognizable from the development of lignocellulose based bioethanol production and will leverage what was learned from that significant investment of resources and effort. Other research and development opportunities are unique to biological hydrocarbon production.

Isoprenoids and Terpenes

State of Technology (Background)The terpenes (aka, isoprenoids) and terpenoids are a large family of natural compounds represented by over 55,000 known members (Koksal et al, 2012). Terpenes are hydrocarbons consisting of multiples of five carbons, most commonly 5-40 carbons but sometimes more. The terpenoids are directly related to terpenes but they have additional functional groups, most frequently hydroxyls and carbonyls added through the action of cytochrome P450s (Hefner et al, 1996; Jennewein et al, 2004). The terpenes and terpenoids are ubiquitous in nature, as they are found in all three domains of life, bacteria, archaea and eukarya. The terpenes have a C5nH8n

formula (where n = the number of five-carbon units), since they are assembled by condensation of the five carbon unit isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate. The hemiterpenes, monoterpenes, sesquiterpenes and diterpenes are classes of compounds with 5, 10, 15 and 20 carbons respectively, which represent the most likely candidates for biologically derived fuels or chemicals, although the larger terpenes (triterpenes, C30; tetraterpenes, C40) could be brought into the liquid fuel range by cracking (Tracy et al, 2011). Among the terpenes there are a limited number of branched olefinic compounds including isoprene, myrcene, ocimene and farnesene that could be hydrogenated to isoparaffins. There are a large number of monocyclic, bicyclic and aromatic terpene structures, a few examples of which are: limonene, pinene, p-cymene, bisabolene and aristolochene.

There are two pathways leading to the production of the precursor IPP, the mevalonic acid (MVA) pathway and the more recently characterized deoxyxylulose-5-phosphate (DXP) pathway, which is also known as the methylerythritol phosphate (MEP) pathway (Lange et al, 2000). The mevalonate pathway incorporates three molecules of acetyl-CoA (each of which is derived from the three-carbon compound pyruvate arising from glycolysis) for every molecule of IPP produced, hence nine carbons (1.5 glucose) are required to produce the five-carbon IPP giving a theoretical carbon efficiency of 56%, or stated alternatively, a 25% (w/w) yield of terpene from glucose. The DXP pathway condenses two three-carbon molecules from glycolysis, pyruvate and D-glyceraldehyde-3-phosphate, and after additional steps in which a carbon is lost, forms IPP. Therefore, the DXP pathway has a theoretical carbon efficiency of 5/6, or 83%, giving a 38% (w/w) yield from glucose based on simple stoichiometry of the carbon skeletons. However, this does not account for the higher net energy cost of the DXP pathway that arises in part due to siphoning off glyceraldehyde-3-phosphate before it enters the energy yielding reactions of the lower glycolytic pathway and the higher demand for reducing equivalents in the unique reactions of the DXP pathway. Therefore, with all other needs such as biosynthesis of cell mass and energy requirements for the MVA and DXP pathways being equal, actual yields from the DXP pathway should be lower than 38%. The mevalonate pathway is found in all archaea and eukaryotes, as well as some prokaryotes. The DXP pathway is found in the majority of bacteria and also represents a second route to IPP in many phototrophic eukaryotes (e.g., plants and algae), as a plastid associated pathway that reflects its bacterial origins. Using the conversion of the substrate glucose to the monoterpene product myrcene as an example, the balance for the two pathways (neglecting water and protons) is:

MVA Pathway: 3 Glucose + 6 NAD + 4 NADPH Myrcene + 6 NADH + 4 NADP + 8 CO2

DXP Pathway: 2 Glucose + 2 NAD + 4 NADPH + 4 Fdred + 2 ATP + 2 CTP Myrcene + 2 NADH + 4 NADP + 4 Fdox + 2 ADP + 2 CMP + 6 Pi (Fd = ferredoxin)

Thus, the MVA pathway is a net producer of redox equivalents (due to more carbon oxidized to CO2) while the DXP pathway is a net consumer of both redox equivalents and NTPs. Regardless, both pathways have been manipulated for terpene production (Carter et al, 2003; DeJong et al, 2006; Kim & Keasling, 2001; Kirby & Keasling, 2009; Pitera et al, 2007).

Terpene synthases are a large and evolutionarily complex family of enzymes that catalyze the formation of terpenes from the precursor pyrophosphate compounds, e.g., isoprene from IPP, monoterpenes from geranyl pyrophosphate, sesquiterpenes from farnesyl pyrophosphate and diterpenes from geranylgeranyl pyrophosphate (Bouvier et al, 2005; Chen et al, 2011; Christianson, 2008; Hillwig et al, 2011). They are mechanistically fascinating enzymes that catalyze an electrophilic cascade reaction. Many of the biochemical characteristics of terpene synthases (aka terpene cyclases, EC # 4.2.3._) were elucidated by Rodney Croteau with his students and collaborators over the last few decades. This includes three-dimensional structures of terpene synthases (Hyatt et al, 2007), product profiles of these enzymes that often produce more than one terpene product (Bohlmann et al, 1997), investigations to identify active site amino acid residues that help determine this specificity (Little & Croteau, 2002), as well as extensive work on the elucidation of the mechanisms of various enzymes within this broad class (Lin et al, 1996; Peters & Croteau, 2002; Peters et al, 2001; Rajaonarivony et al, 1992). Terpene synthases are readily recognized by amino acid sequence similarity but their substrate specificity and product profile cannot be predicted from sequence similarity. Therefore, gene expression analysis must be utilized to define the products of individual terpene synthases.

There are already terpene products in development as biofuels or commodity scale chemicals. Amyris has developed a Saccharomyces cerevisiae bioprocess using the MVA pathway for the production of the sesquiterpene farnesene, which upon reduction with four moles of hydrogen becomes the isoparaffin farnesane (aka, 2,6,10-trimethyldodecane). Although not published in the peer reviewed literature, it was reported in 2010 that the Amyris process can give a final titer of 104 g/L, at a rate of 17 g/L/d at 13% yield (basis unclear) (Pray, 2010). This is an impressive achievement strongly indicating technical feasibility of a terpene bioprocess, though economic feasibility cannot be determined from the data provided. Similarly, bioprocess organisms (both Escherichia coli and S. cerevisiae) have been engineered by researchers at the Joint BioEnergy Institute to produce the monocyclic sesquiterpene bisabolene at about 1 g/L (Peralta-Yahya et al, 2011). Bisabolene is a precursor to the saturated fuel candidate bisabolane. Both of these sesquiterpenes are suitable for the diesel range, most likely as blendstocks. Genencor is pursuing development of a bioprocess for the production of isoprene. This is a common chemical building block that can be used for synthetic rubber production or further reacted to produce other chemicals and materials of interest. Isoprene presents unique opportunities and challenges as a gaseous product. It can be recovered from the gas phase, which is appealing from a purification standpoint but it means the product is likely present at a low mole fraction of the gas phase. The fermentation design and product recovery aspects of this process will be quite distinct from those processes aimed at the liquid monoterpenes and sesquiterpenes. The branched (acyclic) monoterpene olefins myrcene and ocimene represent a number of stereoisomers that are potential isoparaffin precursors but they have not been

pursued to date. After complete hydrogenation, these isomers converge to the single isoparaffin 2,6-dimethyloctane, which is in the right carbon range to be used as a blendstock for any of the three principal liquid transportation fuels. Similarly, the various isomers of the monocyclic monoterpenes limonene, terpinene and phellandrene, when fully reduced, yield the single product 1-isopropyl-4-methyl-cyclohexane. Given the diversity of terpenes and isoprenoids in nature and their potential not only as biofuels, but also as aroma compounds, bulk chemicals and chemical precursors, it seems that the terpenes are a good potential source of the bioproducts that are considered to be crucial to the economic success of the biofuels industry.

Challenges and OpportunitiesChallenges facing the development of bioprocesses for the production of terpenes or terpenoids include the normal issues facing bioconversion in general—increasing titer, rate and yield. This includes directing carbon flux to the desired precursors, IPP and DMAP, in a balanced manner. Given the small intracellular pool of nicotinamides (NAD/NADH, NADP/NADPH) and nucleotide triphosphates (ATP and CTP) it is critical that intermediates in a pathway involving redox reactions, or NTP usage do not accumulate, thus depleting the pool of nicotinamides and NTPs and creating redox or energy imbalances. In other words, the organism needs to be designed to avoid kinetic or thermodynamic roadblocks for the pathways involved as well as the overall process from substrate to product. Avoiding the formation of side-products and avoiding toxicity effects arising from accumulation of those side-products, or even the desired terpene product, are other considerations.

As discussed, terpene synthases are easy to recognize by sequence homology but their products cannot be predicted based on primary structure of the gene or protein alone. Hundreds of candidate terpene synthase genes have been identified in plants, fungi, bacteria, etc. and their products need to be identified. In order to mine the tremendous diversity of terpenes and terpenoids, methods need to be developed or refined to increase the throughput of constructing organisms expressing the terpene synthases and identifying the resulting products.

Excellent progress has been made but continued efforts are needed to solve additional 3D structures of terpene synthases (Hyatt et al, 2007; Koksal et al, 2011; McAndrew et al, 2011; Whittington et al, 2002) and identify critical amino acid residues that form the basis of the catalytic sites and direct the formation of particular terpene or terpenoid products (Little & Croteau, 2002; Wilderman & Peters, 2007). Such efforts could increase the ability to remodel terpene synthases to make new products and increase or decrease their product fidelity.

Choosing the best platform organisms for high titer, rate and yield of these compounds will include a number of factors. Organisms must: be resistant to the potentially toxic aspects of the products, have the ability to secrete the products, avoid breaking down the products, etc. The organisms must be able to utilize the array of sugars and other carbon compounds found in the lignocellulosic hydrolysates, as well as resistantto the inhibitory compounds found in the hydrolysates. Most of the work to date on terpene production has been in E. coli or S. cerevisiae platforms, which have the advantages of being well understood and easily manipulated

genetically. However, other bacterial and fungal systems that can also be manipulated genetically should be considered with regard to the above criteria. Systems biology should be applied to understand and refine these platform organisms.

PolyketidesPolyketides belong to a vast group of natural products known as secondary metabolites (SM) that are not required for the growth and reproduction of the organism that produces them. Rather, they are synthesized only under special circumstances to confer a selective advantage to the producing organism (Rohlfs et al, 2007). A subset of these natural products has been produced for decades as high value compounds such as antibiotics or therapeutic agents (Keller et al, 2005). Polyketides are organic compounds produced by plants and microbes which have yet to be exploited for biofuel production. Polyketides contain from ten to fifty carbon atoms and vary widely with regard to degree of oxygenation (hydroxyl and ketone groups), saturation, and cyclization. Like, fatty acids and isoprenoids, polyketides are also built upon a hydrocarbon backbone, but they vary widely in their degree of oxygenation (via hydroxyl and ketone groups), saturation, and cyclization. All polyketides are biosynthesized by large multidomain enzymes called polyketide synthases (PKS) which are structurally similar to fatty acid synthases (Schumann & Hertweck, 2006; Smith, 1994). These large enzymes contain multiple catalytic domains that can alone synthesize complex compounds. For example PKS enzymes catalyze repetitive Claisen condensations from acyl-CoA starter units and malonyl-CoA (or other carboxylated) elongation units. Polyketide synthases contain ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) domains. In addition, optional β-keto processing reactions may be catalyzed by the keto reductase (KR), dehydratase (DH), and enoyl reductase (ER) domains similar to that of fatty acids. In this manner PKSs are capable of producing complex organic compounds with lower oxygen content which may be suitable for fuels. Some examples of potential fuel precursor molecules that are derived from PKSs are found among toxins produced by fungi. For example both altaneric acid and fumonisin contain long hydrocarbon chains. Small genetic changes may allow for strains of fungi that normally produce these toxins to generate these fuel precursors. The genes for polyketide synthesis are often contained within clusters of other genes that all function in the production of a specific organic compound/metabolite (Osbourn, 2010; Palmer & Keller, 2010). These accessory enzymes may also be exploited to change the characteristics of a given compound to produce more complex molecules or to economically prepare precursor molecules for downstream processing.

The potential for increased production of polyketides is similar to or greater than that of fatty acids. Technology for overproduction of polyketides has been proven in the pharmaceutical industry for compounds such as penicillin. Through traditional strain improvement and process engineering, scientists made rapid progress toward increasing production of this very important antibiotic. One major advantage that polyketides may have over fatty acids is the fact that they are often secreted from the organism. This may aid in separations but is also likely to reduce the potential for inhibitive levels of accumulation within the cells. Given the similarity to fatty acid synthases and the large diversity of compounds that can be produced by polyketides, it is likely only a matter of time before they are exploited in the pursuit of replacements for chemicals derived from petroleum.

Fatty Acids and Triacylglycerides (TAGs)

State of Technology (Background)Utilizing the metabolic capabilities of microorganisms coupled with the ability to engineer novel microbial strains, a variety of biofuel molecules (or their precursors) can be derived from the fatty acid biosynthetic pathway. Most attention has focused on the production of fatty esters and alkanes with properties that allow for the replacement of petroleum derived gasoline, diesel, and jet fuels (Fortman et al., 2008, Keasling, 2010). Although fatty alcohols (Steen et al., 2010) and alkenes (Metzger and Largeau, 2005) can also be generated using microbial catalysts from fatty acid pathway intermediates. Fatty acids in microbes are utilized in the production of cell membranes and not for the storage of cellular energy. However, fatty acids are the foundation of triacylglycerides (TAG), which become the primary energy storage molecules for many species of algae, fungi, and bacteria (Shi et al., 2011).

The steps in fatty acid synthesis (FAS) are conserved among microbes with differences in enzyme organization. In E. coli and other bacteria the catalysis occurs by several distinct proteins (referred to as type II FAS), while multi-subunit protein complexes are found in fungi and other eukaryotes (type I FAS). Utilizing sugar as the carbon source, fatty acid synthesis begins with acetyl-coenzyme A (CoA) molecules produced via glycolysis. In the first committed step of fatty acid biosynthesis acetyl-CoA is carboxylated to produce malonyl-CoA. In E. coli this reaction is catalyzed by acetyl-CoA carboxylase (ACC). Over-expression of the four genes that encode ACC in E.coli resulted in a six fold increase in free fatty acid production in comparison to wild type as well as exhibiting an increased malonyl-CoA concentration (Davis et al., 2000). Malonyl from malonyl-CoA is then covalently attached to acyl carrier protein (ACP) by a transacylase enzyme for subsequent steps in FAS.

A transacylase enzyme also covalently attaches the acetyl group from acetyl-CoA to ACP. In the first round of carbon chain elongation in FAS, an acetyl group from malonyl-ACP is transferred to acetyl-ACP by a condensation reaction. The product of this reaction is acetoacetyl-ACP, with the release of a molecule of carbon dioxide and ACP. Reduction of the β-ketone in acetoacetyl-ACP to a hydroxyl is followed by dehydration and an additional reduction reaction to produce butyryl-ACP. Butyryl-ACP is then utilized as substrate for the condensation with malonyl-ACP for the addition of two more carbons for the second round of elongation. The chain length of fatty acid produced is determined by the number of elongation rounds performed. The profile of fatty acids synthesized varies among species, but typically microbes produce chain lengths between 14 and 20. The most prevalent fatty acid in E.coli is palmitic (Weinbaum and Panos, 1966). Long chain acyl-ACP moieties are substrates for phospholipid synthesis or can be degraded by β-oxidation to produce acetyl-CoA for entry into the tricarboxylic acid cycle. Free fatty acids can be released from ACP by thioesterase enzymes.

The regulation of fatty acid synthesis is probably most understood in E. coli (reviewed in Rock and Cronan, 1996). Synthesis of fatty acids is repressed at the transcript level upon import of extracellular fatty acids. There is also evidence that long chain acyl-ACPs are involved in

feedback inhibition of fatty acid production. The arrest of fatty acid synthesis in cells at stationary phase is not observed when E. coli is engineered to expression thioesterase enzymes in the cytoplasm. In addition, the introduction of heterologous plant thioesterase resulted in the export of free fatty acids from the cell (Voelker and Davies, 1994). Excretion of fatty acids was also observed when E. coli thioesterase was over-expressed (Jiang and Cronan, Jr., 1994). Utilizing heterologous thioesterase genes to engineer fatty acid secretion from cells has also been demonstrated in cyanobacteria (Liu et al., 2011).

Strategies to increase fatty acids production have included the disruption of genes to prevent fatty acids from being converted into phospholipids or degraded via β -oxidation, increasing the malonyl-CoA precursor by over-expressing ACC, and engineering the export of fatty acids to prevent feedback inhibition of the pathway. To date the most effective engineered strains have used a combination of these strategies and engineered multiple genetic changes in E. coli. Titers of 2.5 g/L, (Lu et al., 2008) 4.5 g/L (Liu et al., 2010) and more recently a 4.8 g/L titer have been reported (Liu et al., 2012). LS9, Inc. has been active in this area of research.

In the mid-eighties fungal strains producing high levels of polyunsaturated fatty acids such as γ-linolenic acid for nutritional applications as an alternative source to plant or fish (Ratledge, 2004). Unsaturated fatty acids are produced from saturated precursors that are modified by enzymes with desaturase and elongase activities by conventional FAS. In addition, there is also evidence that in some bacteria unsaturated fatty acids can also be produced via a polyketide synthase pathway.

While microbial fatty acids can be chemically converted to fatty acid methyl esters (FAMEs) and/or fatty acid ethyl esters (FAEEs) for biodiesel, biological catalysis using engineered E. coli has also been demonstrated (Kalscheuer et al., 2006). This was accomplished by introducing a pathway for the production of ethanol using genes from Zymomonas mobilis and a gene encoding a wax ester synthase/acyl-CoA diacylglycerol acyltransferase enzyme (WS/DGAT) from Acinetobacter baylyi. This demonstration was done with the addition of exogenous oleic acid, which produced a final titer of 1.28 g of FAEEs per L. A similar strategy was later used with several additional gene alterations to assemble an E.coli strain, which could yield 400 mg/L of FAEEs on glucose without the addition of fatty acid (Steen et al., 2010). The microbial conversion of fatty acids to FAMEs has also been demonstrated in E. coli engineered with fatty acid methyltransferases from Mycobacterium species (Nawabi et al., 2011). Although the titers produced were forty fold lower than for biological FAEEs production.

Similarly, fatty acids can either be chemically (Lennen et al., 2010) or biologically converted into alkanes. E. coli engineered to express two proteins from cyanobacteria has been demonstrated to produce alkanes from fatty acid intermediates at a reported titer of 300 mg per liter with the majority being secreted from the cell (Schirmer et al., 2010).

