Advanced Biofuels and Biorefinery Platforms

Wednesday, October 10, 2012 - 8:30am-10:00am Forestry Companies Discovering the Biorefinery Within Moderator: Laura McIlveen, Alberta Innovates - Technology Futures

Robert Jost, Alberta Innovates - Technology Futures Geoff Clarke, Alberta-Pacific Forest Industries Inc. Rod Albers, West Fraser Timber Co. Ltd. Martin Feng, FPInnovations

Abstract

In order to remain competitive and expand their current offering of products, some forward thinking forestry companies are utilizing their byproduct streams to produce new value added materials from biomass. The same way that a multitude of chemicals and fuels can be produced from petroleum refineries, forestry companies are now set to produce chemicals, fuels and high value products from their pulp mills. Breaking down biomass into its fundamental building blocks can produce materials that fuel cars, make paints thick, provide strength to flat screen televisions or power electronics. A lot of these products can be produced as byproducts of already existing pulp mills. This panel is a mixture of industry representatives and applied researchers discussing current and future projects aimed at expanding the products that pulp mills produce. Companies like West Fraser Timber and Alberta-Pacific Forest Industries, have innovative, forward thinking leaders and access to research and development that makes these new products viable on a commercial scale. Because of this, both the West Fraser and Alberta-Pacific mills are leaders in the pulp sector. For example, West Fraser‘s pulp mill in Slave Lake, Alberta is currently looking at converting pulp mill effluent to biogas. It will produce electricity for their mill and reduce dependency on natural gas. Alberta-Pacific pulp mill in Athabasca, Alberta is commercializing chemical byproducts from its mill and looking at higher value products made from both cellulose and lignin. These companies work closely with research organizations like FPInnovations and Alberta Innovates-Technology Futures to develop new products and processes. These two applied research organizations are working in the areas of cellulose, hemicellulose and lignin derivatives. Materials like nano-crystalline cellulose and lignin based resins are being developed and will be discussed. The modern pulp mill is a biorefinery and this panel will illustrate how companies and researchers are working hard to capitalize on this.

Wednesday, October 10, 2012 - 10:30am-12:00pm Financing Biorefinery Projects Moderator: Alan Propp, Merrick & Company

Bond Financing as a Source of Capital for Bioproduct Projects John May, Stern Brothers & Co.

Fast Track Project Strategies Alan Propp, Merrick & Company

Andrew Soare, Lux Research

Abstracts

John May Bio-product companies (fuels and chemicals) can secure project financing from the institutional capital markets through bond placements. Institutional investors are willing and able to buy bonds that provide a single vehicle for construction and operations of bioproduct projects. Unlike banks, these investors can finance non- investment grade projects. Using a partial USDA loan guarantee, Stern Brothers closed a transaction for Myriant to fund a bio-chemical plant in Louisiana.

Alan Propp As an engineering firm, we are almost always asked to fast track projects for our clients. Project fast tracking is a misunderstood science. It consists of much more than simply hurrying and working nights and weekends. Rather, there are certain, time-proven techniques for successfully completing a project on a tight schedule without sacrificing quality or safety. This talk will define fast track projects at both the pilot and commercial scales and will outline the methods that can be employed to expedite their delivery. I will present statistically-based evidence for best practices in fast-tracking as well as specific recommendations on fast tracking techniques. I believe many attendees will find this information very useful in planning their technology development programs.

Wednesday, October 10, 2012 - 2:30pm-4:00pm Biogas: Surprising New Opportunities and Approaches Moderator: Michael Weedon, BC Bioenergy

Managing Organic Materials in Urban Environments: Harvest's Organics Operating System Paul Sellew, Harvest Power

Tertiary Biogenic Gas Creation:The Road from Hunter Gatherer to "Gas" Farmer William Mahaffey, Luca Technologies, Inc.

Bio-Upgrading of Syngas Into Renewable Natural Gas (Methane) Serge Guiot, National Research Council of Canada

Technology Meets Permaculture, Advanced Integrated Resource Recovery Christopher Bush, Catalyst Agri-Innovation Society

Abstracts

Paul Sellew The nascent biogas industry in North America could be compared to a growing toddler: it is young, growing quickly, and just beginning to learn how to play well with others. For example, the U.S. has just over 2,200 sites producing biogas compared to Europe‘s 10,000 operating digesters. North America, in turn, is like a new parent figuring out rules (regulations), feeding schedules (feedstock procurement) and play dates (developers) to help this biogas toddler grow. North America still has much to learn about efficiencies: for example, only a fraction of the operating digesters actually use the gas they produce (ibid). Indeed, less than three percent of the 34 million tons of food waste generated in the US in 2009 was recovered and recycled. Tapping into this feedstock is just the beginning: the U.S. Billion-Ton Update projects that ―the U.S. will have between 1.1 and 1.6 billion tons of available, sustainable biomass for industrial bio-processing by 2030.‖ In a quickly changing marketplace, this presentation explores the challenges and opportunities associated with project development in North America including: an overview of waste-to-energy markets; legislative developments and incentives for new plants; challenges in energy markets including natural gas; cultural differences in managing organic materials; and development opportunities in key regions. This presentation will draw heavily from hands-on experience building the first commercial-scale Energy Garden near Vancouver, Canada, and from Harvest Power Inc.‘s experiences in the North American biogas market. In sum, North America is redefining its relationship with organic materials. From apple cores to compost, pizza crusts to power, and farms to forks, this presentation illustrates shifting trends and attitudes towards managing organic materials using anaerobic digestion technologies. Attendees will learn about: • Policies that have stimulated and stymied anaerobic digestion development; • Practices that lead to lower operating costs and higher value products; and • Partnerships that add value at every turn.

William Mahaffey Biogenic methane has enormous potential as a sustainable energy source and is found in a wide variety of subsurface, anaerobic, hydrocarbon bearing environments. An area of great interest for biogenic methane is the Powder River Basin in Wyoming, United States. This area has been previously developed for Coalbed Methane production with over 30,000 wells drilled basin wide. The PRB has been shown to be an active ―geobioreactor‖ based on the gas isotopic signatures of produced methane (δ13C-methane -57‰, δD-Methane -320‰). That coal beds of the PRB, and many other basins, are ―alive‖ with active methanogenic

