Conversion Technologies for Advanced BiofuelsPreliminary Roadmap & Workshop Report

December, 6–8 2011

Arlington, VA

Conversion Technologies for Advanced Biofuels Workshop Executive Summary

Introduction

The 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 annually[footnoteRef:1]—all the while maintaining price parity with foreign fossil imports. In a recent speech, the President affirmed: [1: http://www.eia.gov/dnav/pet/pet_move_imp_dc_NUS-Z00_mbblpd_a.htm]

“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.

Background

There 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-Oils

Production 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 Derivatives

Technical 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 Contents

Crosscutting Issues in the Processing of Biomass to Transportation Fuels9

Feedstock Handling9

Catalysis and Biocatalysis9

Separation and Purification9

Techno-economics and Process Data10

Production of Cellulosic Sugars and Carbohydrate Derivatives from Biomass and their Upgrading to Hydrocarbon Biofuels and Oxygenate Blends11

Pretreatment and Enzymatic Saccharification of Lignocellulosic Biomass11

Nonenzymatic Routes to Sugars and Carbohydrate Derivatives from Lignocellulosic Biomass36

Chemical Conversion of Sugars and Carbohydrate Derivatives to Hydrocarbon Fuels38

Biological Conversion of Sugars and Carbohydrate Derivatives: Isoprenoid, Polyketide, Fatty Acid, and Oleaginous Pathways51

Production of Bio-Oils via Direct Liquefaction of Biomass and Upgrading to Hydrocarbon Biofuels and Oxygenate Blends67

Fast Pyrolysis68

In-situ vapor phase initial catalytic upgrading of the pyrolysis vapors68

Ex-situ vapor phase initial catalytic upgrading of the pyrolysis vapors68

Hydropyrolysis69

Hydrothermal Liquefaction69

Special Topics Areas79

Hybrid Biochemical/Thermochemical Processes79

Direct Microbial Conversion to Fuels from Unconventional Sources81

Conversion Systems for Genetically Modified/Optimized Feedstocks85

Lignin Utilization88

Separation Systems96

Solvent Systems101

Crosscutting Issues in the Processing of Biomass to Transportation FuelsFeedstock Handling

Biomass 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 Biocatalysis

Catalysis 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 Purification

Separations 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 Data

Techno-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 BlendsPretreatment and Enzymatic Saccharification of BiomassBackground

The 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 Plants

Plant 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 Explosion

Steam 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 Pretreatments

In 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 Pretreatment

Alkali 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 Pretreatment

In 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 Pretreatment

Pretreatment 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 Solvents

Cellulose 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 Processes

Oxidative 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 Walls

Following 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-4glucanohydrolases (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 Dglucose from beta-(1,4)-D-glucans and hydrolyze Dcellobiose 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 Dglucose 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 Enzymes

The 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 Fermentation

Conversion 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 Directions

For 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 sugar concentrations

· Lignin derived species hinder enzyme efficiencies

· Cellulase enzyme loading with high solids (>20% w/w) is cost prohibitive

· 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

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 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

· 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

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