Specific, Reoccurring Barriers to Production/PathwayThe most significant barrier to production in the use of fatty acids and their intermediates are current yields. The highest reported engineered microbial strain has a 6% yield with glucose as a carbon source, which is less than 15% of the predicted theoretical yield. While metabolic

engineering strategies have been applied to increase fatty acid yields forty-fold in E. coli over wild-type levels, they have been confounded by “the interconnectedness and finite nature of microbial resources” (Solomon and Prather, 2011). Unexpectedly, fatty acid production decreased when E.coli was engineered in an attempt to obtain additional acetyl-CoA for incorporation into fatty acid synthesis by disrupting two genes that convert acetyl-CoA into acetate or ethanol. The complexity of metabolic regulation is further illustrated by the improvement of an E. coli fatty acid production strain from 2.5 g/L to 4.5 g/L which was accomplished by tuning the expression levels of genes introduced in the first generation strain rather than introducing or modifying an additional gene (Liu et al, 2010). The creation of accurate and predictive metabolic models to inform strain construction is a significant barrier not only to improvement of fatty acid production but for any hydrocarbon fuel precursor produced biochemically.

An additional barrier to production is toxicity of fatty acids to the microbial catalyst. Over-expression of medium chain length fatty acids in E.coli resulted in reductions of cell viability. Both cell morphology and membrane permeability exhibited significant changes from the parental strain (Lennen et al., 2011). These effects were not observed when fatty acids were added to the growth media. Presumably, toxicity issues will be observed with significant production of any type of fatty acid molecule. The fatty acid secretion pathway, which is not well described has been proposed as an area that is ripe for investigation and engineered improvements in this area (Fischer et al., 2008).

Controlling the degree of saturation and carbon chain length of fatty acids produced by microorganisms will also be required in order to design the most effective biofuel mixture (Knothe, 2008).

R&D Being Conducted to Overcome Barriers Significant efforts with metabolic engineering and systems biology approaches are being applied in order to improve biofuel yields and reduce toxicity effects. Results from these experiments while not always successful, inform investigators about additional pathways and regulatory elements that are involved in fatty acid biosynthesis. And consequently enable the construction of improved metabolic models. These studies can be assisted by high-throughput methods to examine fatty acid production for identification of microbial strains with phenotypes of interest (Hoover et al., 2012).

In contrast to directed approaches, some organisms can be utilized for genome wide recombinant methods to modify many genes and then select strains with the phenotype of interest. PCR and sequencing can then identify the genetic modification introduced. Unbiased genome scale genetic manipulation techniques have been successfully applied to improve the tolerance to ethanol (Alper and Stephanopoulos, 2007) and lignocellulosic hydrolase (Warner et al., 2010) in E. coli. These methods have yet to be applied in an attempt to optimize fatty acid biosynthesis.

Determining the carbon chain length of fatty acids produced in microbes appears to be possible through the use of heterologous thioesterase enzymes from plants. Several plant thioesterases

have demonstrated acyl-ACP chain length substrate specificities when expressed in E. coli (Handke, et al., 2011). Thioesterase discovery efforts should produce additional options to design fatty acid with defined carbon chain lengths. There has also been some work done to alter the specificity of desaturase enzymes (Whittle and Shanklin, 2001), although additional studies will be required in order to enable the assembly of fatty acids with controlled carbon bond unsaturation.

While significant barriers exist, the contribution of fatty acid pathways to future production of advanced biofuels and chemicals was perhaps best summarized by Handke et al. (2011)”Given...the close structural relationship of many fatty-acid molecules to petrochemical molecules, this pathway has the potential to serve as an important platform for many more products in the future. To do so, new metabolic engineering strategies that push performance closer to maximum yields, emphasize the integration with inorganic catalytic approaches to broaden product possibilities, and enable the production or co-production of higher-value molecules are needed.”

Lipid Metabolism for Biofuels in Non-Algal Strains

State of Technology (background)The recent progress in the areas of synthetic and systems biology have heightened the precision to which researchers can analyze and implement metabolic pathways into microbial species (Gibson et al., 2010; Tyo et al., 2007; Stephanopoulos et al., 2004). Lipid biosynthesis from various substrates, including sugars and organic acids has been observed in both prokaryotic and eukaryotic microorganisms. The production of energy storage compounds such as triacylglycerol (TAG) by oleaginous bacteria is especially profound, and is of interest to those wishing to use naturally derived lipids for the feedstock of biofuel production, namely biodiesel. Companies such as ExxonMobil Corp., Dow Chemical Co., LS9 Inc., Codexis Inc., BP and Martek Biosciences are believed to be actively pursuing microbial biodiesel production (Shi et al., 2011).

Microorganisms are being explored as an ideal platform for biodiesel production, as in silico metabolic models enable systematic elucidation and design of biological systems allowing for the promotion of desired biodiesel production properties in microbes including enhanced lipid accumulation or de novo pathways for biodiesel production in vivo (Holtz et al., 2010; Kim et al., 2008; Senger, 2010). Targeting increased lipid biosynthesis as a measure of cellular dry weight (CDW), the use of synthetic biology has particular promise. By sequencing microorganism genomes such as Rhodococcus opacus PD360, an exemplary organism for bacterial lipid biosynthesis, which can accumulate over 85% of its CDW in lipid, scientist will achieve a greater understanding of microorganism lipid metabolism and potentially be able to isolate or enhance and transform these key genetic signals for the creation of super oil producing microbes.

In order to survive starvation conditions and limited nutrient environments, microorganisms have developed several strategies including spore formation and accumulation of storage lipids. TAGs and other energy storage compounds serve as exceptional reserve materials due to their hydrophobic properties which allow for large accumulation in the cytoplasm without altering

the osmolarity (Alvarez and Steinbüchel, 2002). Bacterial TAG accumulation does not serve as a simple depot for inert lipids, but has been shown to play a much more vital role in cellular functions and metabolism (Murphy 2001). Triacylglycerols (TAG) are non-polar, water-insoluble triesters of glycerol with fatty acids. As an ideal energy storage compound for carbon, TAGs have a high energy value because they are oxidized less and have a higher caloric value than proteins or carbohydrates, netting much greater return on energy when subsequently oxidized (Alvarez & Steinbüchel, 2002). Biosynthesis of intracellular TAG and subsequent storage have been noted in both Gram-positive and Gram-negative bacteria, but the actinomycetes group seems to be especially productive of these molecules, with species such as Mycobacterium, Streptomyces, Rhodococcus, and Nocardia (Alvarez & Steinbüchel, 2002). Production and accumulation of TAG by these bacteria is most often observed during an excess of source in the growth medium combined with a limitation of nitrogen during the stationary growth phase (Olukoshi and Packter, 1994; Alvarez, 2002).

Through genome sequencing of several phylogenetically related genera from the actinobacteria group, it was discovered that there are multiple pathways for lipid biosynthesis which use a unique combinations of fatty acid synthases (FAS) to generate linear and branched-chain fatty acids (Holder et al., 2011). Lipid synthesis in Rhodococcus and Streptomyces species occur as insoluble cytoplasmic inclusions, which are believed to have a membrane allowing them to remain stable in the aqueous environment of the cytoplasm and can be seen via electron micrographs. Bacterial TAG inclusions where isolated from cells of R. opacus PD630 through centrifugation in discontinuous glycerol and sucrose density gradients (Alvarez et al., 1996) and in general have been shown to possess similar properties as the lipid inclusions from the seeds of oleaginous plants. The lipid droplets that were isolated from R. opacus PD630 contained 2% w/w phospholipids and 0.8 % w/w proteins besides the neutral lipids with TAG as principal compounds (Alvarez et al., 1996; Kalscheuer et al, 2001).

Several strains/species of the bacteria genus Rhodococcus are known to be oleaginous in nature and have the ability to accumulate >20% of their CDW in TAGs and other energy storage compounds. The research model for oleaginous Rhodococcus is R. opacus PD630. In the case of R. opacus PD630, the carbon source will determine the content and composition of accumulated TAGs (Alvarez et al., 1996, 1997). There appears to be an inverse relationship between triacylglycerol and glycogen content within oleaginous bacteria like R. opacus PD630. A substrate containing gluconate at the primary carbon source supports higher amounts of triacylglycerol by a strain of PD630 cells than other carbon sources (Alvarez et al., 1996). A known metabolic inhibitor for the de novo fatty acid biosynthesis pathway is cerulenin, and in operates by binding to the active sites of ketoacyl synthases I and II (Funabashi et al., 1989). The addition of cerulenin to the growth medium in general will cause an increase in polyhydroxyalkanoates and glycogen content in cells (Alvarez & Hernández, 2010).

Biosynthesis of TAG is understood to be a three step process including: production of fatty acyl-compounds, formation of glycerol intermediates, and sequential esterification of the glycerol moiety with fatty acyl-residues (Alvarez & Steinbüchel, 2002). The enzyme DGAT works to catalyze the final acylation step in the TAG production pathway and has been detected in both the Streptomyces (Olukoshi and Packter, 1994) and Mycobacterium (Akao and Kusaka, 1976)

species, which have the ability to produce and accumulate large amounts of intracellular TAG. In Streptomyces the increases in TAG hydrolase activity concurrently signaled a decrease in DGAT activity (Olukoshi and Packter, 1994). Although DGAT is believed to be the enzyme for major pathway bacterial production of TAG, it has been hypothesized that it is not the only pathway, with the enzyme phospholipid:diacylglycerol acyltransferase (PDAT) possibly playing a minor role, notably utilizing phospholipids as acyl donor plants and yeasts (Dahlqvist et al., 2000). Fatty acid biosynthesis for TAG could play a role in inhibiting key enzymes for central cellular metabolism, allowing the bacteria to balance production of TAGs depending on environmental conditions through a feedback mechanism (Alvarez and Steinbüchel, 2002). To date, all TAG accumulating bacteria thus far described are aerobic (Alvarez and Steinbüchel, 2002).

Synthesis of storage lipids is a common feature shared by many microorganisms in response to a nutrient limited environment (Manilla-Pérez et al., 2010). Synthesis of polymeric lipids including polyhydroxylkanoates (PHAs), wax esters (WEs), and TAGs by obligate hydrocarbonclastic marine bacteria (OHCB) such as A. borkumensis has been observed (Wälterman and Steinbüchel 2005). Using these lipophilic compounds for carbon storage is ideal, as they are compact, anhydrous and posses a higher caloric value than proteins and carbohydrates. Nutrient limitation with minerals such as nitrogen, phosphorus, sulfur, magnesium and potassium while having in combination with an excess carbon source will result in the synthesis of storage lipids (PHAs) and can account for up to 80% cell dry weight (CDW) (Anderson and Dawes, 1990). Accumulation of TAGs in bacterial cells most notably increases within the stationary growth phase (phase variant) on unbalanced mineral salts medium (MSM) that also has a high carbon-to-nitrogen ration (Wälterman and Steinbüchel 2006).

By studying the production and accumulation of TAG and PHA in R. ruber, it is believed that biosynthesis of these energy storage compounds is regulated independently and potentially phase dependent. PHA accumulation begins in the exponential growth phase and will reach a maximum when the nitrogen source is depleted. The bacteria will then switch to biosynthesis of TAG during stationary growth phase, and the separation of these pathways may be related to competition for the same precursors, acetyl-CoA and propionyl-CoA (Alvarez et al., 1997a). Being able to distinguish and direct the creation of different lipid storage compounds through the manipulation or inhibition of de novo fatty acid biosynthesis to yield higher CDW percentages of TAG or PHA has important biotechnological implications, and gives promise to biofuel production (Alvarez and Steinbüchel, 2002).

E. coli has been metabolically engineered to produce biodiesel and fatty acid derivatives. Fatty acid biosynthesis was accomplished through the overexpression of several genes encoding enzymes including thioesterase (tesA), acyl-CoA synthase (fadD), acetyl-CoA carboxylase (accABCD), fatty acid synthase (fabH, fabD, fabG, fabF) acyl carrier protein (acpP), wax synthase (atfA), alcohol acyltransferase, alcohol dehydrogenase, as well as several alcohol forming acyl-CoA reductases (Knothe, 2008). This represents a pivotal step in understanding of fatty acid metabolic pathways, and how synthetic biology can be utilized to introduce desired pathways into microbes, in effect creating cell factories. Fatty acid production was further enhanced through the addition of aceEF, attenuating glycerol-3-phosphate dehydrogenase (gpsA), lactate

dehydrogenase (ldhA), pyruvate formate lyase I(pflB), phosphate O-acyltransferase (pta), pyruvate oxidase (poxB), acetate kinase (ackA), and glycerol-3-phosphate O-acyltransferase (plsB) (Knothe, 2008). Under U.S. patent publication 2010/0071259 it was shown that adding a mixed alcohol solution to a medium containing the engineered E. coli at least two different types of fatty esters could be produced. Furthermore, it was demonstrated that by selecting the various combinations of type and amount of alcohols in the solution it was possible to produce a desired fatty ester end product, or designed biodiesel, which could have improved physical fuel properties including cloud point, cetane number, viscosity, and lubricity (Knothe, 2008).

However E. coli is not a natural producer of ethanol, a key substrate for biodiesel, and would require a heterologous ethanol biosynthesis pathway in order to be an effective biodiesel producer. A more tested solution may be utilizing S. cerevisiae, which is well known as an ethanol fermenter in dry grind, starch-based Midwestern ethanol production facilities (Bro et al., 2006). Utilizing the same principles that turned E. coli into an engineered lipid producer, S. cerevisiae H1246 was able to produce fatty acid ethyl esters (FAEE) and fatty acid isoamyl esters (FAIE) from oleic acid while expressing the A. baylyi bifunctional WS/DGAT enzyme (Kalscheuer et al., 2004).

Specific, Reoccurring Barriers to Production/PathwayAs discussed there has been varying degrees of research conducted into biosynthetic production of lipids by microbes, however, there still is not a complete knowledge base for production by any microorganism, with partial knowledge from several sources being collated and assumptions applied. Due to the high degree of substrate specificity (i.e. crude oil or similar complex hydrocarbon mix), genetic modifications to extend substrate utilization range may be necessary for economic feasibility of a fermentation process that involves OHCBs (Stephanopoulos et al., 1998). It has been demonstrated that Rhodococcus species possess intracellular lipases and polyhydroxalkonoic acid (PHA) depolymerase, but it is still unclear whether these bacterial lipase are expressed constitutively, if their formation is induced by environmental or cellular cues, or if they are activated to an active state during stationary growth phase after initial inactivity during exponential growth phase (Alvarez & Steinbüchel, 2002). Despite recent advancements, the understanding of the structure of TAG inclusions in bacteria is very limited and will require more studies to determine the mechanisms responsible for the formation of TAG inclusions in bacteria as well as to characterize granule-associated proteins (Alvarez and Steinbüchel, 2002). For example, a small protein from R.opacus and R. ruber was found to have high sequence similarity to a ribosomal L7 protein (Kalscheuer et al., 2001), which is a known to be component of the 50S ribosomal subunit (Gudkov 1997).

Still, microbial production of lipids may be advantageous over agriculturally sourced oils, which are dependent on unpredictable factors such as weather, climate and large-scale catastrophes (Alvarez and Steinbüchel, 2002). Also, limited availability and high cost of plant-derived oils is leading to the consideration of other feedstocks for biodiesel production including microbial oils, genetically modified crops, used cooking oils and animal fats (Cankci and Sanli, 2008; Meng et al., 2009; Li et al., 2008: Peralta-Yahya et al, 2010), as well as engineered microbes that could directly produce fatty esters from a simple sugar feedstock to avoid cost (Li et al., 2008; Peralta-Yahya et al., 2010; Steen et al., 2010). The largest barrier to commercial production of microbial

lipid production remains cost. To reduce cost and eliminate a processing step necessary for oil recovery from the bacteria is has been suggested that waste or residual materials be used for the production of lipids by the cells (Lemann 1997). Combining knowledge on the physiological, biochemistry, genetics, and cellular and molecular biology of bacterial TAG production will be necessary for the successful metabolic and process engineering for the desired potential biotechnological applications of microbial lipids (Alvarez and Steinbüchel, 2002).

R&D Being Conducted to Overcome BarriersConventional transesterification processes utilize short chain alcohols like methanol or ethanol and triacylglycerides extracted from dry microbial biomass; however a single-step process involving transesterifies lipids with direct alcoholysis of microbial biomass that does not require the lipids to be first extracted (Mata et al., 2010; Liu et al., 2007). Although this single-step method will still require the pretreatment and drying of the microbial biomass (Shi et al., 2011). Currently chemical catalysis is the most widely accepted method of transesterification, but biocatalytic transesterification using lipases has been recently presented as a less energy intensive and environmentally friendly solution, allowing for the avoidance of treatment of contaminated water and biodiesel ester recovery (Parawira 2009). Advances in systems biology, metabolic engineering and synthetic biology have led to the possibility of using a non-oleaginous bacterium such as E. coli to produce large amounts of storage lipids (Shi et. al., 2011). Through a series of genetic modifications, knocking out of fadD gene, encoding fatty acyl CoA synthetase, and by overexpressing acetyl-CoA carboxylase and thioesterase, E. coli can produce fatty acids at a rate of 2.5g/L (Shi et. al., 2011). Although not as prolific in lipid production as microalgae, bacterial lipid production may have several important advantages including a higher specific growth rate and greater ease of cultivation, along with the potential ability to reach commercial-scale production levels sooner (Shi et. al., 2011).

Research into the structure of TAG inclusions, proteins associated with TAG granules and genes involved in TAG biosynthesis and accumulation still require further study and detailed investigation, with particular emphasis on TAG biosynthesis pathways, enzymes and regulation (Alvarez and Steinbüchel, 2002). Once these mechanisms and molecules are better understand a foundation for the synthetic engineering of a commercial production quality and quantity of oil through biotechnological production may be realized (Alvarez and Steinbüchel, 2002). Through solubilization studies it was determined that there were three types of granule-associated proteins—unspecifically bond proteins, relatively weakly associated proteins, and proteins that resisted strong solubilization treatments (Kalscheuer et al., 2001). Understanding the protein content of these isolated oil bodies will be important moving forward if oleaginous bacteria are to be strongly considered for the production of oils as a feedstock for biofuel production.

More active efforts for screening for bacteria that can produce TAGs at a high amount will need to be pursued as the search for oleaginous microorganisms continues. Despite this fact, the potential of OHCBs to metabolize hydrocarbons almost exclusively and convert them into storage compounds such as TAGs, PHAs, and WEs for the purpose of bulk chemical production in a biotechnological transformation pathway is significant, and could represent a major advantage in downstream processing of said compounds.

Systems Biology Approaches to Advanced Biofuel Production from OrganismsThe combination of metabolic engineering and synthetic biology are methods to allow microbial pathways to be reshaped to make new and existing products and fuels. Pathways and endproducts that have a high potential include long chain fatty acids, isoprenoids, polyketides and lipids. We are still far from understanding the details of all the multiple and coupled processes employed by microbial systems and this can slow our ability to exploit these pathways for fuel blendstocks or precursors.

Systems biology is an approach which can provide insight and accelerate these manipulations. Systems biology has many definitions but generally includes the study of the biological organism as a system and the use of data-rich analytical and computational methods. (see figure) These data-rich analysis or “omics” build from the fundamental model of modern biology where a single gene is transcribed into an mRNA which is translated into protein to carry out reactions of metabolites. The tremendous advances in analytic technology allow the simultaneous sequencing and study of many genes (or genomics). Likewise transcriptomics measure many changes in gene expression, whether by arrays or newer RNA-sequencing. Proteomics by gel or mass spectrometry measures thousands of proteins. Metabolomics attempts to measure most of the metabolites in a biological sample. Beyond these primary analyses, there are also data-rich analyses for lipids and sugars (glycomics).