communities that can be stimulated to create new methane from coal, has been determined by coal conversion studies in laboratory experiments. Experimental data will be presented that shows new methane creation in real-time by activating the microbial communities with specific activation amendment packages and observing headspace methane accumulation in excess of the stoichiometric production from the nutrients. In addition, BESA inhibited cultures exhibit an accumulation of metabolic intermediates in coal slurries prepared with live formation waters but not in controls with active formation water only. Data will be presented showing commercial scale field demonstrations, supporting commercial scale proof-of-concept. Quickly moving to projects of large scale, we have been able to demonstrate production of commercial quantities of new gas in multiple basins, using an enhanced in situ microbial based process we call ―Methane Farming.‖ Understanding the composition and metabolism of the methanogenic consortia has been one of several key steps in commercializing the process of sustainable biogenic methane production. In order to identify microbes present in these methanogenic consortia we performed microbial community analysis using error-correcting barcode pyrosequencing analyzed with the QIIME bioinformatic pipeline. This involved thousands of samples collected from different locations within set geographical areas over several years. Both bacterial and archaeal specific primers were used to amplify these distinct populations from our DNA samples. The QIIME pipeline was used for library demultiplexing, OTU picking, alignment, taxonomic identification, and statistical analysis of community structure using Unifrac. Visualizations of principle coordinate analyses of the QIIME results and metadata from the formation will be used to illustrate the variables most responsible for the shift in community demographics associated with successful gas production.Here we demonstrate the large scale sampling and community analysis of coalbed methane (CBM) wells within a discrete but large area of the Powder River Basin, the data set encompassing areal as well as temporal components. The temporal component is defined as the natural baseline state, followed by a bio- stimulation phase (i.e., Restoration), dwell phase and enhanced methane production phase. Under the baseline conditions, we find distinct bacterial and archaeal populations that vary by coal seam and water chemistry, as well as over time. The community signatures help to determine areas of greatest methanogenic potential as well as identify patterns of ground water recharge, water movement, and potential metabolic bottlenecks.

Serge Guiot The synthesis gas (syngas) resulting from organic waste or biomass gasification can be used directly to power industrial boilers, gas turbines or fuel cells to make electricity. Syngas can also be steam reformed and purified into methane, which could be re-injected in the natural gas grid. Catalytic processes are well established. They normally involve high pressure and/or temperature, may be problematic when impurities are present and tend to have low product specificity. To circumvent these disadvantages and use milder treatments with minimal chemical and energy, we can harness the power of microorganisms to convert the syngas compounds into renewable natural gas (RNG). We have shown that non- adapted natural microbial consortia from industrial anaerobic digesters had a non- negligible carboxidotrophic (CO-consuming) methanogenic potential. Kinetic-activity

tests performed under varying CO partial pressures with specific metabolic inhibitors (e.g. 2-bromoethanesulfonic acid and vancomycin) showed that CO was converted mainly to acetate, which was further transformed to methane. At high CO partial pressure, CO conversion shifted to hydrogen which was then used to reduce CO2 into methane. However because the aqueous solubility of CO and H2 is low, syngas bioconversions are typically limited by the gas-to-liquid mass transfer rate which may represent a major engineering challenge for development of large- scale syngas bioconversion facilities cost-effectively compatible with upstream gasifier productivity. Two reactors were investigated for the conversion of CO on the continuous mode: the bubble column reactor (BCR) and the completely stirred tank reactor (CSTR). In biotic conditions (with mixed culture), the BCR showed a transfer rate (kLa) ranging around 2 h-1, while the completely stirred tank reactor (CSTR) showed a kLa around 20 h-1. The BCR reactor, investigated in more details over a 189 days period (with a CO flow rate > 7L/d and a gas recirculation rate > 1200L/d) showed a maximum volumetric activity of 62 mmol CO/Lrx·d (i.e. 9 mmol CO/gVSS·d) at gas loads (100% CO, 1.62 atm) of 22 L/d (101 mmol CO/Lrx·d or 13.4 mmol CO/g VSS·d), with a 85% efficiency of CO consumption at an average yield of 26±2 % (vol. CH4/vol. CO, i.e. 100% of the stoichiometric yield). The CO conversion process was kinetically limited, rather than mass transfer limited. With the CSTR under transfer optimal conditions (kLa 20 h-1), the CO consumption increased linearly with the CO feeding flow rate up to an optimal operation point (90 mmol CO/gVSS.d), where the efficiency of CO conversion could reach 75%, with a yield of near 25% (vol. CH4/ vol. CO) and a specific CO-consuming potential of 65 mmol CO/gVSS.d. For CO feeding flow rates above the optimal operation point, acetate accumulation and reduction of CO conversion were observed.

Christopher Bush Description and explaination of the A-IRR for agriculture designed at the Agricultural Centre of Excellence in Sustainability (ACES-BC) The ―Technology Meets Permaculture‖ program seeks to take the best of nature, and modify, enhance and amplify natural processes through the use of innovative technological integrations. The foundation of the program is anaerobic digestion, with a focus on managing outputs, by managing inputs. ACES was launched with a project that added pre- screening/prewashing of dairy and poultry manure to insure only feedstock with digestion potential entered the tanks. Biomethane will be harvested from this stage. The digestate enters a greenhouse, and a ―Solar Aquatic System in preparation for Duckweed propagation ponds. The Duckweed is harvested, and enters an ethanol production fermenter and distillation system. Ethanol will be harvested from this stage. The biomass mash is then fed to the animals that started the process, closing the loop. Products are Biomethane, ethanol, animal feed, and reclaimed water.

Thrusday, October 11, 2012 - 2:00pm-3:30pm The Move to Drop-In Fuels Moderator: TBD

Title TBD Tim Zenk, Sapphire Energy

From Mill to Wing-A sustainable pathway to drop in fuels Laurel Harmon, LanzaTech

An Efficient, Liquid-Phase, Catalytic Approach to Cellulosic Biofuels Production Karl Seck, Mercurius Biofuels

Title TBD Paul Beckwith, Butamax Advanced Biofuels LLC

Abstracts

Laurel Harmon, LanzaTech LanzaTech, a company founded in 2005, has developed a novel biological process for carbon capture and reuse. LanzaTech‘s proprietary biological microbe can use a variety of waste gases as a nutrient source- including waste gases from industry. LanzaTech can also use syngas generated from any biomass resource (such as municipal biowaste, organic industrial waste, and agricultural waste) and reformed natural gas. LanzaTech‘s process can convert these gas streams into ethanol and 2,3-butanediol, a C4 dialcohol that can be converted into conventional chemicals such as methyl ethyl ketone and butadiene, as well as drop-in fuels such as gasoline, jet or diesel fuel. LanzaTech is using synthetic biology to enable production of other chemical products such as propanol, n-butanol, and acetone. LanzaTech has multiple technology partnerships for the conversion of these products into downstream petrochemicals and drop-in fuels. This technology enables efficient conversion of industrial carbon into fuel and chemical products at scale, using resources that do not compete with the food supply. Life cycle analysis demonstrates that the LanzaTech technology produces fuels and chemicals with a significant reduction in greenhouse gas footprint when compared to conventional petroleum products.

Karl Seck Mercurius Biorefining aims to process renewable biomass feedstocks into liquid biofuels and high value fine chemicals using REnewable Acid-hydrolysis Condensation Hydrotreating (REACH) process. The REACH process consists of three steps; 1. Production of monomeric intermediates from cellulosic biomass 2. Condensation of monomers into longer chain molecules, and 3. Hydrotreatment of the longer chain molecules to remove oxygen. The benefits of the REACH process include the, • Efficient conversion of renewable biomass into biofuels and fine chemicals without concomitant CO2 production, • Deployment of existing industrial

processing techniques – one from the pulp and paper industry and the other from petroleum refining, • Creation of an intermediate bio-crude product through the use of acid-catalysed hydrolysis (similar to the pulp and paper industry), • Production of diesel, jet fuel, and gasoline through a solid-bed-catalyst process analogous to the way the petroleum industry converts crude oil into the various petroleum products on the market today, and • Avoidance of enzymes or microbes thereby making the process insensitive to feedstock impurities. Mercurius‘ approach to biomass conversion is based on the work of Professor Mark Mascal at the University of California Davis (UC Davis), who showed that 5-chloromethylfurfural (CMF) can be obtained in high yields from various biomass sources, including paper, cotton, corn stover, straw, and wood, upon acid treatment at moderate temperatures using batch processing methods. Concurrently, CSIRO in Australia developed a continuous flow process for producing CMF from refined sugars (fructose, glucose and sucrose). The hydrotreating steps in the process relate to technology previously developed by Pacific Northwest National Lab (PNNL). The cellulosic feedstock is chemically fractionated and converted to CMF and furfural. CMF is converted to ethyl levulinate (EL), which is then condensed with furfural. These condensates are then on-processed to alkane based diesel/jet fuel blends through hydrotreatment. Value Proposition Mercurius has based its commercialization plan on the superior economics of its REACH technology. With operating costs (excluding capital charges) of $0.90/ gallon of fuel and $3-5/annual gallon of fuel capacity capital costs the technology has great advantages over competing technologies. A breakdown of the estimated operating costs is shown in the waterfall diagram to the right. Feedstock costs were set at $50/BDMT for this analysis. Since REACH technology is a liquid phase, catalytic process it is inherently more efficient than other biorefining technologies. In a liquid phase process less volume is handled than a gas phase process, reducing capital costs because equipment sizes are reduced. In addition, catalytic processes tend to be much faster, therefore requiring much lower residence times, again lowering equipment sizes and capital costs. A liquid phase, catalytic process benefits from lower capital costs in both ways. REACH does not depend on enzymes or microbes so there is much more feed flexibility. Sugar isn‘t required as a feedstock. Truly a cellulosic process, the front end of the REACH process converts the raw biomass to non-sugar intermediates. REACH will not be exposed to the high price of sugar or have to rely on an expensive enzymatic or acid based hydrolysis method to convert cellulose into purified sugars.

Thrusday, October 11, 2012 - 4:00pm-5:30pm Microalgae: Advancing to Commercial Applications Moderator: John Benemann, MicroBio Engineering, Inc.

Microalgae: Advancing to Commercial Applications Stan Barnes, Bioalgene

Algae Production: Next Steps Shay L. Simpson, Texas AgriLife Research - Texas A&M University System

Amit Vasavada, General Atomics

Abstracts

Stan Barnes Microalgae are steadily advancing in a range of commercial applications. This panel will cover four topics of particular interest in this space: animal feeds, aquaculture, wastewater treatment, and biofuels. These microalgae products and processes overlap with each other, as exemplified by the four panel presentations, covering these four areas and how they integrate together. Advances in microalgae animal feed production are focused on algal biomass high in omega-3 fatty acids and proteins, and are represented by Cellana, LLC, with offices in San Diego, California, and pilot-scale facilities in Kona Hawaii. Cellana not only produces high value animal feeds but also oil that can be converted to biofuels, thus improving the economics for both products. Their ReNew™ process for omega-3s, feeds, and fuels in large-scale biorefineries will be presented by Martin Zabarsky, CEO. Aquaculture is the focus of LiveFuels, Inc., of Menlo Park, California with production facilities in Texas. The company is extracting value from microalgae biomass growing in large aquaculture ponds, while also removing nutrients, in particular phosphorous, from pond effluents and producing high omega-3 fish in a polyculture system. The goal of Live Fuels, Inc., to be presented by Lissa Morgenthaler-Jones, CEO, is to reclaim fertilizers, prevent eutrophication, most clearly exemplified by the dead zones in the Gulf of Mexico, and produce valuable fish protein and omega-3 oils. Municipal wastewater treatment is the focus of Bioalgene of Seattle, Washington, with facilities in Boardman, Oregon. Bioalgene combines municipal wastewater treatment with CO2 capture from power plants and biofuels production. Stan Barnes, CEO, will present the potential of such an integrated technology for the U.S. Pacific Northwest. MicroBio Engineering, Inc., of Walnut Creek and San Luis Obispo, California, is an engineering consulting company active in both the U.S. and other countries, including Spain and Brazil, and assists in the development of microalgae projects for all applications. John Benemann, CEO, and moderator of this panel, will present examples and case studies of microalgae projects for fuels, feeds, foods, fertilizers and wastewater treatment from around the world, in particular from the Pacific Rim.

Shay L. Simpson At Texas AgriLife Research, a member of the Texas A&M University System, research has been on-going in large-scale algae production both in arid and coastal environments for five and fifteen years, respectively. Arid production takes place at the Pecos Station in West Texas in raceways ranging in size from 200 liters to 23,000+ liters. Coastal production takes place at the Flour Bluff Station near Corpus Christi in raceways 200 liters in size. Collaboration with other universities, organizations, and private industry has resulted in large-scale testing of harvest, de-watering, drying, extraction, and conversion equipment. Economic analyses indicate with a successful technical approach, the number of rural jobs and income would be positively impacted with implementation of large-scale algae production. An update of activities at the Pecos and Flour Bluff Stations will be provided along with findings and next steps for research, collaboration, and industry engagement.

A pathway to commercialization based on operational experience gained at our test beds will be discussed.

Friday, October 12, 2012 - 8:30am-10:00am Biorefinery Platforms: Perspectives from Leading Commercial Developers Moderator: Bob Walsh, ZeaChem Inc.

Processing Recalcitrant Feedstocks in a Biorefinery Johnway Gao, Catchlight Energy

Biorefinery Platforms Bob Walsh, ZeaChem Inc.