Systems biology can provide insights into further improvements from metabolic or pathway engineering by helping to identify bottlenecks, key regulatory mechanisms, and alternative pathways. For example, when a key gene to an undesired product is knocked out, occasionally a previously unutilized pathway will be expressed as the cell attempts to preserve its prior function. In synthetic biology, where an entire de novo pathway may be added, these techniques can help identify where bottleneck or metabolic pools are accumulating and allow the pathway to be tuned. An accepted challenge of using “omic” measurements is the careful experimental procedures including the determination of the “state” of the fermentation and the preparation (or rapid preservation) of the sample. This is particularly important for metabolites and especially transient redox carriers.

Microbial adaptation and evolution are also powerful tools for biocatalyst improvement. In these cases, the evolution is constrained by the experimental design (such as to increase tolerance to an inhibitor product) but the nature of the changes is unknown. Resquencing of the evolved strains has become much easier and provides a catalog of the SNPs (single nucleotide polymorphisms) or other changes. Genomic resequencing illustrates one of the challenges of systems biology – the need to interpret the large amounts of data generated to determine which mutations were important and which were random. The interpretation of the data sets requires both the use of the growing repertory of bioinformatic tools as well as biological insights and the ability to validate the predictions or inferences.

Genome annotation and metabolic pathway reconstruction tools are available and are being applied to many microbes of bioprocess interest. [ref 1]. These are being combined with various data to develop regulatory and flux models. The rate of improvement of the experimental design, the analytical measurements and the bioinformatic tools for interpretation continues at an accelerating pace making these increasing powerful and useable approaches.

While Systems biology approaches have been developed to provide fundamental understanding of the microbe; they are powerful techniques to improve yield, titer and rate – the primary process goals for bioconversion.

Barrier Area 1: Carbon Utilization

Research Activities

C5 utilization from biomass feedstocks

Redox balance in microbes producing a fuel

Increase C5 utilization flux by targeting transporters and regulators

Manage stress in organism-redesign Feed H2-mass transfer improvements Enahnce metabolic engineering and pathway design (e.g.

optimizing NAD-NADH coenzyme chemistry) Analyze metabolic flux Develop high throughput redox measurements Complete thermodynamic modeling

Barrier Area 2: Product Separation

Research Activities

Inefficient and costly product separation processes

Removing metals and other impurities that interfere with downstream upgrading

Steam distillation, evaporation, stripping performance

Increase efforts in fundamental separation science and membrane development, flocculation and coagulation chemistry

Process development scale solvent extraction and recycling, novel/improved extraction from cell bodies (acoustic, ultrasonic, microwave), secretion of product

Complete technoeconomic and lifecycle analysis for separations

Complete process integration and collaboration with upstream processes such as organism or pathway design

Develop in situ separation processes for optimized downstream economicsoPhysical and/or continuous removal of product during

fermentation Focus process development efforts around minimizing

separation costs and needs (i.e. heavier/branched molecular targets are typically better candidates for facile

separation)

Barrier Area 3: Inhibitor and Toxicity Considerations

Research Activities

Quantifying feedstock toxicity limits for organisms and enzymes (poor understanding of product dose and process condition dependence /interdependence)

Understanding and identifying action of specific inhibitors

Design process conditions to reduce inhibitors, process separation, detoxification, harvesting/storage/ preprocessing of feedstocks

Minimize or modify lignin composition in biomass feedstocks

Improve end product tolerance and strain development/discovery for organisms that metabolize inhibitors to product molecules

Evaluate end product concentration impacts on cellular function

Develop analytical tool for low-level (ppm/ppb) inhibitor detection

Barrier Area 4: Engineered Hydrocarbon Discovery

Research Activities

Identification/prioritization of target and novel molecules

Create high throughput screening methods around targets-discovery for 1-2 years, then identify 2-4 strains for genetic systems development

Determine characteristics for strains-toxicity, yield, nutrient/media requirements

Focus on two R&D pipelines: One to identify organisms that efficiently degrade biomass, the other to identify organism that produces target. Use metabolic engineering to merge pipelines into single strain

Target molecules that enable technology rather than being mature

Employ bioinformatics for directed screening procedures Examine the potential for an organism platform to allow a

flexible range of products assuming targets change Examine mixed/co-cultures as a model study to utilize

genetics and systems biology to create single, optimized organism

Develop organisms for consolidated bioprocessing Identify favorable traits in extremophiles for pH,

temperature and inhibitor robustness Utilize lignin and/or lignin degradation-engineering of

lignin-modifying enzymes into organisms Complete secondary metabolite pathway engineering

References

Alper, H. and Stephanopoulos, G. Global transcription machinery engineering: a new approach for improving cellular phenotype. Metab. Eng. 2007, 9, 258-267.

Davis, M.S., Solbiati, J., and Cronan Jr., J.E. Overproduction of acetyl-CoA carboxylase activity increases the rate of fatty acid biosynthesis in Escherichia coli. J. Biol. Chem. 2000, 275, 28593-28598.

Fisher, C.R., Klein-Marcuschamer, D., and Stephanopoulos, G. Selection and optimization of microbial hosts for biofuels production. Metab. Eng. 2008, 10, 295-304.

Fortman, J.L., Chhabra, S., Mukhopadhyay, A., Chou, H., Soon Lee, T., Steen, E., and Keasling, J.D. Biofuel alternatives to ethanol: pumping the microbial well. Trends Biotechnol. 2008, 26, 375-381.

Hooverm S.W., Youngquist, J.Y., Angart, P.A., Withers, S.T., Lennen, R.M., and Pfleger, B.F. Isolation of improved fatty acid overproducing strains of Escherichia coli via Nile red based high-throughput screening. Environ. Prog. Sustain. Energy 2012, 31, 17-23.

Jiang, P., and Cronan, Jr., J.E. Inhibition of fatty acid synthesis in Escherichia coli in the absence of phosopholipid synthesis and the release of inhibition by thioesterase action. J. Bacteriol. 1994, 176, 2814-2821.

Kalscheuer, R., Stölting, T., and Steinbüchel, A. Microdiesel: Escherichia coli engineered for fuel production. Microbiol. 2006, 152, 2529-2536.

Keasling, J.D. Manufacturing molecules through metabolic engineering. Science 2010, 330, 1355-1358.

Knothe, G. “Designer” biodiesel:optimizing fatty ester composition to improve fuel properties. Energy Fuels 2008, 22, 1358-1364.

Lennen, R.M., Braden, D.J., West, R.M., Dumesic, J.A., and Pfleger, B.F. A process for microbial hydrocarbon synthesis: overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes. Biotechnol. Bioeng. 2010, 106, 193-202.

Lennen, R.M., Kruziki, M.A., Kumar, K., Zinkel, R.A., Burnum, K.E., Lipton, M.S., Hoover, S.W., Ranatunga, D.R., Wittkopp, T.M., Marner II, W.D., and Pfleger, B.F. Membrane stresses induced by overproduction of free fatty acids in Escherichia coli. Appl. Environ. Microbiol. 2011, 77, 8114-8128.

Liu, H., Yu, C., Feng, D., Cheng. T., Meng, X., Liu, W., Zou, H., and Xian M. Production of extracellular fatty acid using engineered Escherichia coli. Microb. Cell Fact. 2012, 11, 41-

Liu, T., Vora, H., and Khosla, C. Quantitative analysis and engineering of fatty acids in E. coli. Metab. Eng. 2010, 12, 378-386.

Liu, X., Sheng, J., and Curtiss III, R. Fatty acid production in genetically modified cyanobacteria. Proc. Natl. Acad. Sci. USA, 2011, 108, 6899-6904.

Lu, X., Vora, H., and Khosa, C. Overproduction of free fatty acids in E. coli: implications for biodiesel production. Metab. Eng. 2008, 10, 333-339.

Metzger P., and Largeau, C. Botryococcus braunii: a rich source for hydrocarbons and related ether lipids. Appl. Microbiol. Biotechnol. 2005, 66, 486-496.

Nawabi, P., Bauer, S., Kyrpides, N., and Lykidis, A. Engineering Escherichia coli for biodiesel production utilizing a bacterial fatty acid methyltransferase. Appl.Environ. Microbiol. 2011, 77, 8052-8061.

Ratledge, C. Fatty acid biosynthesis in microorganisms being used for Single Cell Oil production. Biochime, 2004, 86, 807-815.

Rock, C.O., and Cronan, J.E. Escherichia coli as a model for the regulation of dissociable (type II) fatty acid biosynthesis. Biochim. Biophys. Acta 1996, 1302, 1-16.

Shi, S., Octavio Valle-Rodríguez, J., Siewers, V., and Nielsen, J., Prospects for microbial biodiesel production. Biotechnol. J. 2011, 6, 277-285.

Steen, E.J., Kang, Y., Bokinsky, G., Hu, Z., Schirmer, A., McClure, A., del Cardayre, S.B., and Keasling, J.D. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 2010, 463, 559-562.

Solomon, K.V., and Prather, K.L. The zero-sum game of pathway optimization: emerging paradigms for tuning gene expression. Biotechnol. J. 2011, 6, 1064-1070.

Volker, T.A., and Davies, H.M. Alteration of the specificity and regulation of fatty acid synthesis in Escherichia coli by expression of a plant medium chain acyl-acyl carrier protein thioesterase. J. Bacteriol., 1994, 176, 7320-7327.

Warner, J.R., Reeder, P.J., Karimpour-Fard, Woodruff, L.B.A., and Gill, R.T. Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides. Nat. Biotechnol. 2010, 28, 856-862.

Weinbaum, G., and Panos, C. Fatty acid distribution in normal and filamentous Escherichia coli. J. Bacteriol. 1966, 92, 1576-1577.

Bohlmann J, Steele CL, Croteau R (1997) Monoterpene synthases from Grand fir (Abies grandis) - cDNA isolation, characterization, and functional expression of myrcene synthase, (-)(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase. Journal of Biological Chemistry 272: 21784-21792

Bouvier F, Rahier A, Camara B (2005) Biogenesis, molecular regulation and function of plant isoprenoids. Progress in Lipid Research 44: 357-429

Carter OA, Peters RJ, Croteau R (2003) Monoterpene biosynthesis pathway construction in Escherichia coli. Phytochemistry 64: 425-433

Chen F, Tholl D, Bohlmann J, Pichersky E (2011) The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant Journal 66: 212-229

Christianson DW (2008) Unearthing the roots of the terpenome. Current Opinion in Chemical Biology 12: 141-150

DeJong JM, Liu YL, Bollon AP, Long RM, Jennewein S, Williams D, Croteau RB (2006) Genetic engineering of Taxol biosynthetic genes in Saccharomyces cerevisiae. Biotechnology and Bioengineering 93: 212-224

Hefner J, Rubenstein SM, Ketchum REB, Gibson DM, Williams RM, Croteau R (1996) Cytochrome P450-catalyzed hydroxylation of taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5 alpha-ol: The first oxygenation step in taxol biosynthesis. Chemistry & Biology 3: 479-489

Hillwig ML, Xu MM, Toyomasu T, Tiernan MS, Wei G, Cui GH, Huang LQ, Peters RJ (2011) Domain loss has independently occurred multiple times in plant terpene synthase evolution. Plant Journal 68: 1051-1060

Hyatt DC, Youn BY, Zhao YX, Santhamma B, Coates RM, Croteau RB, Kang CH (2007) Structure of limonene synthase, a simple model for terpenoid cyclase catalysis. Proceedings of the National Academy of Sciences of the United States of America 104: 5360-5365

Jennewein S, Long RM, Williams RM, Croteau R (2004) Cytochrome P450 taxadiene 5 alpha-hydroxylase, a mechanistically unusual monooxygenase catalyzing the first oxygenation step of taxol biosynthesis. Chemistry & Biology 11: 379-387

Keller NP, Turner G, Bennett JW (2005) Fungal secondary metabolism - from biochemistry to genomics. Nat Rev Microbiol 3: 937-947

Kim SW, Keasling JD (2001) Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production. Biotechnology and Bioengineering 72: 408-415

Kirby J, Keasling JD (2009) Biosynthesis of Plant Isoprenoids: Perspectives for Microbial Engineering. In Annual Review of Plant Biology Vol. 60, pp 335-355.

Koksal M, Chou WKW, Cane DE, Christianson DW (2012) Structure of 2-Methylisoborneol Synthase from Streptomyces coelicolor and Implications for the Cyclization of a Noncanonical C-Methylated Monoterpenoid Substrate. Biochemistry 51: 3011-3020

Koksal M, Jin YH, Coates RM, Croteau R, Christianson DW (2011) Taxadiene synthase structure and evolution of modular architecture in terpene biosynthesis. Nature 469: 116-U138

Lange BM, Rujan T, Martin W, Croteau R (2000) Isoprenoid biosynthesis: The evolution of two ancient and distinct pathways across genomes. Proceedings of the National Academy of Sciences of the United States of America 97: 13172-13177

Lin XY, Hezari M, Koepp AE, Floss HG, Croteau R (1996) Mechanism of taxadiene synthase, a diterpene cyclase that catalyzes the first step of taxol biosynthesis in Pacific yew. Biochemistry 35: 2968-2977

Little DB, Croteau RB (2002) Alteration of product formation by directed mutagenesis and truncation of the multiple-product sesquiterpene synthases delta-selinene synthase and gamma-humulene synthase. Archives of Biochemistry and Biophysics 402: 120-135

McAndrew RP, Peralta-Yahya PP, DeGiovanni A, Pereira JH, Hadi MZ, Keesling JD, Adams PD (2011) Structure of a Three-Domain Sesquiterpene Synthase: A Prospective Target for Advanced Biofuels Production. Structure 19: 1876-1884

Peralta-Yahya PP, Ouellet M, Chan R, Mukhopadhyay A, Keasling JD, Lee TS (2011) Identification and microbial production of a terpene-based advanced biofuel. Nature Communications 2

Peters RJ, Croteau RB (2002) Abietadiene synthase catalysis: Conserved residues involved in protonation-initiated cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate. Biochemistry 41: 1836-1842

Peters RJ, Ravn MM, Coates RM, Croteau RB (2001) Bifunctional abietadiene synthase: Free diffusive transfer of the (+)-copalyl diphosphate intermediate between two distinct active sites. Journal of the American Chemical Society 123: 8974-8978

Pitera DJ, Paddon CJ, Newman JD, Keasling JD (2007) Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. Metabolic Engineering 9: 193-207

Pray T. (2010) Biomass R&D Technical Advisory Committee: Drop-in Fuels Panel. Vol. 2012, p. Presentation.

Rajaonarivony JIM, Gershenzon J, Croteau R (1992) Characterization and mechanism of (4S)-limonene synthase, a monoterpene cyclase from the glandular tirchomes of peppermint (Mentha x Piperita). Archives of Biochemistry and Biophysics 296: 49-57

Rohlfs M, Albert M, Keller NP, Kempken F (2007) Secondary chemicals protect mould from fungivory. Biology Letters 3: 523-525

Schumann J, Hertweck C (2006) Advances in cloning, functional analysis and heterologous expression of fungal polyketide synthase genes. Journal of Biotechnology 124: 690-703

Smith S (1994) The animal fatty acid synthase: one gene, one polypeptide, seven enzymes. The FASEB Journal 8: 1248-1259

Tracy NI, Crunkleton DW, Price GL (2011) Catalytic cracking of squalene to gasoline-range molecules. Biomass Bioenerg 35: 1060-1065

Whittington DA, Wise ML, Croteau R, Christianson DW (2002) Insights into monoterpene cyclization reactions in biology: crystal structure of (+)-bornyl diphosphate synthase. Biochemistry 41: 8973-8973

Wilderman PR, Peters RJ (2007) A single residue switch converts abietadiene synthase into a pimaradiene specific cyclase. Journal of the American Chemical Society 129: 15736-+

Production of Bio-Oils via Direct Liquefaction of Biomass and Upgrading to Hydrocarbon Biofuels and Oxygenate Blends

There has been significant progress in the myriad of routes to thermochemically or thermocatalytically convert biomass into predominantly a bio-oil intermediate, gases and char in the absence of air or oxygen. There is a broad process space currently bounded by the technologies: i) fast pyrolysis, ii) vapor phase initial catalytic upgrading of the pyrolysis vapors either in-situ or ex-situ, iii) hydropyrolysis with or without catalysts and in the presence of a reductant, and iv) solvothermal liquefaction, where the solvent may be water from the biomass or an additional organic solvent. All bio-oils produced are on a continuum of differing quality and relative amounts of different oxygenated species present and all require additional upgrading in order to form a hydrocarbon fuel blendstock. The routes from biomass through a bio-oil and subsequent upgrading should be examined holistically in order that optimal techno-economic solutions can be identified, an example is the trade-offs that are made in producing a lower oxygen content bio-oil which may require less aggressive additional upgrading to become a fuel blendstock versus a higher water and oxygen content bio-oil that requires significant upgrading.

Optimized Hydrocarbon

Production Strategy

Chemical Composition Modification

Physical Modification

In-Process(vapor phase)

Post-Process (liquid phase)

Vapor-Phase Upgrading (VPU)Catalytic Pyrolysis (CFP)Hydropyrolysis (HYP)

Fast Pyrolysis (FP) + StabilizationHydrothermal Liquefaction (HTL)

CFP Oil RefiningHYP Oil Refining

FP Oil UpgradingHTL Oil Upgrading

Selective FractionationHot Gas Filtration Liquid Filtration

Graphic adapted from Honeywell’s UOP, LLC

Production of Bio-Oils

Fast PyrolysisFast pyrolysis is done at essentially 1 atm and about 500 °C and typically requires a feedstock of ≤ 10 wt % moisture with a particle size of ~2-6 mm. It is a high throughput (< 2 second vapor residence time) with a high yield (~70 wt%) of liquid product with high carbon conversion to liquid (≤ 74 wt% of carbon). Reactor configuration plays a relatively minor role in product liquid quality and composition, if all other process parameters remain constant. The monophasic bio-oil is typically a low energy density (because of the high retained oxygen content and water dissolved in the bio-oil 10-20 MJ/kg, 1.1 - 1.3 g/mL, typically 20 to 35 wt% water, viscosity 10-150 cP at 50 °C). Fast pyrolysis oil is acidic, having a pH in the range of 2.5 to 3.0, and therefore unstabilized pyrolysis oil fuel storage tanks will need to be made of material that will not corrode. Bio-oil has sensitivity toward thermal degradation/polymerization. This technology is moving into the commercial marketplace at scales of 400 tons of dry biomass/day pyrolysis units in contrast to most of the alternative technologies mentioned below which are still predominantly in the laboratory.

Reaction Products from Fast PyrolysisFast pyrolysis can generate high yields of carbonaceous oil (up to 70%) with a short residence time and moderate temperature (< 1 s, 500°C) and is of great interest as means to produce

renewable transportation fuel. However, the chemical compounds found in pyrolysis oil contain a large amount of oxygenates, requiring upgrading of the oil or of the pyrolysis vapor.

Understanding the composition of biomass fast pyrolysis vapors and oils is critical for improving current upgrading processes. Knowing the types of products formed and their rates of formation as a function of reaction conditions will aid in the development of catalytic processes and optimium pyrolysis conditions for high yields of hydrocarbons.