Dan Cummings, INEOS

Title TBD Len Bykowski, Mascoma Corporation Alberta

Abstracts

Johnway Gao Catchlight Energy (CLE) is a joint venture of Chevron and Weyerhaeuser focused on commercializing the production of liquid transportation fuels from sustainable biomass resources. We will discuss the critical success factors associate with our efforts to: a) Supply cellulosic feedstock to third parties and off-take of the resulting fuel products. Catchlight Energy (CLE) leverages the expertise and infrastructure of both parents to accomplish this and is supplying these services to third parties who wish to build and operate plants to produce renewable, cellulosic fuel. b) Develop targeted transformational technologies capable of processing softwood feedstocks and achieving superior economics. CLE is developing both biological and thermochemical routes to achieve this goal. Softwood is the most abundant but also the most recalcitrant feedstock. CLE will discuss its robust process to facilitate efficient hydrolysis of softwood feedstocks to sugar and its thermochemical process based on a novel solvent which produces high yields of a low oxygen content bio-oil that can be upgraded to ―drop-in‖ hydrocarbon fuels.

Bob Walsh ZeaChem Inc., a producer of advanced biofuels and bio-based chemicals from a wide variety of cellulosic biomass resources, began operations of its 250,000 gallons per year (GPY) integrated biorefinery in 2012. The facility is located in Boardman, Oregon and is processing woody biomass, wheat straw, and other biomass resources, into a range of fuel and chemical products. By the end of 2012, the facility will have the capability to produce cellulosic acetic acid, ethyl acetate and ethanol. ZeaChem will further expand the product portfolio by installing additional process modules to convert ethanol into jet and diesel fuel in 2013 and bio-based gasoline in 2015. ZeaChem‘s feedstock flexibility and diverse product

portfolio allow it to deploy its biorefining technology throughout the U.S. and globally. ZeaChem‘s first commercial scale biorefinery of 25MM or more GPY is under development and will be located adjacent to the demonstration facility in Oregon. The USDA announced a conditional loan guarantee of $232.5MM for the project, which is scheduled to begin operations by the end of 2014. All of the feedstock has been contracted, with the primary feedstock producer, GreenWood Resources, providing poplar tree feedstock from their nearby tree farm, and agricultural residues (wheat straw) from local farmer providing supplemental material. Current partners who are helping to drive ZeaChem‘s growth in the US as well as internationally include Procter & Gamble for bio-based chemicals to be used in products and packaging, Chrysler Group for advanced cellulosic ethanol, and Itochu Corp. for fuels and chemicals in Asia and other markets. ZeaChem is exploring commercial production sites along the Pacific Rim including in Australia and Canada.

Len Bykowski Founded in 2005, Mascoma is a renewable fuels company that has developed innovative technology for the low cost conversion of biomass to renewable fuels and chemicals. Mascoma envisions a world where renewable energy and a healthy environment are achieved by combining efficient use of sustainable resources and leading edge innovation Using proprietary consolidated bioprocessing, or CBP, technology platform, Mascoma has developed genetically-modified yeasts and other microorganisms to reduce costs and improve yields in the production of renewable fuels and chemicals. According to the U.S. Department of Energy, CBP technology is the ultimate low- cost configuration for the hydrolysis and fermentation of cellulosic feedstocks. The Mascoma CBP platform utilizes genetically modified yeast and bacteria to convert cellulosic biomass into high-value end-products in a single step that combines hydrolysis and fermentation. Mascoma is currently targeting the large and established first generation corn ethanol industry with a proprietary Mascoma Grain Technology, or MGT,yeast product branded Transferm. This genetically-modified yeast product can be used by corn ethanol producers as a drop-in substitute to conventional yeast while increasing yields. Mascoma is also working with leading industry partners to develop and construct commercial scale biorefining facilities. These facilities in Kinross Michigan and Drayton Valley Alberta will offer compelling economic value to the company and associated collaborators. The initial facility to be constructed in Kinross will convert mixed hardwoods to cellulosic ethanol while the Drayton Valley facility will convert aspen to cellulosic ethanol and biochemicals. In the future, Mascoma plans to expand the application of its CBP technology to develop advanced biorefineries that produce multiple high-value end-products, such as advanced biofuels and chemicals, from many different feedstocks.

Biomass Production and Utilization

Wednesday, October 10, 2012 - 8:30am-10:00am Creating an Acceptable Supply of Biomass Feedstock to Satisfy Project Financing Requirements Moderator: Spencer Swayze, Ceres, Inc.

Chris Roach, Ceres, Inc. John May, Stern Brothers & Co. Bob Avant, Texas AgriLife Paolo Carollo, Chemtex International Inc.

Abstract

As financial support from the public sector continues to diminish, the advanced biofuels and bio-products industries are shifting their focus to traditional project finance for the deployment of commercial scale technologies. Considering the medium to high technology risk and large capital investment common with these types of projects, the project cash flow generation risk exposure to feedstock supply must be minimized to achieve an acceptable risk premium for debt financing. Project developers are compelled to create feedstock supply plans that can survive the scrutiny of financial due diligence and are based in large part on contractual commitments from credit worthy counterparties. These financeable feedstock supply plans must address process risks, third party risks, and market risks. Process risks of a feedstock supply plan include biomass performance in the field, the cost and reliability of the agronomic system to establish, manage, and deliver the material, and the quality and consistency of the material. The design of the feedstock supply plan should be based on statistically verifiable performance of feedstock types under project relevant conditions (land, climate, inputs, etc.), along with agronomic processes and equipment. Inclusion of feedstock diversification by type, source, and seasonal availability will help mitigate short and long term supply interruptions. A quantitative understanding of how feedstock quality impacts conversion characteristics should also be a project development consideration. Third party risks that can impact feedstock cost, availability, or quality, need to be contractually mitigated. To the extent possible, commitments to resources (land) and performance (establishment, harvest, delivery, etc.) that match the length of the debt term should be secured. Obligations to quantity, quality, and direct costs should also be pursued. Third parties that enter into these commitments should be selected based on past experience, adequate funding to perform, and the financial wherewithal to support guarantees. As with feedstock, third party diversification should also be included to further mitigate third party derived risks and enhance the likelihood of improving feedstock supply cost and efficiency through competition. Market risks to feedstock supply can affect both availability and cost. Dedicated energy crops should be preferred to agricultural residues as it is harder to guarantee the availability of agricultural residues, by definition a byproduct of other market activity. Cost uncertainty can be minimized through contracts with fixed or capped direct costs and hedge positions for inputs like fuel and fertilizer.

Financeable feedstock solutions can be developed concurrently to an overall project development effort given an in-depth understanding of the requirements, resources, and activity required. Early communication with financial partners and investors can be invaluable to anticipating due diligence requirements for feedstock plans and keeping an overall project development effort on schedule. Successful non-recourse project financing of biomass based projects will include the same level of rigor in the feedstock solution development process observed in the overall project development effort of successfully financed first generation biofuel and energy projects.

Wednesday, October 10, 2012 - 10:30am-12:00pm Next-Gen Feedstocks: Grass, Reeds, and Macroalgae Moderator: TBD

Sweet and High Biomass Sorghum as Sources for Sustainable Feedstock in Biofuels and Biobased Chemical Production. MJ Maloof, NexSteppe

Tropical Grasses and Weeds from the Coastland of Eastern Mexico as Potential Feedstock for Bioethanol Production Sergio Trejo-Estrada, Instituto Politécnico Nacional - México

Giant Reed (Arundo Donax L.): How a “Noxious Weed” Can Become a Sustainable Energy Crop for 2nd Generation Ethanol in Warm Temperate Climates. Enrica Bargiacchi, University of Pisa

Renewable Products from the Seaweed Biorefinery Ajay Kshatriya, Bio Architecture Lab

Abstracts

MJ Maloof The emergence of the global biofuels and biobased product industries is creating growing demand for renewable feedstocks. Because of their reliability, scalability and consistency, dedicated crops, particularly sweet sorghum, high biomass sorghum and switchgrass, will play a large and important role in meeting this demand. NexSteppe is using a combination of directed conventional breeding, marker assisted breeding and cutting-edge analytical techniques to improve a selected set of economically attractive, high-yielding, broadly adapted, low-input, and non-food dedicated crops. NexSteppe is currently commercializing its sweet and high biomass sorghum hybrids as the lead crops in its respective sugar and biomass platforms. For its sugar platform, NexSteppe has recognized the potential for sweet sorghum to meet rapidly expanding sugar feedstock demand, based upon its adaptability to wide geographic areas of the country, low water, fertilizer, and pesticide inputs, and readily extractable high-quality C6 sugars. Many companies with sugar-derived biobased alternatives for fossil-derived fuels and chemicals are

building their first commercial production capacity. This infrastructure is accumulating around the lowest cost feedstock, currently sugarcane in Brazil. Intense competition and limited expansion capacity for this resource mean that high yield dedicated energy crops will be core to any economical, scalable, and sustainable solution to displace meaningful quantities of fossil-derived fuels and chemicals without competing with food sources. Compared to both sugar cane and sugar beets, sweet sorghum is more economical in the many regions of the world. It is much easier from a crop management and, particularly, harvesting perspective when compared to sugar beets. Further, sweet sorghum has a wide range of geographic application since it can be grown on land less suited to sugar cane or beet production while maintaining comparable levels of fermentable sugars. Sweet Sorghum has the potential to turn the U.S. Southeast into a global hub for low-cost, sustainable manufacturing of biofuels and biobased products in the near term. Using current downstream conversion technology and advanced approaches to breeding and agronomic management of sweet sorghum, it‘s economically viable to build facilities in the U.S. in the near-term and to extend the operating season of mills in Brazil. NexSteppe is also working on cellulosic solutions which have the potential to reach even larger scales and, ultimately lower costs. High biomass sorghum is a broadly adapted, high-yielding crop with strong tolerance to heat and drought. Its range of maturities and ease of establishment make it well suited to providing just-in-time biomass for power production or conversion to cellulosic biofuels. Compared to perennial grasses, high biomass sorghum has the potential for higher yields of overall biomass with significantly lower establishment costs. Further the application of cutting edge breeding technology applied to this crop means that the rate of improvement in this high yielding annual will greatly outstrip that of comparable perennial crops. Beyond yield, NexSteppe is also targeting a number of compositional characteristics that will make it more ideally suited to downstream conversion, thus further reducing the production costs of the ultimate product be it fuels or chemicals.

Sergio Trejo-Estrada Mexico,an important petroleum producer, has delayed the use of anhydrous ethanol as a substitute of ETBE or MTBE in gasoline. Both corn starch and sugarcane juice are too expensive and currently used exclusively for human consumption by law. The coastland of the Gulf of Mexico is rich in a variety of native or introduced grasses wich colonize marginal lands. In the present study, non arable, eroded lands, located near sugarcane fields, with no acces to irrigation and chemical fertilization were sampled to determine the presence of tropical grasses during different seasons. Selected plants from grasses of different genera such as Rottboellia, Eleusine, Panicum, Echinochloa, Cenchrus and Sorghum, were treated with either acid or enzymes to determine the yield of reducing sugars. The content of lignin, hemicellulose and cellulose, and the potential fermentation inhibitors present in the hydrolysate, were also determined, and compared to sugarcane (Saccharum oficinarum). The highest sugar yield was found in samples of Panicum and Rottboelia. The highest ethanol yield was obtained from Panicum hydrolysates. Their widespread distribution and their low water and fertilization requirements, provide a sustainable source of biomass for the production of second generation bioethanol.

Enrica Bargiacchi Giant reed (Adx) is the leading candidate among potential ligno-cellulosic feedstocks for 2nd generation ethanol, under warm temperate climates, due to its high yield of ethanol-per-hectare and low ecological demands. This perennial C3 grass, with high photosynthetic rates and little photoinhibition, is diffuse in natural landscapes in Southern Europe and the Mediterranean areas, where, if undesired, it is easily controlled by glyphosate. Its history as a crop began in Northern Italy in 1930s at Torviscosa, where Adx for the first time was extensively cultivated on a large scale basis (6 070 ha) as an energy crop and a cellulose source for over 20 yr, with no problem of long-term containment in the cultivation area. This mesophyte (easily switching from hydrophyte to xerophytes) tolerates a wide range of soils, including saline and metal contaminated soils. Usually it is referred to as a drought-resistant grass, able to reach early after establishment, and consistently maintain for +10 years, high yields with limited inputs and favorable logistics (extended harvest period, and good bulk density). It can produce 20-30 t ha-1 DM in Southern Europe, with no irrigation supplement. Numerous field studies indicated that Adx modestly responds to Nitrogen fertilization, harvest time and plant density. A positive associations with microorganisms, especially endophytic and micorrhizal fungi is present. The research activity in progress at DAGA, in collaboration with Chemtex Agro, aims at investigating the plant ability to sustain anoxia, heat, freeze, and salinity, in order to better exploit marginal soils, and avoid competition with food crops. A major research issue is also plant water use and stress, as affected by micorrhizal infection. To help plant procurement plans, DAGA collaborates to the development of innovative equipment to harvest both Adx crops, and its stands, naturally present in riparian and natural areas.

Ajay Kshatriya Seaweed is a renewable resource which can be uplifted in value using industrial biotechnology and chemical processing. We have developed a novel ‗wet mill‘ biorefinery concept which extracts the maximum economic value from seaweed with products including fertilizer, animal feed, chemicals, and fuels. Here we present the environmental and economics benefits of seaweed resources, our proprietary technical platform to unlock seaweed‘s potential, and our roadmap and progress towards commercialization.