Much has already been learned about the products from biomass pyrolysis and the mechanisms and kinetics that lead to their formation. A general conclusion appears to be that the individual biopolymers, cellulose, hemicellulose and lignin, react independently so that the products from biomass pyrolysis is roughly a composite of the products of the individual components. Thus, studies of the pyrolysis of the individual biopolymers provide insight into the products from biomass.

A number of studies have shown that the reactivity of the biopolymers increases in the order hemicellulose > cellulose > lignin. The figure to the right shows the results from a typical differential scanning calorimetry (DSC) experiment involving these biomass polymers. As can be seen, the peak for weight loss from hemicellulose occurs at roughly 290°C, cellulose at 340°C and lignin at 400°C. Further, the pyrolysis of hemicellulose and cellulose occur over a fairly narrow temperature range, while lignin pyrolysis is spread over a broad range. This is because hemicellulose and cellulose have fairly uniform labile linkages, while the linkages are stronger and more diverse in lignin. Hemicellulose is amorphous and cellulose is both crystalline and amorphous, accounting for their difference in reactivity. These results suggest that when biomass is pyrolyzed, hemicellulose products will volatilize first in a burst followed by cellulose products. Concurrent with these events, lignin will volatilize at a lower level over a longer period of time. This assumes the constituent biopolymers do not interact during pyrolysis and alter the thermal depolymerization pathways, energetic, and kinetics.

Cellulose Pyrolysis of pure cellulose produces greater than 60% yields of levoglucosan, an anhydrosugar, with little char formation. However, alkali and alkaline earth metals that are found in biomass

or in biomass itself, char formation is catalyzed and levoglucosan yields are dramatically reduced. Biomass pyrolysis oils typically contain less than 10wt% levoglucosan. Other products from cellulose decomposition include glucose, furans, small aldehydes and ketones, and light gasses. Although there general consensus in the literature about the products from cellulose pyrolysis, there is less

agreement about the effects of metals on the cellulose pyrolysis products or kinetics. More

DSC profiles for hemicellulose, cellulose and lignin

Mechanism of Cellulose Pyrolysis

detailed kinetic information is needed for cellulose pyrolysis in the presence of naturally occurring metals and salts.

HemicelluloseThe pyrolysis of hemicellulose has been studied less thoroughly, but, as might be anticipated, similar products are observed. The backbone of hemicellulose is comprised of five carbon sugars such as xylose and side chains containing acetyl groups, five and six carbon sugars and uronic acids. Sugars, anhydrosugars, acetic acid and low molecular weight aldehydes and ketones, and light gases are observed from hemicellulose pyrolysis. As with cellulose, alkali and alkaline earth metals affect the product formation. However, a detailed understanding of the effects of the metals is not available and an understanding of the pyrolysis mechanisms for the side chains is not well developed.

LigninLignin is perhaps the most complex biopolymer in biomass and the thermal decomposition products from this material have a profound impact upon pyrolysis vapor and bio-oil composition. These products include substituted phenolic and aromatic compounds, monolignols, lignin oligomers, condensation products, light gases and char.

A major focus of mechanism research in lignin pyrolysis has been on the b-O4 linkage, which represents up to 50% of the chemical bonds between monolignol residues in this polymer. This bond between the phenoxy oxygen and the middle carbon of the propyl side chain is relatively weak, (D298K ~ 69 kcal mol-1), due to the resonance stabilization of the resulting radical and it has been suggested that lignin decomposition chemistry is dominated by radical processes resulting from homogeneous dissociation of this bond. However, experimental and computational results have suggested that concerted reactions forming non radical species are kinetically more favored (figure to the right). In addition, other reaction pathways are possible. For instance, the C-O bond in the methoxy groups in lignin can dissociate to form methyl radicals and phenoxy radicals since the bond dissociation energy for these bonds is also low, (D298K ~ 65 kcal mol-1). In addition, mechanisms involving quinone methide intermediates similar to those observed during lignin synthesis could also be important. Much work remains to be completed to understand the mechanisms and products from lignin pyrolysis due to the complexity of the biopolymer.

The figure below shows the structures of some typical products from the pyrolysis of biomass polymers.

Rate constants for lignin

In-situ vapor phase initial catalytic upgrading of the pyrolysis vaporsIn-situ vapor phase upgrading is commonly referred to as catalytic biomass pyrolysis. The role of the catalyst in the process is to control the chemistry during biomass pyrolysis to produce a bio-crude that has lower oxygen content and is more thermally stable than conventional biomass fast pyrolysis oil.Consequently, catalytic biomass pyrolysis has the potential to eliminate mild hydrotreatingt to stabilize the liquid intermediate.

Complete deoxygenation can be achieved; however, the more oxygen that is removed the lower the bio-crude yield. Deoxygenation by CO and CO2 removal (decarboxylation and decarbonylation) plus any carbon losses in the form of coke formation on the catalyst lead to lower hydrocarbon liquid yields. Therefore, the objective is to produce a bio-crude intermediate that is thermally stable and recover as much biomass energy in the bio-crude as possible. The assumption is that bio-crude with lower oxygen content will be more stable because the more reactive oxygen functional groups that lead to re-polymerization will be removed.

Ex-situ vapor phase initial catalytic upgrading of the pyrolysis vaporsFast pyrolysis is done at essentially 1 atm and about 500 °C and typically requires a feedstock of ≤ 10 wt % moisture with a particle size of ~2-6 mm. It is a high throughput (< 2 second vapor residence time). Biomass is converted to predominately condensable vapors, non-condensable gas and some char. Char and sand are separated from the vapor in cyclones and subsequently sent to a combustor to burn the char and reheat the sand, which is returned to the pyrolysis reactor. The pyrolysis vapors leaving the cyclone are passed to the vapor phase upgrading reactor. The catalyst converts the vapors to less reactive compounds. Secondary vapor phase reactions are minimized although coking of the higher molecular weight vapors and aerosols on the catalyst surface can be a problem. The biphasic bio-oil includes a typically a higher energy density settled predominantly organic and an organic contaminated predominantly aqueous phase. Ex-situ vapor phase initial catalytic upgrading of the pyrolysis vapors oil is moderately acidic, therefore unstabilized pyrolysis oil fuel storage tanks will need to be made of material that will not corrode, and it is yet to be determined if there is any sensitivity toward thermal degradation/polymerization.

HydropyrolysisHydropyrolysis is another in-situ catalytic biomass pyrolysis process that combines biomass and hydrodeoxygenation catalysts in a high temperature reactor at high pressure and high hydrogen partial pressure. The hydrocarbon-rich product vapors can be quenched and further upgraded or introduced into a second hydroprocessing reactor to finish the deoxygenation and conversion to gasoline and diesel-range hydrocarbons.

The key difference is biomass and hydrogen are simultaneously fed to a pressurized hydropyrolyzer. Pressurized biomass is contacted with hydrogen rich gas (H2/biomass weight ratio = 7%) and is typically done at 300-350 psia and about 400 °C in the presence of a catalyst and requires a feedstock of ≤ 10 wt % moisture with a particle size of ~2-6 mm. It is a high throughput (< 2 second vapor residence time). Char and catalyst are separated from the vapor in cyclones and the hydropyrolysis vapors leaving the cyclone can be passed to a vapor phase upgrading reactor or quenched. The goal of the Hydropyrolysis Strategy Area in the NABC is is to evaluate teh technical viability and economic feasibility of a process to convert biomass into a hydrocarbon-rich bio-crude. The physical and chemical properties of the bio-crude are evaluated as a function of catalyst functionality, hydrogen partial pressure, and temperature to determine the feasibility of processing the resulting bio-crude in a refinery. Preliminary results from the NABC activities have demonstrated the production of a bio-crude intermediate with > 5 wt% oxygen content with a C4

+ yield between 15-25 wt%, depending on the process conditions.

In the GTI process the hydropyrolysis bio-oil vapors are fully integrated with the upgrading of the gaseous intermediate to a finished fuel blendstock. At this time there is very little data available on the quality and quantity of the oil leaving the hydropyrolysis reactor prior to upgrading. A better understanding of the relative capital costs for this high pressure process and the capital and operating costs associated with introducing hydrogen in the beginning of the process compared to other pyrolysis processes is needed. Are increased capital and operating costs offset by the lower oxygen content and higher quality of the hydropyrolysis bio-crude that potentially improves the efficiency of the upgrading step? Comparing the techno-economics of the entire conversion and upgrading process is needed to assess this.

Hydrothermal LiquefactionHydrothermal liquefaction is done at essentially 200 atm and about 350 °C and typically requires a feedstock of ≤ 20 wt % solids with a particle size of ~0.2-2 mm in a water slurry or other solvents and/or feedstocks which may broaden this range of operation. It is a lower throughput (< 30 minutes liquid residence time) with a moderate yield (~35 wt%) of predominantly organic liquid product with high carbon conversion to liquid (≤ 53 wt% of carbon).[refs]. The biphasic bio-oil is typically a higher energy density (because of the high retained oxygen content 30 MJ/kg, 1.0 - 1.2 g/mL, typically 2-10 wt% water, viscosity 3,000 – 17,000 cP at 60 °C). Hydrothermal liquefaction oil is moderately acidic, having a typical TAN of 50, and therefore unstabilized pyrolysis oil fuel storage tanks will need to be made of material that will not corrode. In contrast to fast pyrolysis bio-oil it has a low sensitivity toward thermal degradation/polymerization. There are also variations of the HTL technology that have been developed to different degrees. Solvent liquefaction has been tested in the laboratory wherein the water slurry is replaced with a solvent. The solvent allows the reaction to be performed at lower pressure while still maintaining the liquid phase. The solvent may also allow for better recovery of the bio-oil from the byproduct aqueous phase, which would still be formed through deoxygenation of the biomass.

A summary table like the one below might be nice.Process Process Conditions Intermediate

Oxygen ContentTemperature ©

Pressure (psig)

pH2 Bio-crude yield

??? Other

Fast Pyrolysis 350-500 atmospheric 0 50-75 wt%

40-50 wt%

Catalytic Fast Pyrolysis

350-600 atmospheric 0 5- 25 wt%

Hydropyrolysis 300-500 250-450 25-100%

15-25 wt%

0.1-10 wt%

Hydrothermal Liquefaction

300-500 1500-4000 0 20-40 wt%

15-25 wt%

Upgrading of Bio-Oils

Upgrading of bio-oil is primarily focused on oxygen removal as that is the significant difference between bio-oil and petroleum. Removing oxygen has two components, chemical reaction to separate chemically bound oxygen and physical separations of dissolved/emulsified water in the bio-oil. Removal of the oxygen affects all the important fuel parameters including reduced density, increased volatility, increased energy content, reduced corrosive potential and reduced thermal instability. Upgrading can also include removal of inorganic components or suspended solids, which have been shown to negatively affect the fuel properties. Upgrading process development is currently focused on catalytic hydroprocessing. The requirement for hydroprocessing varies with the quality of the bio-oil product. Conventional fast pyrolysis bio-oil requires the most extensive upgrading and is also the most difficult to process in typical hydroprocessing systems. In-situ, ex-situ and hydropyrolysis as well as hydrothermal liquefaction all produce bio-oils with a less severe requirement for upgrading as some of the deoxygenation is accomplished as part of the initial production process. In all cases the catalytic hydroprocessing has similar characteristics of processing in pressurized hydrogen for considerable processing times at temperatures around 400 °C. Pretreating of fast pyrolysis bio-oil at lower temperature (100-300 °C) prior to finishing the hydrodeoxygenation at higher temperature (400 °C) is a key feature, which may not be required for the other bio-oil types.

Critical Bio-oil Production Challenges in Conversion Technologies for Advanced Biofuels

1. Feedstock variability: Feedstocks are produced from a range of agricultural and silvacultural materials. Even with a specific feedstock, biomass composition, as well as water and ash content, are dependent on the conditions for growth, harvesting, processing, transport, and storage. Yield and reactivity of bio-oil is driven by composition and variability of the feedstock and in order to facilitate commoditization of feedstocks for these technologies specifications of feedstocks and the impacts of variance outside of feedstock specifications on downstream processes need to be determined. Higher ash content leads to lower liquid yields on a wt% basis of input biomass and ash potentially catalyszes cracking reactions and poisons in-situ catalysts.

2. Bio-oil reactivity: Inorganic and organic components of bio-oil in liquid and solid form are reactive and can react with other molecular components in the bio-oil or react with the surface of the container the bio-oil is in at storage- and higher-temperatures. Typical outcomes that can be observed are the sedimentation of

solids, the increased viscosity of the fluid, phase separation of the liquids, and the corrosion of metallic surfaces. Additionally the inorganic and organic reactive components can react with catalysts in a deactivation mode through chemical poisoning, fouling, or leaching of active catalytic species. Identification of reactive species and the kinetics of cross reactions within the bio-oil matrix are in their infancy. Bio-oil thermal stability is a key parameter for downstream processing/upgrading.

3. Bio-oil quality: With the complexity of the bio-oil matrix and the continuum of bio-oils produced via different technologies, the concept of what are the critical species to have in bio-oils and what chemical species are less desirable in order to define the quality of a bio-oil and could lead to the attainment of standards for particular applications or insertion points in subsequent processes is currently undefined. Developing clarity around bio-oil speciation, reactivities of species, and standards are critical to enabling market acceptance of the new bio-intermediates and comparisons between technologies.

4. Biogenic carbon efficiency: With the diversity of phases of products from thermochemical or thermocatalytic routes to bio-oils (diversion of biogenic carbon between: non-condensables, char and bio-oils (both single phase, and the splitting of carbon between a predominantly organic phase and predominantly aqueous phase) the carbon efficiency for the conversion of the relatively expensive and limited biogenic carbon feedstock is an environmental and economic challenge.

5. Efficient Utilization of Hydrogen. Biomass is hydrogen deficient compared to petroleum crude and refined fuels. Therefore, hydrogen is required for biofuels production to upgrade bio-oil intermediates. In process production of hydrogen for upgrading must not reduce the biofuels yields too dramatically. External hydrogen input has negative greenhouse gas implications if natural gas or other fossil fuels are used for hydrogen production.

6. Process Integration. Maximum process integration is required to optimize heat and by-prodcut stream utilization to maximize carbon efficiency and energy recovery of biofuels.

7. Refinery Integration. Optimizing the physical and chemical properties of bio-oil intermediates for integration into existing petroleum refining infrastructure can access existing capital assets and improve process economics.

R&D challenges for the technical barriers are listed in Table 2 along with proposed activities. The time frames for the proposed activities are: evaluate (1-5 yrs.), develop (3-7 yrs.), and demonstrate (5-10 yrs.).

Barrier Area 1: Catalyst and catalytic process comprehension

Research Activities

Lack of understanding of how catalysts interact with the oxygen containing functional groups in the bio-oil vapor or liquid matrix together with how the entities within bio-oil vapor and liquids react intermolecularly.

Incomplete understanding of how to tune the optimal catalytic upgrading conditions for the molecular components of different bio-oils derived from different liquefaction processes and feedstocks in order to efficiently generate an optimal fuel blendstock.

Current biomass/bio-oil catalysts have a short lifespan and are prone to deactivation. Catalyst selectivity, ability to regenerate, and stability are problematic.

Lack of understanding of cross-reactions between species in the vapor stream and on the surface of catalysts for formation of bio-oil, and the relative ratios of components (acyclic molecules versus cyclic and aromatic molecules), and side products (char and volatile organics).

Evaluate deoxygenation/decarboxylation catalyst interactions with multiple oxygenated species in order to develop catalyst active sites flexible to the broad classes of species present in bio-oil vapors (determine the benefits of multi-functional catalysts).

Develop effective catalysts that combine; high activity, optimal residence time, attrition resistance, and reactivation/regeneration.

Conduct parametric testing to evaluate catalyst performance using real feedstocks in order to be representative for large-scale process design.

Develop an understanding of thermal and catalytic deactivation mechanisms with the identification of key reactants. Ultimately facilitating the development of robust catalysts and accelerated aging protocols.

Barrier Area 2: Hydroprocessing of Bio-Oils and Hydrogen Considerations

Research Activities

Process sustainability and economics are strongly influenced by the amount of hydrogen and flexibility of distribution that is needed to simultaneously achieve high carbon yields, and lower GHG emissions.

Evaluate biomass-specific heat and mass transfer correlations for reactor systems.

Evaluate carbon and hydrogen atom efficiency of processes.

Develop an understanding of thermal and catalytic deactivation mechanisms with the identification of key reactants. Ultimately facilitating the development of robust catalysts and accelerated aging protocols.

Evaluate use of H2, H2-donor molecules, and other reductants for in-situ bio-oil reduction, these technologies need to be identified, understood for optimal efficiency, and tested.

Evaluate innovative processes for oxygen removal

mechanisms, including robust catalytic and electrochemical routes that limit the production of COx species.

Evaluate the inorganic species identity and transport through the direct liquefaction and upgrading processes with a particular emphasis correlation of inorganic species with biogenic carbon usage efficiency.

Barrier Area 3: Separation Systems and Selective Fractionation

Research Activities

Solvent recovery and recycle is difficult in many liquefaction processes. Better technologies are needed to improve the cost-competitiveness.

Vapor phase filters: process optimization, robust high-temperature filter and gasket materials, catalytic removal/upgrading of specific process-stream components and permeable selective filters.

The need better understand solids separation from condensable gas streams with the balance of filter efficiency, and regeneration cycles for optimal performance while minimizing the loss of bio-oil.

Liquid phase filters/membranes: process optimization and techniques to improve anti-fouling, aids to stabilize desired product, and process yields.

Lack of knowledge about what fraction(s) do we want to separate (e.g., large oligomers, water, small organics, inorganics) and the trade-offs involved.

Need for improved separation of aerosols and particulates from vapor phase

Evaluate bio-oil quality and contaminant buildup over longer-term testing in recycle scenarios in continuous operation.

Evaluate/develop optimal high-temperature membrane/filtration materials that are compatible with biomass-derived liquids, solids, and gases.

Develop optimal pressure pulse to enable particulate removal from filters/membranes that are compatible with biomass-derived liquids and gases with minimal bio-oil loses.

Demonstrate long-term solids filtration at high temperature.

Evaluate catalytically active filters to balance optimal solid filter cake and upgrade bio-oils.

Demonstrate high-temperature, high-pressure permeable, and selective filters/membranes to remove H2 from off gas, syngas, and recycled gas.

Evaluate process economics for separating solids from bio-oils – compare hot-vapor separation and condensed phase separation

Evaluate the fundamental aspects of degradation mechanisms for hydrocyclones in liquid-phase biomass separation systems.

Evaluate which oxygenates are problematic and develop selective separation processes, e.g., adsorption, extraction, magnetic- and electric-active separations, etc.

Develop/demonstrate energy efficient water removal processes.

Evaluate particle size and shape in order to drive agglomeration or flocculation of entrained

particles to enhance filter efficiency.

Demonstrate filtration technology at engineering relevant scales for extended periods of time in order to reduce risk for market entry.

Barrier Area 4: Aqueous Phase Processing Concerns Research Activities

Too much carbon is lost to the predominantly aqueous phase in some biomass liquefaction processes in order to enable optimal economics.

Evaluate selective condensation, conversion and/or processing to extract useful products/intermediates from aqueous phase or convert the species to a form useable in downstream processes.