Wednesday, October 10, 2012 - 2:30pm-4:00pm The Challenge of Sustainable Feedstock: Certification and Reputation Management for Producers and Brand Owners Moderator: Jack Huttner, Huttner Strategies

Andrew Held, Virent Bob Bernacki, Gevo Mike Schultheis, The Coca Cola Company

Abstract

As biorefineries move into commercial deployment, they will need to address the obligations of corporate social responsibility. This bears particular relevance to the producers of bio derived bulk chemicals and materials. Customers will likely expect suppliers to adhere to their guidelines for sustainable production. This may pose special challenges to the producers intending to begin production using first generation feedstock as they transition to cellulosic sugars. This panel will review the challenges from both the producer and the brand owner side.

Thrusday, October 11, 2012 - 2:00pm-3:30pm Biomass Crops: The Role of Breeding, Genomics, and Plant Biotechnology Moderator: Dilara Ally, SG Biofuels (SGB)

From Seed to Processor: Sustainable Customized Feedstocks John W. Fulcher, Chromatin, Inc.

Bringing a New Commercial Energy Crop to Market Through Breeding, Plant Biotechnology and Genomics Dilara Ally, SG Biofuels (SGB)

The Perennial Search for Improved Biomass Feedstocks Toni Voelker, Monsanto

Lower Cost Fermentable Sugars from Sugarcane Biomass Mark Harrison, Queensland University of Technology

Abstracts

John W. Fulcher The growing need for global non-food feedstocks over the next 20 years will be met by a combination of three primary sources: energy crops; agricultural residues; and woody biomass. By 2030 it is estimated that 70 million acres of agriculture will be need to supply the demand for non-food biomass. By way of comparison: there are

50 million acres of sugarcane grown globally; 20 million acres of canola in Canada; and 70 million acres of rice in China today. By 2020 the projected demand for biomass feedstock will be approximately 600 million tons going to cellulosic ethanol, US heat and power, EU power, chemicals and animal feed. Today‘s markets for purpose-grown biomass for cellulosic and the U.S. power industry are in the $50 - $70 per ton range while the EU power market is $150 - $200 per ton. Global markets for purpose-grown biomass are emerging. In order to meet that demand, rapid increases in feedstock production will require: • Proven genetics • Established cropping systems (agronomics) • Yield quality (cellulose, sugar, starch, etc) • Global cultivation • Existing infrastructure • Economic viability (profit) One of the key economic drivers for the selection of biomass feedstocks will be water. Agriculture accounts for greater than 90% of fresh water consumption. Sorghum is well suited as a purpose-grown biomass given its water use is 85% less than sugarcane and 50% less than corn. Sorghum is also sustainable and scalable. Sorghum can also be grown on marginal land with high yields and has a reduced environmental impact due to fewer chemical inputs (for example herbicide and pesticide use is 40-80% less than corn). Sorghum has a four-month growing season and in Brazil, sweet sorghum is being co-cultivated with sugarcane to extend sugar harvests. Chromatin is a vertically integrated feedstock provider with experience in biotechnology, seed, and feedstock growth, harvest, and delivery. Chromatin‘s core enabling biotech and traits platform is built on the mini- chromosome platform. In the future this will enable others and Chromatin to stack multiple traits of interest into select feedstocks. Another component of vertical feedstock integration is Chromatin‘s sorghum operating capabilities: • R&D pipelines: 1) conventional 2) molecular breeding and 3) transgenic • Product pipelines: greater than 30 grain and forage commercial hybrids, leveraging 40 plus years of breeding, seed production and brand recognition. • Seed production: 30 million pounds of seed annually. • Supply chain: export to greater than 20 countries including Brazil and Argentina. Products on greater than 3 million acres • Relationships: Domestic and international grower, dealer, distributor and agent networks • Synthetic biology: Enabling technology and traits to reshape feedstock composition Customers are looking for sustainable customized feedstock solutions to meet their needs in power, transportation fuel, and chemical markets. Biomass production must be economically produced, harvested, stored ,and transported. A combination of purpose-grown energy crops along with crop residues may be required to provide adequate biomasss supplies year round while minimizing transportation and storage costs. Through strategic alliances Chromatin can harvest, store, and transport a wide variety of biomass to provide customers with the right feedstock solution at the right time.

Dr. Dilara Ally, SG Biofuels (SGB) SGB Vice President & Chief Technologist Eric Mathur will explain how SGB‘s global breeding, plant biotechnology and genomics platform is accelerating the development of improved genetics and greater yields for the subtropical energy crop Jatropha curcas. SGB is leveraging refined and optimized plant biotechnology tools developed within the past 10 years for 1/100th of the cost, and in the process bringing an entirely new commercial crop to the global market.

Jatropha curcas (―Jatropha‖) is a non-edible, subtropical crop that produces oil bearing seeds, which can be used for a wide variety of bio-based materials including biodiesel, biojet fuel and specialty chemicals. Because it is a non-edible feedstock and can be effectively harvested on sub-prime land that is considered undesirable for food crops, it does not compete with global food supplies. The spread of a few Jatropha cultivars from its center of origin in Central America to foreign subtropical and tropical regions such as Africa, India and Southeast Asia effectively created a ―genetic bottleneck‖ in the diversity of Jatropha found in those locations. This resulted in a restricted germplasm base, limiting the success of those attempting genetic improvement through traditional breeding. Since the crop had not yet undergone professional domestication, genetic diversity was limited and agronomic best practices were not properly developed, and many early projects failed to produce expected yields. While this ad hoc approach to Jatropha commercialization was going through the painful process of trial and error, SGB has pursued a different, more methodical, approach. SGB began developing a proper breeding and biotechnology platform based on a robust germplasm foundation. SGB has developed a large-scale collection of wild J. curcas ecotypes from Central America, the reputed origin of the species. The collection comprises approximately 12,000 genotypes grouped into about 600 accession families. This germplasm resource is the basis of the company‘s domestication program, which combines traditional breeding methods with the latest molecular breeding techniques. The germplasm collection is maintained at field research centers located at four different geo-climatic regions in Guatemala. The genotypes exhibit high levels of phenotypic diversity including variation in flowering time, oilseed content, fruit yield, plant architecture, susceptibility to fungal pathogens, pest resistance, drought, heat, flood and cold tolerance SGB is using intensive selection and breeding programs to identify and develop the most productive strains from its germplasm collection. Through outcrosses which combine important traits and inbreeding to improve uniformity of parental lines, hundreds of Jatropha hybrids have already been produced and are being advanced at hybrid trial locations located in Guatemala, Brazil and India. In parallel and in support of its breeding activities, SGB is applying sophisticated genotyping and molecular breeding tools designed to shorten the breeding cycle and accelerate the time to marketplace of elite planting material. Through its partnership with Life Technologies, SGB sequenced the Jatropha curcas genome, using the SOLiD™ 4.0 System in combination with their Ion Torrent sequencing platform. SGB scientists have developed a state of the art, high throughput genotyping pipeline which has revealed remarkable genotypic diversity correlating with the extensive phenotypic variation observed in its germplasm collection. The genotyping pipeline includes a novel high throughput High Resolution Melt technology coupled with real time DNA sequence confirmation of molecular markers for definitive allele confirmation. This genotyping platform enables rapid, digital DNA fingerprinting of parental and hybrid lines which enables SGB breeders to maximize hybrid vigor. The genotyping platform combined with genomic selection and genome wide association studies serves as the foundation for an aggressive molecular breeding program.

Technologies include both marker assisted selection which links molecular markers to economically important plant trait genes and innovative genome selection methods which incorporate genome-wide marker information in a breeding value prediction model that calculates breeding gain of our hybrid parental lines. Breeding gain can be predicted through a training population which is genotyped and phenotyped in targeted regional environments. SGB‘s laboratory also includes a tissue culture component where plant regeneration, transformation and dihaploid plant technologies are under development. Following more than four years of intensive research, scientists at SGB have also developed proprietary hybrid seed production technology. Hybrid seed production requires 1/50th the land needed to produce clonal plants by vegetative propagation, with the added benefits of hybrid vigor, strong tap roots and overall improved plant health, all at a fraction of the cost of tissue culture or other methods.

Toni Voelker Governments, academia and private enterprises, from small start-ups to the oil and agriculture giants, are pursuing a vast variety of feed stocks for the production of liquid biofuels and other renewables. Monsanto, as the world‘s leading seed company, is developing and applying tools and processes for accelerating yield gains of crops with the goal of conserving resources and minimizing inputs. I will give an overview of the current state of crop yield improvement at Monsanto and discuss some aspects of crop domestication. I will also share results from our ongoing evaluation of established, new or alternative energy crops.

Mark Harrison Sugarcane is one of the best biomass resources for the sustainable production of renewable fuels and chemicals. Advantages of sugarcane bagasse for biofuel and green chemical production include the availability of large quantities of biomass at existing industrial facilities, existing infrastructure for steam and electricity generation, process services and established feedstock and product handling systems. As a result, many biotechnology innovation companies are targeting sugarcane as the lead feedstock for commercialization. A multi-faceted biotechnology approach to improve sugarcane traits will result in lower cost fermentable sugars. Sugarcane biotechnology developments at the Queensland University of Technology in Australia include improved sugarcane transformation technologies, new molecular tools for controlling transgene expression, abiotic stress tolerance to enhance crop range and yield, and cellulase expression in planta. These developments in sugarcane biotechnology in combination with novel bioprocessing technologies and unique capability in pilot scale bioethanol production are driving the development and demonstration of lower cost fermentable sugars from sugarcane in Australia.

Thrusday, October 11, 2012 - 4:00pm-5:30pm Biomass Pretreatment and Fractionation - Fundamentals and Application Moderator: Richard Chandra, University of British Columbia

Perspectives of Wet Explosion Pretreatment Birgitte K. Ahring, Washington State University

The Effect of Biomass Moisture Content on Ethanol Yields from Steam Pretreated Hybrid Poplar Renata Bura, University of Washington

TBD Brad Seville, University of Toronto

Steam Pretreatment; How the Nature of the Biomass Substrate Influences the Fractionation and Recovery of the Hemicellulose, Lignin and Cellulose Fractions and the Effective Enzymatic Hydrolysis of the Cellulose Component Linoj Kumar, University of British Columbia

Abstracts Nature has designed lignocellulosic biomass to be highly resistant toward deconstruction and the fractionation/recovery of its cellulose, hemicellulose and lignin components in a usable form. An effective pretreatment process is essential and has a ―domino effect‖ on all the subsequent down-stream processing steps of a biomass-to-fuels/chemicals facility. The pretreatment step must allow the clean fractionation and recovery of all of the biomass components that can be used for various co-products, while producing a cellulosic substrate that is readily hydrolysed at minimal protein loadings and at maximum solids loadings. Papers in this session will discuss recent developments in pretreatment processes including the changes to the biomass structure imparted by the pretreatment and the potential for the processes to improve cost-effective biochemical conversion.

Birgitte K. Ahring Wet explosion (WEx) pretreatment is a steam-explosion based pretreatment where oxygen or air is added to catalyze the deconstruction of lignocellulosic materials. No chemicals such as SO2, sulfite or solvents are added to the process. The actual amounts of lignin which will be hydrolyzed and solubilized into the aqueous phase depend on the concentration of oxygen added, the process temperature and duration of the pretreatment. For production of sugars moderate temperatures (between 170 and 185oC), pressure (less than 10 bars) and short reaction times (less than 30 minutes) are often chosen to avoid further reactions of mainly the hemicellulose fraction resulting in loss of sugars and production of fermentation inhibitors such as HMF and furfural. Wet explosion pretreatment has been tested on a large variety of lingo-cellulosic raw materials with great success. Lately or group has focused on softwood raw materials such as loblolly pine and Douglas fir which has been found difficult to pretreat by other methods. Surprisingly, the results show that after wet explosion

pretreatment these woody materials are ready for enzymatic hydrolysis with a high convertibility and a total sugar yield of over 90%. Enzyme needs with commercial cellulases were further lower than previously reported for woody materials but still higher than our findings with agricultural residues. Techno-economic evaluation shows that Wex pretreatment has several advantages compared to for instance dilute acid pretreatment demanding the use of high-ranked steel and further prevention of sulfur emission when the lignin residue from the biorefinery is used for combustion. In the presentation we will discuss the results obtained with WEx pretreatment and the future perspectives for the large scale implementation of this pretreatment technology.

Renata Bura The role of physical and chemical characteristics of biomass has been extensively studied in relation to production of bioethanol and other products. Equally important, however, is the role of biomass processing prior to pretreatment. This study aimed to determine the effect of moisture content of hybrid poplar on overall ethanol yield via steam pretreatment and simultaneous saccharification and fermentation process (SSF). Hybrid poplar were either soaked in water (~80% moisture) or left dry (~12% moisture), and all were steam pretreated. The overall ethanol yield after simultaneous saccharification and fermentation of hexoses was higher in samples that were soaked prior to SO2 addition than in SO2-catalysed samples that were not soaked. The implications of this work cannot be understated. By simply increasing the moisture content of biomass prior to SO2-catalysis and steam pretreatment, the yield of ethanol can be greatly increased. This represents a promising means of increasing commercial ethanol yields through simply monitoring and altering moisture of biomass as it enters the process. Improved solids digestibility also represents a potential cost savings in that reduced enzyme loadings are required for the same ethanol yield. These results also go a long way towards explaining discrepancies in the literature in overall ethanol yields from similar feedstocks in different labs. Unless the moisture content of the starting biomass is the same, it is difficult to compare the results of experiments utilizing the same biomass.