Barrier Area 5: Bio-oil Composition Research Activities

There is no standard definition of what constitutes an acceptable bio-oil intermediate. This includes physical properties (density, viscosity, etc.) as well as chemical properties (hydrocarbon range, stability, speciation etc.).

Develop an understanding and control the relative ratios of the different molecular functional groups in bio-oil vapors and liquids in order to facilitate more efficient upgrading

Evaluate gas phase versus aerosol reaction pathways in order to better-understand intermolecular reactions of bio-oils.

Evaluate analytical techniques to characterize the complex oxygenated hydrocarbon mixtures.

Evaluate the characterization of primary thermal liquefaction products and subsequently develop an understanding of the intermolecular vapor phase and liquid phase reactions.

Barrier Area 6: Refinery Integration and Specifications

Research Activities

Lack of established minimum requirements for acceptable refinery inputs at specific points of integration

Increasing involvement and collaboration with the petro industry

Characterize and evaluate the properties of bio-oils and develop optimal insertion points for intermediate bio-oil products.

Understand refinery insertion point requirements and specifications

Defining quality traits for suites of refinery produces

Optimizing carbon distribution in the C10-C18 range

Analyze the impact of finished fuel and new specs for biofuels (ASTM, etc.)

Barrier Area 7: Improved Processes Research Activities

Lack of detailed understanding of the tradeoff between optimizing bio-oil yield,

Develop and evaluate process modeling with comprehensive techno-economic analyses to

and bio-oil quality. A clear definition of what constitutes “quality” in the product needs to be addressed.

The fundamental thermochemical /thermocatalytic mechanisms of biomass decomposition and initial upgrading are not well understood.

Lack of data for demonstration of quench areas assumed in thermal liquefaction designs and distribution or accumulation of species in the quenching fluids.

Need to reduce capital costs through the identification of positive process synergies and process intensification.

The need for robust real time, on-line analysis to enable process control.

guide the selection of feasible intermediate products, optimal scale, and trade-offs for distributed direct liquefaction and central upgrading.

Evaluate the development of well-defined “benchmark” processes, feeds, and catalysts to provide a common measure to compare emerging technologies.

Develop in situ, on-line analyses to improve process monitoring, reduction of deactivation and excursion events and ultimately enhanced process control.

Demonstrate the operation a pilot-scale research facility to study integrated heat transfer, internal recycle (e.g., solvent and dense-phase recovery), continuous operation, refine process models, and reduce risk for scale up.

Barrier Area 8: Supply Chain Research Activities

Need process robustness across biomass sources Understand the wide array of bio-oil to utilize (animal fat, seed oil, waste cooking oil, pyrolysis oil, algae lipid)

Enhance biomass pretreatment Study the physical deconstruction/homogenization or

fractionation effects on oil qualityBarrier Area 9: Material Handling Issues

Current biomass feed-systems are inadequate for the volumes, conditions, and types of feedstocks that are representative for large-scale process design.

Lack of data for demonstration of quench areas assumed in thermal liquefaction designs and distribution or accumulation of species in the quenching fluids.

Need for low(er) cost materials of construction (i.e., cheaper than stainless steel)

Corrosion of structural material in processing systems

Detail mechanisms of bio-oil corrosion on materials and the species formed under ambient and reaction conditions

Membranes for separations of bio-oil components Expose materials in laboratory tests to simulated

environments or to samples of actual environments

References

FP

Bridgwater, A. V.; Peacocke, G. V. C.; “Fast Pyrolysis Processes for Biomass” Renewable and Sustainable Energy Reviews 2000, 4, 1-73.

Bridgwater, A. V.; Meier, D.; Radlein, D. “An Overview of Fast Pyrolysis of Biomass” Organic Geochemistry 1999, 30, 1479-1493.

Czernik, S.; Bridgwater, A. V. “Overview of applications of biomass fast pyrolysis oil.” Energy & Fuel 2004, 18, 590–598.

Diebold, J. P., Scahill, J. W., Czernik, S., Phillips, S. D. & Feik, C. J. Progress in the production of hotgas filtered biocrude oil at NREL. 1997. In: Bridgwater, A. V. & Hogan, E. N. (eds.). Proc. 2nd EU Canada Workshop on Thermal Biomass Processing. Newbury: CPL Scientific Information Services Ltd. Pp. 66–81.

Oasmaa, A.; Solantausta, Y.; Arpiainen, V.; Kuoppala, E.; Sipila, K. “Fast Pyrolysis Bio-Oils from Wood and Agricultural Residues” Energy & Fuels 2010, 24, 1380-1388.

Bridgwater, A. V. “Review of Fast Pyrolysis and Product Upgrading” Biomass and Bioenergy 2012, 38, 68-94.

Mullen, C. A.; Boateng, A. A. “Chemical Composition of Bio-Oils Produced by Fast Pyrolysis of Two Energy Crops” Energy & Fuels 2008, 22, 2104

Oasmaa, A.; Elliott, D. C.; Korhonen, J. “Acidity of Biomass Fast Pyrolysis Bio-oils” Energy Fuels 2010, 24, 6548-6554

Elliott, D. C.; Oasmaa, A.; Preto, F.; Meier, D.; Bridgwater, A. V. “Results of the IEA Round Robin on Viscosity and Stability of Fast Pyrolysis Bio-oils” Energy Fuels 2012, 26, 3769-3776.

Mourant, D.; Wang, Z.; He, M.; Wang, X. S.; Garcia-Perez, M.; Ling, K.; Li, C.-Z.; Malle wood fast pyrolysis: Effects of Alkali and alkaline earth metallic Species on the yield and composition of bio-oil Fuel, 2011, 90, 2915-2922.

Keiser, J. R.; Howell, M.; Connatser, R. M.; Lewis, S. A. Sr.; Elliott, D, C. “Corrosivity and Composition of Raw and Treated Pyrolysis Oils, TAPPI, 2012, manuscript submitted.

Wang, X. “Biomass Fast Pyrolysis in a Fluidized Bed, Product Cleaning by in-situ filtration” Ph. D. Thesis University Twente February 9, 2006.

Czernik, S. 1994. Storage of biomass pyrolysis oils. In: Proc. Specialist Workshop on Biomass Pyrolysis Oil Properties and Combustion, Estes Park, CO, 26–28 Sept. 1994. Golden, CO: NREL, pp. 67–76. Report No. CP-430-7215.

VPUPODayton, D..; “Catalytic Deoxygenation of Biomass Pyrolysis Vapors to Improve Bio-Oil Stability” RTI International presentation at DOE/OBP Thermochemical Platform Review. February 16,2011

Diebold, J.; Phillips, S.; Tyndall, D.; Scahill, J.; Feik, C.; Czernik, S.; “Catalytic Upgrading of Biocrude Oil Vapors to Produce Hydrocarbons for Oil Refinery Applications” Preprint from the 1994 Fall American Chemical Society Energy & Fuels Division Conference. Washington DC, August 1994. http://web.anl.gov/PCS/acsfuel/preprint%20archive/Files/39_4_WASHINGTON%20DC_08-94_1043.pdf

Mante, O.; Agblevor, F. “Catalytic Conversion of Biomass to Bio-Syncrude Oil “ Biomass Conv. Bioref.; Vol. 1, 2011; pp. 203-215.

Stefanidis, S.; Kalogiannis, K.; Iliopouulou, E.; Lappas, A.; Pilavachi, P.; “In-situ Upgrading of Biomass Pyrolysis Vapors: Catalyst Screening on a Fixed Bed Reactor”. Bioresource Technology Vol. 102, 2011. pp. 8261-8267.

Zacher, A.; Santosa, D.; Elliott, D. Brown, C.; Beckman D.; “Mild Catalytic Fast Pyrolysis of Biomass and Catalytic Hydrotreating to Liquid Transportation Fuels” TCSBiomass 2011 Presentation, Chicago Il; PNNL-SA-82908

Cheng, Y.-T.; Jae, J.; Shi, J.; Fan, W.; Huber, G. W. “Production of Renewable Aromatic Compounds by Catalytic Fast Pyrolysis of Lignocellulosic Biomass with Bifunctional Ga/ZSM-5 Catalysts” Angew. Chem. Int. Ed. 2011, 50, 1-5.

Agblevor, F. A.; Mante, O.; Abdoulmoumine, N.; McClung, R. “Production of Stable Biomass Pyrolysis Oils Using Fractional Catalytic Pyrolysis” Energy Fuels, 2010, 24, 4087-4089.

HTLToor, S. S.; Rosendahl, L.; Rudolf, A. “Hydrothermal Liquefaction of Biomass: A Review of Subcritical Water Technologies” Energy 2011, 36, 2328-2342.

Akhtar, J.; Amin, N. A. S. “A Review on Process Conditions for Optimum Bio-oil Yield in Hydrothermal Liquefaction of Biomass” Renewable and Sustainable Energy Reviews 2011, 15, 1615-1624.National Advanced Biofuels Consortium “Hydrothermal Liquefaction Process Strategy” http://www.nabcprojects.org/hydrothermal_liquefaction.html

Zhu, Y.; Biddy, M.; Jones, S.; Elliott, D.; Schmidt, A.; “Gasoline and Diesel Production from Woody Biomass via Hydrothermal Liquefaction (HTL) and Upgrading – A Techno-Economic Analysis based on Experimental Tests”; National Advanced Biofuels Consortium draft journal publication currently in NABC review, 2012.

Knežević, D.; van Swaaji, W.; Kersten, S. “Hydrothermal Conversion of Biomass II. Conversion of Wood, Pyrolysis Oil, and Glucose in Hot Compressed Water. Ind. Eng. Chem Res. 2010, 49, 104-112.

Okuda, K.; Man, X.; Umetsu, M.; Takami, S.; Adschiri, T. Efficient Conversion of Lignin into Single Chemical Species by Solvothermal Reaction in Water-p-Cresol solvent. J. Phys.: Condens. Matter 2004, 16, S1325-S1330.

Bio-Oil UpgradingFrench, R. J.; Hrdlicka, J.; Baldwin, R.; “Mild Hydrotreating of Biomass Pyrolysis Oils to Produce a Suitable Refinery Feedstock” Environmental Progress & Sustainable Energy 2010, 29, 142-150.

Christensen, E. D.; Chupka, G.; Luecke, J.; Smurthwaite, T.; Alleman, T. L.; Iisa, K.; Franz, J. A.; Elliott, D. C.; McCormick, R. L. “ Analysis of Oxygenated Compounds in Hydrotreated Biomass Fast Pyrolysis Oil Distillate Fractions” Energy & Fuels, 2011, 25, 5462-5471.

Elliott, D. C.; “Historical Developments in Hydroprocessing Bio-oils” Energy & Fuels 2007, 21, 1792-1815.

Oasmaa, A.; Kuoppala, E.; Elliott, D. C.; “Development of the Basis for an Analytical Protocol for Feeds and Products of Bio-oil Hydrotreatment” Energy & Fuels, 2012, 26, 2454-2460.

Elliott, D. C.; Hart, T. R.; Neuenschwander, G. G.; Rotness, L. J.; Olarte, M. V.; Zacher, A. H.; Solantusta, Y. “Catalytic Hydroprocessing of Fast Pyrolysis Bio-oil from Pine Sawdust” Energy & Fuels 2012, 26, 3891-3896.

HYPRTI 2011; “Hydropyrolysis Catalyst Development: A Lab-Industry Partnership” National Advanced Biofuels Consortium Highlights, May 12, 2011; ww.nabcprojects.org/pdfs/hydropyrolysis_catalyst_development.pdf

Marker, T.; Felix. L.; Linck, M.; “Integrated Hydropyrolysis and Hydroconversion (IH2) Process for Production of Gasoline and Diesel Fuel from Biomass”, Gas Technology Institute presentation at TC Biomass 2009, Chicago, Il.

Marker, T.; Felix, L.; Linck, M.; “Hydropyrolysis of Biomass for Producing High Quality Liquid Fuels,” U. S. Patent Application No. 2010/0256428 A1, October 2010.

Marker, T. L.; Felix, L. G.; Linck, M. B.; Roberts, M. J. ; “Integrated Hydropyrolysis and Hydroconversion (IH2) for the Direct Production of Gasoline and Diesel Fuels or Blending Components from Biomass, Part 1: Proof of Principle Testing”; Progress and Sustainable Energy 2011, 31, 191-199.

Special Topics Areas

Hybrid Biochemical/Thermochemical Processes Biochemical routes offer opportunities to extremely selective conversions and a less energetically intensive pathway to initial carbon-carbon bond formation while thermochemical routes typically are operated in continuous modes for higher throughput and have ability to readily make distributions of molecules to enable the emulation of petroleum fuel blendstocks. Thermochemical processes also facilitate the conversion of lignin-bound carbon to fuel and chemical products. Unique combinations of biochemical reactions with thermochemical and/or thermocatalytic reactions may offer opportunities that offer a greater positive synergy than just a linear combination of the two processes. The following table indicates attributes commonly associated with biochemical and thermochemical or thermocatalytic processes that become positive or negative in the context of a biorefinery. It also highlights some potential synergies that might be captured by using hybrid processing.

Table 3 – Why Hybrid Processing Might be Considered

Biochemical Processes: Thermochemical and/or Thermocatalytic Processes:

Exhibit high levels of chemical and stereochemical selectivity

Typically operate in continuous configurations

Are less energetically-challenged with initial C-C bond formation

Offer product distributions aligned with distributions of molecules required in fuel blendstocks

Can produce intermediates with functionalities well suited for chemical catalysis

Exhibit high efficiencies

Are prone to reaction deactivation at product yields <20%

Lack selectivity at yields above 20%

Typically operate in batch configurations Are thermodynamically challenged with initial C-C bond formation

Several hybrid technologies are currently under development. Some configurations are biochemical conversion followed by thermocatalytic processing. Permutations of this approach might also involve chemical deconstruction of biomass to make simple carbohydrates available to organisms. Other configurations under development employ thermochemical processing followed by biochemical conversion to desired products.

Biomass pretreatments for making simple carbohydrates available to biochemical processes and chemical catalysis for fuel synthesis and/or upgrading represent the largest cost contributors to the production of biofuels apart from the cost feedstocks. Implementing a thermochemical conversion process in place of the chemical deconstruction of biomass (e.g., utilizing a synthesis gas intermediate) has the potential to improve costs by increasing the amount of carbon that is available for biological conversion, hence yield. The biological process for synthesis gas conversion has the potential to be less susceptible than chemical catalysis to contaminants typically present in synthesis gas, the biological entities would benefit from the presence of sulfur compounds. This is only one example of pairing the synergies of biochemical conversion with thermochemical conversion processes wherein thermochemical conversion is followed by biochemical processing.

Conversely, biochemical front-end technologies can yield highly homogeneous intermediate streams (e.g., isoprenoids, fatty acids, organic acids, and polyketides) ideal for catalytic conversion to non-oxygenated hydrocarbons suitable for fuel blendstocksthat are currently not readily arrived at via biological approaches. This scenario also pairs the positive synergies of biological and thermochemical conversion processes by implementing the energetically-favored initial C-C bond formation and selectivity of biology followed by leveraging the efficiency of chemical catalysis.

Hybrid processes may turn out to be more economical, utilize more carbon, and be capable of producing products that are challenging to synthesize through a non-hybrid process. However, hybrid processing may also introduce more unit operations than standalone biochemical or thermochemical processing systems, thereby increasing the risks and cost.

The most notable barriers to economical and efficient hybrid processes include the risk of unproven process configurations; energy and flow integration; lack of catalysts and enzymes that are tolerant to contaminants; separating intermediates, products, and poisons; and challenges in communication between scientists traditionally working in the biochemical and thermochemical areas. Activities to address these barriers include using techno-economic modeling to optimize the process flow; research into new catalysts, enzymes, and separation steps; laboratory and demonstration-scale testing.

References

Chia, M.; Schwartz, T.J.; Shanks, B.H.; Dumesic, J.A.; “Triacetic acid lactone as a potential biorenewable platform chemical”; Green Chemistry 2012, 14, 1850-1853.

Werpy, T.; Petersen, G.; “Top Value Added Chemicals From Biomass, Volume I: Results of Screening for Potential Candidates from Sugars and Synthesis Gas”; U.S. Department of Energy, Energy Efficiency and Renewable Energy, Biomass Program 2004.

Holladay, J.E.; White, J.F.; Bozell, J.J.; Johnson, D.; “Top Value Added Chemicals From Biomass, Volume II: Results of Screening for Potential Candidates from Biorefinery Lignin”; U.S. Department of Energy, Energy Efficiency and Renewable Energy, Biomass Program 2007.

Bozell, J.J.; Petersen, G.R.; “Technology development for the production of biobased products from biorefinery carbohydrates – The U.S. Department of Energy’s “Top 10” revisited”; Green Chemistry 2010, 12, 539-554.

Simmons, B.; “Chemical and Biochemical Catalysis for Next Generation Biofuels”; RSC Energy and Environment Series No. 4, Royal Society of Chemistry 2011.

Barrier Area 1: Separations Research Activities Need for multi-pronged

approach to separations/fractionation of products – distillation, extract

New selective separation methods that target products, intermediates, and poisons

Separations as a step to link biochemical and thermochemical processes (e.g., recovery of sugars from thermochemical conversion)

Barrier Area 2: Economics/Techno-Economic Analysis

Research Activities

Overall economics of a combined biochemical/thermochemical process is not known

Target intermediate molecules and fuel blendstocks based on techno-economic analyses.

Perform GAP analysis: what has been done, what might be done, and screening cost of each.

Barrier Area 3: Catalysts: Inorganic or Organic

Research Activities

Enzymatic selective heteroatom removal for catalyst feed pretreatment

It is still unknown whether we have thermochemical sugar production methods

Engineer and screen organisms/catalysts to make specific intermediates than can be converted to fuels and products.

Develop and improve catalyst/organism tolerance/resistance.

that will produce a feed which does not contain contaminants poisonous to organisms.

Inorganic catalysts that are tolerant to the aqueous media used in earlier biochemical processing as well as contaminants

Rational design of new enzymes and catalysts.

Barrier Area 4 Feedstock Interface

Research Activities

Biochemical conversion allows the use of wet feedstocks. Are there comparable thermochemical conversion routes?

Feedstock anatomy and fractionation is presumably different for thermochemical and biochemical processes are there opportunities for a hybrid process?

Generate process compatible feedstocks for hybrid processing.

Modify feedstocks to take advantage of a hybrid process.

Assess the techno-economic impact over the entire process for the use of wet feedstocks and the potential for energy savings.

Barrier Area 4: Interdisciplinary Communication

Research Activities

The challenges of collaboration between biochemical and thermochemical conversion researchers

Hold a multidisciplinary workshop on the hybrid concept with cross fertilization of presentations and breakout sessions

Emphasize interesting hybrid research initiatives to encourage collaboration.

Barrier Area 4: Integration Research Activities Energy integration.

Thermochemical processes are generally higher temperature and potentially high pressure, while biochemical processes generally have milder conditions. Heat integration

Publish more screening techno-economic analyses of different hybrid options and explore integration options.

Energy “pinch” analysis to intelligently stage energy transfer.

Counter current process flow concepts can potentially “conserve/transfer” thermal energy from thermocatalysts to thermogenic biocatalysts.

TEA tools to enable rational evaluation of hybrid process

and temperature swings may be challenging.

Balancing the Weight Hourly Space Velocity of thermochemical/thermocatalytic and biochemical processes.

options, including screening new hybrid options, assessing economics outlook, and determining potential operational ranges.

Barrier Area 4: Risks/Unknown Research Activities Proliferation of unit

operations. Use techno-economic analysis to address unit operation

proliferation Techno-economic tools to enable rational evaluation of

hybrid process options, including screening new hybrid options, assessing economic outlook, and determining potential operational ranges.

Direct Microbial Conversion to Fuels from Unconventional SourcesExciting new platform technologies are emerging whereby fuels can be synthesized directly without the input of sugars, or at least those used commonly in heterotrophic fermentation schemes. These processes, taking place in algal or bacterial cells, are fueled by photosynthesis or by electrical input that alters the redox state of compounds (metals or other inorganic substances) that the organisms use to grow. In the photoautotrophic case, biochemical pathways are being engineered within algae or cyanobacteria that divert pathways that normally lead to lipid production. In different variations on this theme, fatty acids or hydrocarbons accumulate inside the cells or are excreted into the fermentation broth where they are found to aggregate and, in some cases, float to the top of the culture vessel for ease in separations. In the electrically-driven lithotrophic case, reducing equivalents are introduced by the formation of an electrochemical potential at the surface of an electrode or by the utilization of soluble electron transfer carriers, some of which are abiotic and designed specifically for use in these production strategies. Among these latter approaches, ammonia-, sulfur, and iron-reducing organisms are being pursued in (re)directing electron flow to reduce carbon dioxide into short-chain hydrocarbons and alcohols, at least initially, that can be used as replacement fuels.

Direct conversion strategies offer many advantages, including minimalized media formulations, little competition for valuable resources and food crops, low environmental impact, direct CO2

utilization, and biomass production decoupled from hydrocarbon production. In remote locations, electrical demands for lithotrophic approaches can be met by the output of relatively simple photovoltaic devices. In contrast, these strategies are challenged by the need to maintain refined culture conditions, relatively sluggish production rates, the requirement for elaborate (photo) bioreactor designs with capability for mass transport of gasses, the implementation of organisms without tractable genetic systems, poor performance in economic feasibility studies, and the potential for exotic waste streams.

The pursuit of these technologies, while challenged, is encouraged since the majority of these approaches are in their infancy. All of these approaches would benefit from in-depth techno-economic and life-cycle analyses. Photoautotrophic approaches will benefit from all of the on-going basic and applied research that utilizes photoheterotrophic strategies. Additional challenges that have been identified for future research and development efforts involve bioreactor design and engineering to facilitate the mass transport of gases and increased light penetration required in cultures of high cell density. Additionally, if secretion of the fuel proves to be the most viable separations path forward, then basic research on secretion mechanisms will be warranted. Research aimed at advancing the lithographic electrofuels approach will be, comparatively, at a more fundamental level. Organisms and strains will need to be discovered and their genomes sequenced, in addition to the development and testing of new catalysts and electrodes.

Consolidated BioprocessingThe conversion of lignocellulosic feedstocks to fuels is a complicated process that involves many steps, with the breakdown of feedstocks into sugars and subsequent fermentation into higher-value feedstocks being the two most significant steps in the process. In biofuel production strategies that have shown the most promise to date, these steps operate in isolation and future research plans are to optimize them independently. However, efficiencies may be realized if and when several of the steps in the process are grouped and work in concert to produce bio-based hydrocarbon fuels from coarsely treated or raw feedstock materials. The term that has been coined for this research area is Consolidated BioProcessing (CBP), and large industrial efforts, as well as several focused, interdisciplinary research teams, are looking to make this once thought-to-be dream a reality. This integrated approach for the production of biofuels can be considered a direct conversion pathway to fuel from rather unconventional sources, in its loosest definition, since most strategies that are being discussed to date involve distinct input of energy and carbon reserves, most commonly in the form of C5 and C6 sugars. In nature, fungi and bacteria are the workhorse organisms that breakdown lignocellulosic materials. These microorganisms, or pathways derived from them, will be key players in CBP conversion technologies through the use of highly engineered strains in monoculture or, more likely, by unique fermentation routes involving a mixture or community of natural or

engineered organisms. The key distinction of the CBP approaches is that large carbohydrate reserves at any stage in the process are not likely to be observed because any carbohydrate generated is immediately consumed.

These particular direct conversion strategies offer many process advantages, however, these strategies are challenged by rates of conversion that are expected to be very low. Since feedstock materials input into this method are raw or will have minimal pretreatment, the process will need to proceed in an environment of extremely high solids. The organisms that accomplish these tasks may not have been identified or isolated yet and getting them to work in an environment in the presence of other dedicated organisms will prove challenging. In addition, the nutrient and media requirements will likely need to be tailored to the specific blend of feedstock being employed and could vary from batch to batch of the input material. As with any of the direct processes, there are concerns about the requirements for elaborate reactor designs with capability for mass transport of gasses, the implementation of organisms without tractable genetic systems, poor performance in economic feasibility studies, and the unique separations challenges that present themselves with high-solids fermentation processes and hydrocarbon production.

The pursuit CBP technologies, while challenged, are encouraged since the majority of these approaches are in their infancy. In-depth techno-economic and life-cycle analyses are desperately needed but are likely to be extremely difficulty due to the nature of the state of the research. Additional research areas that may help to bring CBP to the mainstream or determine whether or not it is an economical route for biofuels production include rheology for such systems, an understanding of what hydrocarbons may be outputs of such approaches, analytical tools to measure microbe biomass on surfaces, and genome sequencing and culture methodologies to understand more readily how and what organisms degrade cellulosic materials directly in nature.

Barrier Area 1: Photoautotrophic Organisms

Research Activities

C5 obtaining/utilizing light Product secretion

Engineer strains with smaller light harvesting antennae for greater light penetration

Introduce modified antennae systems that also broaden range of spectrum

Photobioreactor engineering Develop new/better gene regulation

cassettes/schemes Decide if secretion of product is good Survey economics of both strategies

o Intracellular retention

o Extracellular secretionBarrier Area 2: Electrofuels Research Activities Reaction rates Comparative energetics in

relation to CO2 capture and utilization

Develop fundamental approacheso Screen strainso Search for extremophiles

Find better catalysts/electrodes/bioelectrodes Improve proton transport membranes

o Side by side comparisonso Kinetic & thermodynamic modeling, a ‘whole’

systems approacho Examine ruminant systems

Barrier Area 3: Consolidated Bioprocessing

Research Activities

Improved deconstruction mechanisms

Integrate deconstruction methodologies Incorporate physical deconstruction process Develop analytical tools for complex systems [Insert a verb here] Microbes growing on dense

biomass

Conversion Systems for Genetically Modified/Optimized FeedstocksGenetic modification of feedstocks will enable engineering of crops with desirable traits for downstream processing and conversion to advanced biofuels. However, significant challenges will exist, from research to biofuel production. Recent research has focused on reducing the recalcitrance of cell walls to downstream deconstruction processes, reducing inhibitors to processing and conversion, shifting sugar content to be more amenable for fermentation, utilizing plants to produce cellulases and other enzymes for deconstruction, improved traits for processing and storage, and more general fitness traits for success as energy crops.

Potential Modifications for Improved Downstream ProcessingGenetic manipulation of lignin content has been pursued vigorously to reduce recalcitrance of cell walls to deconstruction thereby improving access to polysaccharides during hydrolysis. Biosynthesis of lignin monomers and polymerization is well understood, allowing for advances in genetic modification of cell walls in both energy crops and model plants. For example, downregulation of monolignol biosynthetic enzymes has resulted in reduced lignin content but increased cellulose composition, saccharification and fermentation efficiency in both poplar and

switchgrass (Leple et al., 2007; Fu et al., 2011). When tested in field trials of poplar, the reduction in lignin content was confirmed and an increase in ethanol production was reported (VIB News, 2011). Other lignin reduction strategies have resulted in increased release of cell wall sugars as demonstrated in the model dicot, Arabidopsis thaliana. Modifying lignin polymerization to include “stoppers” (units that decrease the degree of polymerization)(Eudes et al., 2012), other phenolic compounds resembling monolignols (Tobimatsu et al., 2012), or secondary metabolites (Grabber et al., 2012) has shown increases in saccharification efficiency and fermentation with minimal effects on growth, development, and fitness. Over 90 potential lignin monomers have been identified that could enable specific metabolic engineering of lignin tailored to specific downstream applications (Vanholme et al., 2012).

Other cell wall polymers are targets for reducing recalcitrance, but also tailoring sugar content for fermentation. Cellulose, a glucose polymer present in the cell wall, is an important source of carbon for downstream conversion and upgrading. Loss-of-function mutations in cellulose synthases often have deleterious effects on plant morphology (Somerville, 2006), indicating a more controlled manipulation of cellulose synthesis is required for effective genetic modification of feedstocks. For example, specific point mutations in an Arabidopsis cellulose synthase, CESA1, results in the formation of cellulose, but with reduced crystallinity and enhanced glucose release after enzymatic hydrolysis (Harris et al., 2012). Reduced cellulose crystallinity is desirable, as it allows for better access to cellulose for cellulases (Abramson et al., 2010). Other sugar biosynthetic proteins also play a role in cellulose accumulation. Overexpression of a cotton sucrose synthase in poplar leads increased cell wall thickening and cellulose accumulation, but as a result increased crystallinity (Coleman et al., 2009).

Hemicellulosic and pectic (or matrix) polysaccharides present a greater challenge for genetic modification due to the complexity of their structure and the likely number of enzymes involved. Some glycosyltransferases that assemble various matrix polysaccharides have been identified, including those for xylan and xyloglucan, but the understanding of biosynthesis of less common polysaccharides is still lacking (Scheller and Ulvskov, 2010). Many of these enzymes have been identified through mutant screens, where loss of function results in developmental defects. Regulation of the substrates used for polymerization may be another route to controlling the content of matrix polysaccharides. Nucleotide sugar transporters move sugar monomers from their site of synthesis in the cytosol to the Golgi apparatus, where they are then incorporated into polymers before being secreted to the plasma membrane and cell wall (Reyes and Orellana, 2008). Matching transporters with their nucleotide sugar substrates would enable future modification of transporter activity to control the flux of nucleotide sugars into the Golgi and allow for better control of matrix polysaccharide content for downstream processing and fermentation events.

Recently, identification of transcription factors that regulate secondary wall biosynthesis of cellulose, hemicellulose, and lignin have been identified (Demura and Ye, 2010; Wang and Dixon, 2011). A set of “master switches” initiates a cascade of transcriptional activation that then finely tunes the deposition of polymers. Current research is focusing on how to tease apart the different transcription factors responsible for cellulose, hemicellulose, and lignin synthesis and polymerization in order to maintain integrity of necessary tissues, such as fibers, while simultaneously reducing the overall recalcitrance of cell walls. This approach may be more promising than constitutive cell wall polymer modification as timing and levels of expression could be more tightly controlled. Additionally this approach could minimize the developmental problems inherent in constitutive modification of the cell wall.

Other potential methods of cell wall modification for compatibility with deconstruction methods include expression of proteins that modify the cell wall in planta. Expression of cellulases, other glycosyl hydrolases, ligninases, or other proteins could begin the process of cell wall loosening or degradation in advance of preprocessing. Another class of enzymes, the expansins, are thought to loosen cell walls by cleaving hydrogen bonds (Cosgrove, 2000; Whitney et al., 2001). Controlled overexpression of expansins could result in exposing more cellulose to cellulases with less pretreatment. Many researchers have successfully expressed enzymes in planta, particularly thermostable glycosyl hydrolases that are inactive at room temperature, but can be activated at higher temperatures during processing (Sainz, 2009; Jung et al., 2012). Alternatively, enzymes can be expressed in plants and then total protein extracted during processing, for later use during hydrolysis (Sticklen, 2008). Often, expression of desirable enzymes is targeted to organelles to insure proper folding, modification, and to restrict potential enzymatic activity away from the cell wall. Some of the benefits to expressing cellulases and other hydrolases in plants include reduced cost over current production methods and proper folding and modification.

Beyond recalcitrance, research efforts have focused on removing inhibitors to downstream processing from the cell wall. Certain cell wall constituents, such as acetyl groups, are released during enzymatic hydrolysis of cell wall materials and can be inhibitory to the fermenting organisms involved in upgrading these sugars into fuels. Two separate gene families have been identified that control aspects of acetylation of cell wall polysaccharides. While knockout mutations in members of these families result in less O-acetylation of hemicelluloses (Gille et al., 2011; Manabe et al., 2011), large reductions can only be achieved with multiple mutations present, which results in plants with less stem strength and reduced cell wall thickness (Lee et al., 2011). Interestingly, an Arabidopsis accession, Ty-0, with three single nucleotide polymorphisms in one acetyltransferase was identified, suggesting that a decrease in acetylation has little cost to overall fitness (Gille et al., 2011). Research into modifying inhibitor

content will have to include understanding the roles of these molecules in plant growth and development, as well as consequences to the production of biofuels.

While much of the current bench-scale research is being performed on C3 carbon fixing plants, many of the proposed biofuel feedstocks are C4 plants, which have more efficient photosynthetic systems, better water and nutrient usage, and produce comparatively higher yields (Jakob et al., 2009). However, the genetic backbones of many of these crops are still poorly understood and lacking the tools for modification available for model species. There are significant barriers to be overcome with potential C4 crop plants, including the fact that Miscanthus and switchgrass are not yet domesticated as crop plants, the complex genomic structure of these plants, a lack of genetic tools similar to those available in model organisms, difficulty of transformation and other modification techniques, and ease of propagation.

Another particular concern for thermochemical processing, is the ash content of feedstock crops. Inorganics, such as silica, alkali metals, chlorine, and sulfur can constitute up to 10% of biomass. These inorganics end up as waste during processing and complicate processing, (Sannigrahi et al., 2010). Potential feedstocks may be modified to reduce the ash content and thereby improve conversion processes. Reduction of inorganics may have developmental consequences, as was observed in rice, where mutants with reduced silica uptake had reduced growth (Ma et al., 2002).

It is imaginable that more general improvements in crop traits will be considered as well for genetically modified (GMO) biofuels feedstocks, including such traits as increased yield, pathogen resistance, abiotic stress tolerance, improved nutrient usage and potential for long-term storage will also be considered. Many of these traits have been or are under development for food and feed crops, and crops with desirable agronomic traits are already being successfully farmed.

Unintended Consequences and New Logistical Problems with GMO FeedstocksWhile GMO feedstocks hold great promise to reduce the cost and difficulties of downstream processing events, the unintended consequences of such changes must be considered. Many of these potential modifications are still being developed in bench-scale experiments and translation to the field holds many unknowns about the fitness and yield costs of modifications. It is possible that size, morphology, strength, water and nutrient usage, and other important agronomic traits will be negatively affected by novel genetic modifications.

Modifications geared towards improved biofuels feedstocks present new challenges to existing logistical, processing, conversion, and upgrading technologies. Mechanical processing of biomass could be affected by alterations in strength due to either softening cell walls for easier deconstruction or by increasing strength, either for long-term storage reasons or because of

greater glucose accumulation. It would also be reasonable to expect these same alterations in the cell wall impacting both chemical and biochemical hydrolysis post-processing. Access to certain bonds or structures would be altered in plants with relaxed cellulose crystallinity or with altered matrix polysaccharide content. As enzymes have high specificity for the structures they bind to, enzymatic cocktails may need to be tailored to the specific feedstock and its sugar composition and content. Engineering plants to express their own cell wall-degrading enzymes may affect the stability of the cell walls for long term storage as well. Additional concerns that come with expressing cell wall-degrading enzymes in planta include restricting activity of the enzymes so as not to affect the cell wall until necessary and maintaining enzymatic activity during processing and until hydrolysis.

It is also possible that by altering the composition of the cell wall, for example by using novel lignin units, or the changing metabolite profile of a feedstock via changes in gene expression, new and unknown inhibitors will be introduced. With so many proposed monomers for metabolic engineering of lignin, it is conceivable some may be detrimental to deconstruction, conversion, or upgrading processes. Another source of new inhibitors could be the alteration of transcriptional regulation of cell wall biosynthesis. While transcription factors can finely control biosynthetic pathways, it is also possible that untargeted pathways would also be altered by the cell wall regulatory transcription factors. Metabolic, transcriptomic, and proteomic profiling are rarely done to understand the effect of genetic modification, often because of the assumption that such modification is targeted and limited in effect.

Other aspects to consider in GMO biofuels feedstocks processing and conversion is the impact on current separation technologies. A shift in the amount of sugars, lignin, or ash may present difficulties for existing systems or introduce new molecules to separate into different streams. Additionally, some potential modifications, such as reducing acetylation, may end up decreasing the amount of valuable byproduct, in this case acetic acid, produced during biofuels production. New separation technologies or the loss of co-products may increase associated costs.

Plants with altered cell walls may also be less resistant to certain pathogens. In the case of snl6, a rice CCR mutant, reduced lignin and increased sugar extractability comes with the cost of increased susceptibility to certain pathogens (Bart et al., 2010). It is believed that some pathogen resistance signaling is mediated by the cell wall, and while the mechanisms are not yet well identified, alteration in the cell wall could also lead to alteration in pathogen detection and response (Wolf et al., 2012).

More general crop improvement will need to be considered for effects on the entire biofuels production process. Traits desirable in food or feed crops could have negative impacts on traits desirable in a biofuels feedstock.

Regulation of GMO feedstocks will be a key component to their implementation. USDA, EPA, and FDA have been the traditional regulators of GMO crops and will continue to be. How future biofuels crops will be regulated will influence the logistics of adoption, planting, harvesting, transporting, tracking, etc. Consideration of the regulatory aspects will need to be included in development of pathways and research goals.

Barrier Area 1: Unintended Consequences of Genetic Modification

Research Activities

Narrowing feedstock optimization of a conversion facility increases supply risks or dramatically increases cost if non-optimal varieties have to be used

New agronomic risks resulting from secondary effects of modification

Lack of understanding of the effect of modified feedstocks on soil/carbon

Will modified feedstocks need to depend on commodity-based system for economic viability, and if so, can they be integrated into a uniform format that OBP is pursuing?

Develop standard pipeline assay for feedstock evaluation Facilitate integration of modified feedstocks into logistics

and processing

Barrier Area 2: Supply Chain Considerations

Research Activities

Chain of custody tracking to meet regulatory or custom labeling requirements

Impact of GMO on harvesting and processing

New collection systems may be required

Traits desirable for storage may not be compatible with downstream processing

Identify traits for improved processing Integrate modified feedstocks at pilot scale (INL PDU?) to

validate performance and identify problems/future improvements

Scale-up/deployment

Barrier Area 3: Process Design Engineering

Research Activities

Current MO’s target ethanol as final product but new focus is on

Identify desirable traits for process and benefits/costs Integrate modified feedstocks to validate performance and

hydrocarbon fuels Modified feedstocks may behave

differently in conversion processes than non-modified equivalents

Separation systems may need to be modified

Potential need to tailor catalysts (catalyst requirements will depend on genetic modifications, e.g. poisons, coking agents)

Novel components/by-products associated with modified feedstocks

Some new inhibitors to established processes

Enzyme stability through pretreatment

As varieties are optimized as feedstocks for niche conversion technologies, the UFF commodity model becomes challenged.

Difference in time-scale between GMOs and the conversion/fuel process

Lack of understanding of resulting makeup of bio-oils or other products

identify problems/future improvements at the pilot scale Scale-up/deployments

Abramson, M., Shoseyov, O., Shani, Z., 2010. Plant cell wall reconstruction toward improved lignocellulosic production and processability. Plant Science 178, 61–72.

Bart, R.S., Chern, M., Vega-Sánchez, M.E., Canlas, P., Ronald, P.C., 2010. Rice Snl6, a Cinnamoyl-CoA Reductase-Like Gene Family Member, Is Required for NH1-Mediated Immunity to Xanthomonas oryzae pv. oryzae. PLoS Genetics 6, e1001123.

Coleman, H.D., Yan, J., Mansfield, S.D., 2009. Sucrose synthase affects carbon partitioning to increase cellulose production and altered cell wall ultrastructure. Proceedings of the National Academy of Sciences 106, 13118–13123.

Cosgrove, D.J., 2000. Loosening of plant cell walls by expansins. Nature 407, 321–326.

Demura, T., Ye, Z.-H., 2010. Regulation of plant biomass production. Current Opinion in Plant Biology 13, 298–303.

Eudes, A., George, A., Mukerjee, P., Kim, J.S., Pollet, B., Benke, P.I., Yang, F., Mitra, P., Sun, L., Çetinkol, Ö.P., Chabout, S., Mouille, G., Soubigou-Taconnat, L., Balzergue, S., Singh, S., Holmes, B.M., Mukhopadhyay, A., Keasling, J.D., Simmons, B.A., Lapierre, C., Ralph, J., Loqué, D., 2012. Biosynthesis and incorporation of side-chain-truncated lignin monomers to reduce lignin polymerization and enhance saccharification. Plant Biotechnology Journal 10, 609–620.

Fu, C., Mielenz, J.R., Xiao, X., Ge, Y., Hamilton, C.Y., Rodriguez, M., Chen, F., Foston, M., Ragauskas, A., Bouton, J., Dixon, R.A., Wang, Z.-Y., 2011. Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass. Proceedings of the National Academy of Sciences 108, 3803–3808.

Gille, S., de Souza, A., Xiong, G., Benz, M., Cheng, K., Schultink, A., Reca, I.-B., Pauly, M., 2011. O-Acetylation of Arabidopsis Hemicellulose Xyloglucan Requires AXY4 or AXY4L, Proteins with a TBL and DUF231 Domain. The Plant Cell 23, 4041–4053.

Grabber, J.H., Ress, D., Ralph, J., 2012. Identifying new lignin bioengineering targets: impact of epicatechin, quercetin glycoside, and gallate derivatives on the lignification and fermentation of maize cell walls. J. Agric. Food Chem. 60, 5152–5160.

Harris, D.M., Corbin, K., Wang, T., Gutierrez, R., Bertolo, A.L., Petti, C., Smilgies, D.M., Estevez, J.M., Bonetta, D., Urbanowicz, B.R., others, 2012. Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase. Proceedings of the National Academy of Sciences 109, 4098–4103.

Jakob, K., Zhou, F., Paterson, A.H., 2009. Genetic improvement of C4 grasses as cellulosic biofuel feedstocks. In Vitro Cellular & Developmental Biology - Plant 45, 291–305.

Jung, S.-K., Parisutham, V., Jeong, S.H., Lee, S.K., 2012. Heterologous Expression of Plant Cell Wall Degrading Enzymes for Effective Production of Cellulosic Biofuels. Journal of Biomedicine and Biotechnology 2012, 1–10.

Lee, C., Teng, Q., Zhong, R., Ye, Z.-H., 2011. The Four Arabidopsis REDUCED WALL ACETYLATION Genes are Expressed in Secondary Wall-Containing Cells and Required for the Acetylation of Xylan. Plant and Cell Physiology 52, 1289–1301.

Leple, J.-C., Dauwe, R., Morreel, K., Storme, V., Lapierre, C., Pollet, B., Naumann, A., Kang, K.-Y., Kim, H., Ruel, K., Lefebvre, A., Joseleau, J.-P., Grima-Pettenati, J., De Rycke, R., Andersson-Gunneras, S., Erban, A., Fehrle, I., Petit-Conil, M., Kopka, J., Polle, A., Messens, E., Sundberg, B., Mansfield, S.D., Ralph, J., Pilate, G., Boerjan, W., 2007. Downregulation of Cinnamoyl-Coenzyme A Reductase in Poplar: Multiple-Level Phenotyping Reveals Effects on Cell Wall Polymer Metabolism and Structure. THE PLANT CELL ONLINE 19, 3669–3691.

Ma, J.F., Tamai, K., Ichii, M., Wu, G.F., 2002. A rice mutant defective in Si uptake. Plant Physiol. 130, 2111–2117.

Manabe, Y., Nafisi, M., Verhertbruggen, Y., Orfila, C., Gille, S., Rautengarten, C., Cherk, C., Marcus, S.E., Somerville, S., Pauly, M., Knox, J.P., Sakuragi, Y., Scheller, H.V., 2011. Loss-of-Function Mutation of REDUCED WALL ACETYLATION2 in Arabidopsis Leads to Reduced Cell Wall Acetylation and Increased Resistance to Botrytis cinerea. PLANT PHYSIOLOGY 155, 1068–1078.

Reyes, F., Orellana, A., 2008. Golgi transporters: opening the gate to cell wall polysaccharide biosynthesis. Current Opinion in Plant Biology 11, 244–251.

Sainz, M.B., 2009. Commercial cellulosic ethanol: The role of plant-expressed enzymes. In Vitro Cellular & Developmental Biology - Plant 45, 314–329.

Sannigrahi, P., Ragauskas, A.J., Tuskan, G.A., 2010. Poplar as a feedstock for biofuels: A review of compositional characteristics. Biofuels, Bioproducts and Biorefining 4, 209–226.

Scheller, H.V., Ulvskov, P., 2010. Hemicelluloses. Annual Review of Plant Biology 61, 263–289.

Somerville, C., 2006. Cellulose Synthesis in Higher Plants. Annual Review of Cell and Developmental Biology 22, 53–78.

Sticklen, M.B., 2008. Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol. Nature Reviews Genetics 9, 433–443.

Tobimatsu, Y., Elumalai, S., Grabber, J.H., Davidson, C.L., Pan, X., Ralph, J., 2012. Hydroxycinnamate Conjugates as Potential Monolignol Replacements: In vitro Lignification and Cell Wall Studies with Rosmarinic Acid. ChemSusChem 5, 676–686.

Vanholme, R., Morreel, K., Darrah, C., Oyarce, P., Grabber, J.H., Ralph, J., Boerjan, W., 2012. Metabolic engineering of novel lignin in biomass crops. New Phytologist n/a–n/a.

VIB News, 2011. Initial field test results GM poplars: bioethanol yield almost doubled [WWW Document]. URL http://www.vib.be/en/news/Pages/Initial-field-test-results-GM-poplars-bioethanol-yield-almost-doubled.aspx

Wang, H.-Z., Dixon, R.A., 2011. On-Off Switches for Secondary Cell Wall Biosynthesis. Molecular Plant 5, 297–303.

Whitney, S.E.C., Gidley, M.J., McQueen-Mason, S.J., 2001. Probing expansin action using cellulose/hemicellulose composites. The Plant Journal 22, 327–334.

Wolf, S., Hématy, K., Höfte, H., 2012. Growth Control and Cell Wall Signaling in Plants. Annual Review of Plant Biology 63, 381–407.

Lignin UtilizationLignin is a heterogeneous, alkyl-aromatic polymer comprised of 3 monomeric phenylpropane units with 0, 1, or 2 methoxyl groups on the aromatic rings. These monomers are linked together by C-O and C-C bonds (Figure 1) formed via oxidative coupling mechanisms during plant cell wall formation (1). In fermentation-based conversion processes, such as biochemical conversion of lignocellulosic biomass to ethanol, lignin is currently burned for process heat and power because selective and cost effective conversion strategies have not yet been developed to maturity and demonstrated at scale in an integrated biorefinery context. In thermochemical conversion processes, such as pyrolysis, lignin produces reactive phenolic species, which are difficult to stabilize and lead to undesired side reactions and pyrolytic lignin. Despite significant challenges with lignin utilization in current biomass conversion technologies, lignin offers new potential value streams from biomass for fuels, commodity chemicals, and fine chemicals. Lignin is the most energy dense polymer in biomass and in some feedstocks, it represents up to 30% of the plant cell wall by mass, which is a substantial fraction of usable carbon for the production of fuels or chemicals. To harness this significant potential, lignin utilization will require substantial research investments to develop integrated processes that will enable deconstruction and utilization of lignin in concert with carbohydrate streams, and development of markets of commensurate scale to fuels or chemicals produced from carbohydrate streams. To this end, translational research efforts are warranted (and many are underway) to examine the following key aspects of enhanced lignin utilization:

Understand genetic variation in lignin recalcitrance and structure in potential energy crops, and the development of genetically modified feedstocks that will enable more homogeneous and less recalcitrant lignin polymers for simpler fractionation or deconstruction processes (2-8),

Enhance processes to separate lignin from carbohydrate streams (9-11) for independent upgrading processes or development of enhanced thermal (12-15) or biological schemes (16,17) that can deconstruct both sugars and lignin simultaneously,

Develop new, enhanced catalytic (18-21) or biological (16,22) routes for lignin depolymerization and upgrading,

Develop new analytical tools to more effectively characterize and understand lignin structure from the plant cell wall context through deconstruction and upgrading, which will enable more efficient design of catalytic and separation processes,

Identifiy of chemical and biological pathways for the production of fuels and/or chemicals from lignin.

Development of new pathways and processes for lignin utilization that are fully integrated into a biorefinery context with simultaneous carbohydrate usage will rely heavily on the application of techno-economic and life-cycle analysis to quantify the efficacy of a given pathway over others. Here, some of the primary challenges associated with lignin utilization are discussed and research areas are identified that merit further investigation to enable the utilization of the lignin fraction of the plant cell wall.

Characterizing and Engineering Lignin for Energy CropsLignin is a heterogeneous biopolymer, which unlike cellulose and hemicellulose, is not yet easily broken down into constituent, stable monomers with the application of mineral acids or enzymes at industrial scales. Even when lignin is deconstructed to its constituent monomeric species, there exist few upgrading strategies to date that have been identified to convert those monomeric species to products of higher value in a cost-effective manner. To that end, one of the most important research activities to enable lignin utilization is the characterization of lignin

Figure 1. A representative structure of a lignin polymer, which is connected via C-C and C-O bonds between

recalcitrance as a function of natural diversity in a given feedstock as well as homogenization of lignin via genetic engineering of putative energy crops such as poplar and switchgrass. A recent study on natural variation in poplar demonstrated that lignin content and chemistry can vary significantly across a natural, undomesticated population (23). More study is warranted to quantify natural variation in other feedstocks and connect lignin structure and hemistry to ascertain traits that enable simpler, cheaper conversions of lignin to monomeric species. Additionally, genetic modification studies in biomass feedstocks to date have been primarily focused on aiding release of carbohydrates in a more efficient manner, which will reduce pretreatment and enzyme costs. However, an added benefit of these efforts is that modification of lignin by, e.g., the introduction of non-natural monolignols (6,7) or homogenization of lignins (4,5) in some cases provides potential for simpler and inexpensive fractionation processes. In addition, more homogeneous streams of oxygenated aromatic monomers derived from genetically modified lignins will enable more straightforward upgrading processes downstream and identification of realistic pathways for production of value-added products from lignin.

Current Strategies for Lignin Isolation and Depolymerization and Research NeedsThe vast majority of research into low-temperature routes for biomass conversion to date has historically focused on utilization of carbohydrates, such that the most developed low-temperature biomass conversion processes (e.g., biochemical fermentation processes for ethanol production) have not typically been designed with lignin utilization in mind. Other processes that are able to separate lignin from the carbohydrate fraction of the plant cell wall are less well-developed at large scales and in an integrated fashion (10,11), and further development and demonstration are warranted. Several primary strategies for selective lignin removal from biomass to date can be broadly classified as:

Carbohydrate removal from intact lignin Examples of this strategy are the many pathways that use thermochemical pretreatment (e.g., AFEX, dilute acid, hot water, steam explosion, lime, etc. (24,25)) to make the cell wall more accessible and (for some technologies) to remove hemicellulose, followed by enzymatic hydrolysis and fermentation or catalytic upgrading of the resulting carbohydrates. Most of these pathways leave some or all of the lignin in the cell wall (although it is usually chemically and physically modified), and ultimately burned for heat and power generation. Elucidating the fate of lignin in these various processes is important to understanding if it can be utilized via downstream thermochemical or biochemical conversion and upgrading strategies. Concentrated acid hydrolysis also falls into this category, although higher severity acidic conditions, especially in a continuous flow-through scheme, may liberate a substantial fraction of lignin with the carbohydrates as acid catalysis is known to readily cleave C-O linkages.

Lignin deconstruction from (mostly) intact carbohydratesThese types of processes typically revolve around pulping or bleaching of biomass, wherein lignin (and in some cases, some hemicellulose) is removed from the carbohydrate fraction of biomass. Many variations of paper and pulp milling processes have been examined, especially for woody feedstocks, for translation of these technologies to biomass conversion for fuels, but to date these types of processes are not economically viable for that purpose. Further

development is needed around understanding the chemistry required for lignin depolymerization and optimizing these types of processes such that removal of lignin in a form that is chemically useful or upgradeable can be achieved in a cost-effective manner without sacrificing yields of carbohydrates.

Lignin/Carbohydrate fractionationFractionation strategies are broadly characterized by the use of solvent-based systems such as the Organosolv processes (9,10,26) or the use of specialized solvents like ionic liquids that can dissolve plant cell walls completely (11,27,28). Organosolv processes typically employ mixtures of water and organic solvents (e.g., ethanol and MIBK or acetone), sometimes with a low concentration of a mineral acid present, such that lignin can be partially deconstructed and partitioned to the organic phase and hemicellulose and cellulose partitions to the aqueous phase. The cellulose and hemicellulose in Organosolv processes can be partially deconstructed to monomeric or oligomeric sugars (hemicellulose, being less recalcitrant than cellulose, is typically deconstructed in these types of processes when acid is present). Both Organosolv and ionic liquid-based fractionation processes require solvent separation and recycle processes to recover the solvent materials, which add significantly to capital and operating costs, but they enable the use of a separated lignin stream for downstream conversion and upgrading. For Organosolv processes, significantly more research and analysis is warranted to understand the chemical composition of the resulting organic lignin stream as a function of a given feedstock and fractionation process (including operating conditions and types and ratios of solvents). For fractionation processes to be industrially viable for fuels or chemical production, fully integrated upgrading and solvent recovery processes must be designed in the context of a biorefinery, which will require substantial research efforts tightly coupled to techno-economic and life-cycle analysis, as well as a viable end-use for the resulting lignin and carbohydrate streams.

Additionally, thermochemical routes such as pyrolysis and liquefaction are able to depolymerize both the lignin and carbohydrate streams simultaneously (12-15). The resulting streams from these types of processes typically are quite reactive, and indeed stabilization of the resulting bio-oils is a topic of current major research efforts. Lignin depolymerization products from high-temperature thermochemical deconstruction routes primarily consist of reactive oxygenated phenolics. In the condensed phase, it is common for these products to form C-C bonds, and thus convert into even more recalcitrant species that are of little to no value. However, the vapor phase products from pyrolytic depolymerization of lignin are often monomeric species, and are thus excellent targets for upgrading. Significant research is needed to deoxygenate and upgrade lignin deconstruction products, especially in pyrolysis vapors.

As discussed directly above, some processes already exist to (partially) depolymerize lignin in a process context with the aim to separate it from carbohydrates, or to depolymerize both carbohydrates and lignin simultaneously via thermochemical routes such as liquefaction and pyrolysis (12-15). In the case of fractionating lignin from carbohydrates, in many cases, lignin may subsequently require additional depolymerization before upgrading. For this purpose, acid or base treatments are both known to cleave the more labile lignin inter-monomer linkages (29-31). For isolated lignins, base-catalyzed depolymerization has been shown to be a potentially

attractive means to both reduce the molecular weight of the lignin polymers and to remove oxygen (typically via the methoxy groups on the aryl rings) (32). Additional work to depolymerize lignin by catalytic means using base or acid chemistry has been recently reviewed by Zakzeski et al. (20). To date, many catalytic conversion processes using base or acid for isolated lignins suffer from low yields of monomeric species. Recent work from Roberts et al., (19) demonstrated that the addition of boric acid in base-catalyzed depolymerization of lignin acts as a protecting agent and shifts the molecular weight distribution of these reaction products to yield more monomeric species. More research is warranted to develop cost effective and commercially viable routes for increasing yields of monomeric species from lignin depolymerization, potentially in concert with fractionation. Further, there are tremendous opportunities and economic incentives to find conversion pathways that would allow for efficient and integrated processes for lignin deconstruction and upgrading. A key aspect of all of this work will include the need to characterize how “clean” the lignin will need to be in terms of impacts on downstream conversion processes, including understanding catalyst lifetimes and biological toxicity.

With respect to lignin depolymerization via biological routes, various species of fungi and bacteria are known to employ cocktails of oxidative enzymes for lignin depolymerization in the biosphere (17,33). The ability to depolymerize and eventually “mineralize” lignin (i.e. convert it to CO2) is thought to enable access to carbohydrates (17), which is quite similar to the objective of many biomass pretreatment options wherein lignin is simply “in the way” of accessing carbohydrates. The genomes of a number of these lignin-degrading organisms, especially white-rot fungi, have been sequenced recently at the Department of Energy Joint Genome Institute, so that the proteins putatively involved in the lignin-degrading machinery have been identified at an organism level (17,34). However, utilization of these types of enzymes in a process context is far from developed, and as cellulase and hemicellulase enzymes have matured into an industrial context with significant investment from DOE, further study of lignin-degrading enzymes may lead to the development of “ligninase” cocktails. However, a major difference between cellulases and lignin-degrading cocktails, meriting further investigation is the need for enzyme co-factors required for the oxidative mechanisms in lignin depolymerization, which are potentially expensive, if not derived from biomass directly. An additional potential pitfall of lignin-degrading enzyme cocktail utilization is that much of the known oxidative chemistry employed leads to the repolymerization of lignin, as it is non-specific and employs radical chemistry (which may attack cellulases, leading to lower conversion due to enzyme deactivation). Also, the rates of lignin-degrading enzymes have not been directly compared to cellulase enzymes, but for economical deconstruction of lignocellulosic biomass, the performance of these types of enzyme cocktails will need to be characterized in more detail.

Lastly, it is noted that lignin streams from most fractionation process or after pretreatment and enzymatic hydrolysis can be gasified directly (35). As Bozell, et al. discuss, gasification of lignin streams offers a near-term utilization pathway beyond direct combustion that may lead to products of higher value than boiler fuel (36). However, gasification of lignin residues from biochemical conversion processes often suffers from high amounts of char formation, and thus research is needed to more efficiently utilize these streams. Another near term strategy for lignin utilization is pelletization for use as low-ash, “green” coal (37), and it is likely that many

other uses will be developed. Further investigation of these routes should be technically demonstrated to understand any potential barriers and confirm the economic viability of scaling up these processes.

Current Strategies for Lignin Upgrading and Research Needs Lastly, given biological, catalytic, or chemical strategies for lignin depolymerization, a further technical hurdle that must be overcome is the upgrading and use of the resulting small-molecule lignin deconstruction products. The ability to utilize lignin for value-added products to date has primarily been limited to small-market chemicals (primarily vanillin and DMSO), as well as the production of lignosulfonates from the pulp and paper industry (38). A report by Bozell et al. entitled the “Top Ten Value-Added Chemical from Lignin” (35) and subsequent follow-up reviews from this original report (39-41) describe a portfolio of compounds that could potentially be produced from lignin. The classes of molecules that could be produced from lignin can generally be categorized as fuels, aromatic chemicals, and macromolecules for specialty applications. Significant research efforts utilizing TEA are needed to identify routes to products of higher value or reasonable routes to produce fuel molecules from lignin, which should be coupled to life-cycle analysis to understand the impacts of reduced heat integration and increased power demands. For fuels purposes, studies have demonstrated that the small-molecule deconstruction products of lignin depolymerization (oxygenated aromatics) can be converted to fuel additives via several catalytic deoxygenation routes (32,35). Many studies have been conducted with heterogeneous catalysts using either biomass-derived lignin deconstruction products or model compounds, which have been extensively reviewed recently (20). These catalytic routes are typically focused on cracking and oxygen removal for the production of fuels and commodity aromatic chemicals or catalytic oxidation reactions for the production of fine chemicals (20). For fuels and aromatic chemicals that lack oxygen, additional research is warranted in the development of heterogeneous catalysts tailored to remove methoxyl and hydroxyl groups, which will be quite prevalent in lignin deconstruction products. Biological routes for upgrading of lignin deconstruction products represent potentially interesting options as well, although significant technical challenges will need to be overcome to convert lignin to valuable molecules including investigations into the feasibility of producing streams of lignin deconstruction products without significant inhibitory properties to organisms.

References

1. Boerjan, W., Ralph, J., and Baucher, M. (2003) Annu. Rev. Plant Biol. 54, 519-546

2. Chen, F., and Dixon, R. A. (2007) Nat. Biotechnol. 25, 759-761

3. Carpita, N. C. (2012) Curr. Opin. Biotechnol. 23, 330-337

4. Mansfield, S. D., Kang, K. Y., and Chapple, C. (2012) New Phytol. 194, 91-101

5. Mizrachi, E., Mansfield, S. D., and Myburg, A. A. (2012) New Phytol. 194, 54-62

6. Eudes, A., George, A., Mukerjee, P., Kim, J. S., Pollet, B., Benke, P. I., Yang, F., Mitra, P., Sun, L., Cetinkol, O. P., Chabout, S., Mouille, G., Soubigou-Taconnat, L., Balzergue, S., Singh, S., Holmes, B. M., Mukhopadhyay, A., Keasling, J. D., Simmons, B. A., Lapierre, C., Ralph, J., and Loque, D. (2012) Plant Biotechnol. J. 10, 609-620

7. Vanholme, R., Ralph, J., Akiyama, T., Lu, F. C., Pazo, J. R., Kim, H., Christensen, J. H., Van Reusel, B., Storme, V., De Rycke, R., Rohde, A., Morreel, K., and Boerjan, W. (2010) Plant J. 64, 885-897

8. Vanholme, R., Morreel, K., Ralph, J., and Boerjan, W. (2008) Curr. Opin. Plant Biol. 11, 278-285

9. Bozell, J. J., O'Lenick, C. J., and Warwick, S. (2011) J. Agric. Food Chem. 59, 9232-9242

10. Bozell, J. J., Black, S. K., Myers, M., Cahill, D., Miller, W. P., and Park, S. (2011) Biomass Bioenerg. 35, 4197-4208

11. Fort, D. A., Remsing, R. C., Swatloski, R. P., Moyna, P., Moyna, G., and Rogers, R. D. (2007) Green Chem. 9, 63-69

12. Czernik, S., and Bridgwater, A. V. (2004) Energy Fuels 18, 590-598

13. Mohan, D., Pittman, C. U., and Steele, P. H. (2006) Energy Fuels 20, 848-889

14. Elliott, D. C. (2007) Energy Fuels 21, 1792-1815

15. Peterson, A. A., Vogel, F., Lachance, R. P., Froling, M., Antal, M. J., and Tester, J. W. (2008) Energy Environ. Sci. 1, 32-65

16. Bugg, T. D. H., Ahmad, M., Hardiman, E. M., and Rahmanpour, R. (2011) Nat. Prod. Rep. 28, 1883-1896

17. Floudas, D., et al. (2012) Science 336, 1715-1719

18. Hicks, J. C. (2011) J. Phys. Chem. Lett. 2, 2280-2287

19. Roberts, V. M., Stein, V., Reiner, T., Lemonidou, A., Li, X. B., and Lercher, J. A. (2011) Chem.-Eur. J. 17, 5939-5948

20. Zakzeski, J., Bruijnincx, P. C. A., Jongerius, A. L., and Weckhuysen, B. M. (2010) Chem. Rev. 110, 3552-3599

21. Vigneault, A., Johnson, D. K., and Chornet, E. (2007) Can. J. Chem. Eng. 85, 906-916

22. Fuchs, G., Boll, M., and Heider, J. (2011) Nat. Rev. Microbiol. 9, 803-816

23. Studer, M. H., DeMartini, J. D., Davis, M. F., Sykes, R. W., Davison, B., Keller, M., Tuskan, G. A., and Wyman, C. E. (2011) Proc. Natl. Acad. Sci. U. S. A. 108, 6300-6305

24. Tao, L., Aden, A., Elander, R. T., Pallapolu, V. R., Lee, Y. Y., Garlock, R. J., Balan, V., Dale, B. E., Kim, Y., Mosier, N. S., Ladisch, M. R., Falls, M., Holtzapple, M. T., Sierra, R., Shi, J., Ebrik, M. A., Redmond, T., Yang, B., Wyman, C. E., Hames, B., Thomas, S., and Warner, R. E. (2011) Bioresour. Technol. 102, 11105-11114

25. Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y. Y., Holtzapple, M., and Ladisch, M. (2005) Bioresour. Technol. 96, 673-686

26. Doherty, W. O. S., Mousavioun, P., and Fellows, C. M. (2011) Ind. Crop. Prod. 33, 259-276

27. Li, C. L., Knierim, B., Manisseri, C., Arora, R., Scheller, H. V., Auer, M., Vogel, K. P., Simmons, B. A., and Singh, S. (2010) Bioresour. Technol. 101, 4900-4906

28. Sun, N., Rahman, M., Qin, Y., Maxim, M. L., Rodriguez, H., and Rogers, R. D. (2009) Green Chem. 11, 646-655

29. Kobayashi, T., Kohn, B., Holmes, L., Faulkner, R., Davis, M., and Maciel, G. E. (2011) Energy Fuels 25, 1790-1797

30. Kohn, B., Davis, M., and Maciel, G. E. (2011) Energy Fuels 25, 2301-2313

31. Shimizu, S., Yokoyama, T., Akiyama, T., and Matsumoto, Y. (2012) J. Agric. Food Chem. 60, 6471-6476

32. Johnson, D. K., Chornet, E., Zmierczak, W., and Shabtai, J. (2002) Abstr. Pap. Am. Chem. Soc. 223, U583-U584

33. Bugg, T. D. H., Ahmad, M., Hardiman, E. M., and Singh, R. (2011) Curr. Opin. Biotechnol. 22, 394-400

34. Fernandez-Fueyo, E., et al. (2012) Proceedings of the National Academy of Sciences 109, 5458-5463

35. Bozell, J. J., Holladay, J. E., White, J. F., and Johnson, D. K. (2007) Top Value-Added Chemicals from Biomass.

36. Yung, M. M., Jablonski, W. S., and Magrini-Bair, K. A. (2009) Energy Fuels 23, 1874-1887

37. Kollaras, A., Koutouridis, P., Biddy, M. J., and McMillan, J. D. (2012) Multiple Coproducts Needed to Establish Cellulosic Ethanol Industry. in Ethanol Producer Magazine

38. Zhang, X., Tu, M. B., and Paice, M. G. (2011) BioEnergy Res. 4, 246-257

39. Bozell, J. J. (2006) Feedstocks for the future using technology development as a guide to product identification. in Feedstocks for the Future: Renewables for the Production of Chemicals and Materials (Bozell, J. J., and Patel, M. K. eds.), Amer Chemical Soc, Washington. pp 1-12

40. Bozell, J. J. (2008) Clean-Soil Air Water 36, 641-647

41. Bozell, J. J., and Petersen, G. R. (2010) Green Chem. 12, 539-554

Separation SystemsFrom the biochemical conversion end, there are several areas in product recovery that could benefit from improved separation mechanisms. After saccharifcation and fermentation, solids from distillation bottoms (containing mostly lignin) need to be dried after they are precipitated from the raw stillage beer. In a majority of current designs, thermal inefficiencies exist as the bottoms need to be cooled before pressure filter let-down removes stillage water from the solids. Ideally, this should take place without cooling at temperatures above 47C. The beer its self is then separated in a three stage process into produce, carbon dioxide and water. During the first step, the CO2 and most of the water is removed in a vapor distillation column. The product then goes to another distillation column in the in the second step, where the produce is further purified. In the third step, vapor-phase molecular sieve adsorption is used to concentrate the product stream to 99.5% pure ethanol. All these areas could hypothetically benefit from optimization or simplification of unit operations. Molecular sieve adsorption could potentially be replaced with another, more efficient mesoporous separation media such as a pervaporation membrane or a different type of zeolite.

Additionally, there is a need to remove both organic and mineral acids during biochemical conversion. After dilute acid pretreatment, both mineral acids (sulfuric acid) and organic acids (formic, acetic and other carboxylic acids) need to be removed to avoid contaminating the saccharification enzyme stream and acting as inhibitors to the fermentation. After fermentation, there is potentially a need to remove fermentation derived acetic acid from raw products. This applies not only to conventional fermentation, but to syngas fermentations as well. And for all such considerations, polymer and vapor-based membranes can remain thermally stable at high temperatures (120–130°C) and can efficiently separate desired charged species from the fermentation broth or product stream are coveted.

From the thermochemical conversion end, gasification and pyrolysis can both potentially benefit from improved separation unit ops. Hot/Warm gas cleanup and filtration (T < 350°C)

can be improved to optimize for thermal efficiencies in gasification, which are lost any time a synthesis gas stream must be cooled to meet operating conditions of current unit operations. The ability to remove particulates, tars, trace contaminants and acid gases at higher temperatures would reduce the negative impact on thermal efficiency, hence cost. Fouling resistant hot gas filters (ceramics) are desirable. Other opportunities for oil and aqueous based extraction of syngas particulates and tars exist as well. Central challenges to these tasks are reducing water/oil consumption and water cleanup and recycling. Improved adsorbent materials for chloride, sulfur, and acid gas removal are also of interest.

Current liquefaction efforts have been investigating fractionation of pyrolysis oils by collecting various fractions under different thermal conditions. This could present an opportunity to volumetrically size reduce the fraction that needs to be hydrotreated (and hence reduce hydrogen consumption) while making intermediate fractions available for extraction of valuable co-products. Submicron particulate removal from pyrolysis oil presents an interesting opportunity that could enable removal of char from liquid phases. Char acts as a catalyst for condensation of high molecular weight molecules in pyrolysis oils and thereby increases the instability and aging rates of pyrolysis oil. Additionally, there is a need to better understand the fate of trace biomass components (e.g. alkali, chlorides, non H2S sulfur species and other inorganics) and formulate strategies for their removal and/or management as waste streams.

Separation technologies were categorized by the application of specific techniques to both liquid and vapor phase systems – see Table 4.

Table 4 - Separation Techniques

Separation Basis

Liquid Phase System Vapor Phase System

Rheology/Hydrodynamics Hydrocyclone Cyclone

Size/Morphology/Surface Chemistry

Membrane Filter

Electrical Potential Electro-deionization (EDI) Electrostatic Precipitation

Thermal Distillation Condensation

Catalytic Membrane-bound Filter-bound

Chemical Nature Solvent extraction

Adsorption

Magnetic (New) Sorption/Covalent coupling

Barrier Area 1: Membrane/Filtration Separation Processes

Research Activities

Vapor Phase Filters: process optimization, high-temperature filter and gasket materials, catalytic removal/upgrading of specific process-stream components, and permselective filters.

Liquid Phase Membranes: process optimization and techniques to improve anti-fouling, aids to stabilize desired product, and process yields.

Demonstrate long-term char filtration.

Develop optimal pressure pulse to enable particulate removal from filters that are compatible with biomass-derived liquids and gases.

Evaluate/develop optimal high-temperature filter materials compatible with biomass-derived liquids and gases.

Evaluate/develop optimal high-temperature gasket materials compatible with biomass-derived liquids and gases.

Evaluate/develop catalytically active filters to remove solids and upgrade pyrolysis oils.

(10 yrs) Develop high-temperature, high-pressure permselective filters to remove H2 from off gas, syngas, and recycled gas.

Develop optimal pressure pulse process to enable particulate removal from membranes that are compatible with biomass-derived liquids.

Evaluate/develop anti-fouling membranes compatible with biomass-derived liquids. Focus on materials and system (e.g., flow pattern design to develop pressure-driven flow pattern; membrane surface chemistry to control wetting; magnetic- and electric-active separations).

Evaluate/develop anti-bacterial membranes compatible with biomass-derived liquids.

Evaluate/develop membrane process to remove char from bio-oil with low liquid organic losses.

Evaluate process economics for separating solids from bio-oils – compare hot-vapor separation and condensed phase separation

Barrier Area 2: Hydrocyclone/Cyclone Separation Processes

Research Activities

Optimize processes and establish process limits

Equipment development

Optimize hydrocyclone and cyclone separation processes. Can these processes: selectively remove solids, minimize process stream recycling, and operate at process conditions and on commercial scale?

Conduct fundamental study of degradation mechanisms for cyclones in high-temperature gas applications for biomass separation systems.

Conduct fundamental study of degradation mechanisms for hydrocyclones in liquid-phase biomass separation systems.

Evaluate/develop hydrocyclone process to separate mineral matter from hydrothermal media – equipment development and validation.

Barrier Area 3: New Separation Processes

Research Activities

Acid removal

Organics and char removal

Water removal

Determine which acids are problematic and develop selective separation processes, e.g., adsorption, extraction, magnetic- and electric-active separations, etc.

Develop/evaluate process to esterify acids with alcohols.

Develop/demonstrate selective separation of acids by electrodeionization technology coupled with optimized membrane design

Evaluate/develop high- or medium-temperature sulfur removal processes, and sulfur absorbents that may be regenerated.

Evaluate/develop organic (aldehyde) extraction processes, e.g., organic solvents that can blend in refinery stream, sorbents that adsorb organic and may be removed from process stream, etc.

Evaluate/develop solvents or sorbents that may remove

oxygenates from process stream.

Develop char-surface science to enable aggregation or sorbent extraction from process stream.

Quantify value proposition for vapor/char separation versus pyrolysis oil/char separation – define pre- or post-condensation strategy

Develop/demonstrate energy efficient water removal process

Develop/demonstrate an integrated water/acid removal during pyrolysis vapor collection

Develop a vapor/vapor separation filter/membrane for oil/water separation

Develop/demonstrate separation process based on thermodynamics of prospects for “fractionation”

Solvent SystemsSolvent systems are an area on both continued and new interest in the processing of biomass (e.g., organosolv and ionic liquids). The major area was the deconstruction and fractionation of biomass into usable components via solvents. The power of these methods is due to preservation of the monomers and increased accessibility to further processing. The use of solvents in downstream operations (e.g., purification) and in biocrude fractionation and upgrading were also discussed.

The primary barriers identified were clustered in the overlapping arenas of solvent properties and in the solvent use in the overall process. Critical solvent properties that are barriers are: solvent toxicity and compatibility, solvent costs, and solvent specificity for the desired fractions. Critical related process barriers were solvent recovery, challenges for fractionation on biomass, and challenges for solvent use in bio-oil (biocrude) treatment and stabilization. Needs to overcome these barriers were discussed and ranged from fundamental properties of the new or existing solvents to the desired for better cost models and process demonstrations. Solvent recovery (beyond distillation) was seen as a major barrier to cost-effective use—for example due to residual solubility, entrainment of ash, solids, and particulates, and impacts of downstream process (i.e., fermentation of carbohydrates or catalytic upgrading of biocrude).

Barrier Area 1: Solvent recovery Research Activities Development of fluid modifiers

for increased specificity and Develop technology whereby SC fluids are used for

solvent recovery Study particulate/solvent system dynamics

improved recovery Develop cost-effective schemes for cleaning solvents used in recovery (something other than distillation)

Develop materials used in productions processes that are not wetted by solvent to maximize its recovery

Barrier Area 2: Solvent Toxicity Research Activities Improving fundamental

understanding of non-traditional solvent toxicity issues

Identify toxins and their levels in solvents Improved solvent recovery systems that remain

economically viable Investigate use of non-traditional volatile solvents

Barrier Area 3: Downstream process compatibility

Research Activities

Identifying and categorizing solvent impact on intermediate streams and product slate

Reconsider the solvent/re-engineer/reformulate solvent selection

Develop novel separation mechanisms Develop solvent manufacturing process

Barrier Area 4: Solvent deconstruction and fractionation

Research Activities

Transfer of knowledge from existing industrial uses of cellulose solvents (dissolving pulp)

Ash removal-focus on reasonable targets: silicates, non-integral ash (dirt), alkali metals, density separation, chelation

Solvent-enhanced pretreatment that can produce soluble fragments of cellulose and xylose oligomers without degradation

Barrier Area 5: Understanding Dissolution Chemistry

Research Activities

Understanding biomass reactivity as a function of solvent properties

Create a thermodynamic database as a function of relevant biomass variables, e.g., DP, chemistry, etc.

Develope processes for generating biomass based solvents (i.e. from lignin or degradation products) in-situ

Prioritize a screening program based on solvent cost and manufacturing scale

Design solvents that disrupt hydrogen bonding advance knowledge of structure-function

Barrier Area 6: Solvents for Research Activities

biocrude separations Recovering and recycling

solvent streams from biocrude Demonstrate feasibility of recycling solvent in a PDU need to identify the optimal solvents for product

recovery Develop a rational design—base case does not exist

looking at efficiency and cost comparison within same reactor set-up

Understand chemistry of what’s going on and why solvents work

Barrier Area 7: Solvents for biocrude separations

Research Activities

Characterizing feed handling issues

Understand mechanical design and feed coordination and scale up

Demonstrate the technology at a scale sufficient for piloting

Achieve desired solids loading Understand rheology of liquefaction reactors to

maximize efficiency of feeder system Study behavior of different feedstock and impact

of rheology