Brad Seville The role of physical and chemical characteristics of biomass has been extensively studied in relation to production of bioethanol and other products. Equally important, however, is the role of biomass processing prior to pretreatment. This study aimed to determine the effect of moisture content of hybrid poplar on overall ethanol yield via steam pretreatment and simultaneous saccharification and fermentation process (SSF). Hybrid poplar were either soaked in water (~80% moisture) or left dry (~12% moisture), and all were steam pretreated. The overall ethanol yield after simultaneous saccharification and fermentation of hexoses was higher in samples that were soaked prior to SO2 addition than in SO2-catalysed samples that were not soaked. The implications of this work cannot be understated. By simply increasing the moisture content of biomass prior to SO2-catalysis and steam pretreatment, the yield of ethanol can be greatly increased. This represents a promising means of increasing commercial ethanol yields through simply monitoring and altering moisture of biomass as it enters the process. Improved

solids digestibility also represents a potential cost savings in that reduced enzyme loadings are required for the same ethanol yield. These results also go a long way towards explaining discrepancies in the literature in overall ethanol yields from similar feedstocks in different labs. Unless the moisture content of the starting biomass is the same, it is difficult to compare the results of experiments utilizing the same biomass.

Linoj Kumar Steam pretreatment has been studied for many years as a relatively low capital and operating cost method for pretreating, fractionating and enhancing the enzymatic hydrolysis of the cellulosic fraction of a wide range of lignocellulosic substrates. Although ―predictability methods‖ such as the ―severity factor‖ have been used to try and estimate the conditions that would provide the optimum recovery of the starting material and enhanced cellulose hydrolysis, these conditions will always be a compromise due to the often counterproductive mechanisms involved in maximising hemicellulose and ―reactive lignin‖ recovery while enhancing accessibility of the cellulose to the cellulase enzymes. We have looked at a wide range of lignocellulosic substrates, factors such as particle size, moisture content, etc., and the role that catalysts such as H2SO4 and SO2 might play in enhancing the pre-and-post treatment of these substrates. It is apparent that, although steam pretreatment can be effectively applied to a wide range of biomass substrates, the pretreatment conditions used for a more labile and accessible substrate such as corn fibre will be quite different from more recalcitrant substrates such as many types of softwoods. When more heterogeneous biomass feedstock‘s such as hog- fuels are used, the identification of ―optimum‖ pretreatment conditions becomes even more challenging! In this presentation we will describe the application of steam pretreatment to a range of substrates and how it can be ―tailored-to-fit‖ specific biomass feedstocks.

Friday, October 12, 2012 - 8:30am-10:00am Advances in Algal Biomass Production and Processing Moderator: Tim Zenk, Sapphire Energy

Algal Biofuels & Bioproducts in a Cold Climate - A Canadian Perspective Patrick C. Hallenbeck, Université de Montréal

Co-cultivation of Algae and Bacteria for Improved Productivity and Metabolic Versatility Alexander Beliaev, Pacific Northwest National Laboratory

Pall Applications in Algae BioMass Processing Membrane Harvesting Technology Algae Separation & Concentration Doug DiLillo, Pall Corporation

The Potential for Integrating Algal Carbon Capture, Wastewater Nutrient Removal and the Production of Bioenergy and Bioproducts by Testing Municipal Wastewater from the Annacis Island Wastewater Treatment Plant

Paul Kadotam, Metro Vancouver

Abstracts

Patrick C. Hallenbeck Under the Canadian Renewable Fuel Standard, all diesel fuel and home-heating oil must contain an average of 2% biodiesel, leading to an annual demand of 600 million liters. The production of biofuels using microalgae is promising and could become an important sustainable alternative production system for producing liquid fuels that is independent of food crops. A critical factor in the use of microalgae for the production of biofuels is the use of fertilizers. A particular challenge for countries like Canada in developing algal biofuels is the cold climate and lower solar irradiation. The panel will present ongoing research efforts on a number of possible partial solutions to these problems and describe some of the major efforts on algal biofuels presently underway in Canada. Dr. Patrick C. Hallenbeck will present The Laboratory of Advanced Biofuels Development at the University of Montreal, which is developing an extensive collection of algae native to Quebec. Using this collection, 100 strains have been assessed for the ability to grow at either 20°C or 8°C on the secondary stream of a municipal wastewater treatment plant. Fifteen different isolates showed promising growth rates, 9 at 20°C and 6 at 8°C. Moreover, strains showing maximum growth at 8°C had similar cell densities to those with a 20°C optimum, suggesting that good biomass production could be achieved in cold climate regions with systems coupled to wastewater treatment plants. In addition unialgal non-axenic cultures from this collection are being assessed for the ability to grow mixotrophically, using arabinose, xylose, molasses or waste glycerol from biodiesel production, as alternative sources of carbon. Dr. Pascale Champagne will discuss a multidisciplinary collaborative research project that she is leading which is exploring the enhanced production of bio-oil in freshwater micro-algae and the extraction of the bio-oil using a novel green solvent process. This project will employ flue gas, CO2, organic materials and nutrients derived from waste streams. The novel extraction process will employ switchable solvents and be applicable to "wet" biomass eliminating the need for energy intensive biomass drying, as well as costly and environmentally detrimental solvent recovery processes. Many industries are looking for sustainable alternatives to fossil consumption. Most of these industries have multiple waste streams; CO2, nutrients and heat, that can be used to produce lipid-rich algae biomass for obtaining biofuel, other forms of bioenergy, and coproducts for in-house or local uses. Thus, one promising approach for Canada would be to co-locate algal production facilities with these industries. Dr. Simon Barnabé will present and discuss the most promising projects. For biofuel and bioenergy production, high amounts of algal biomass must be produced to ensure profitability. This could be achieved through heterotrophic- mixotrophic production. Of course, a cold climate could present a major constraint for heterotrophic-mixotrophic production. However, producing algal biomass in proximity to an active industrial plant can overcome this issue. The plant can supply the algae producers with waste energy to reduce the cost of heating and agitation. This is one of the many advantages of adopting a co-location approach for algae fuel and energy production in cold climates.

Alexander Beliaev Oxygenic microalgae and cyanobacteria, which utilize solar energy, H2O, and CO2 to produce biomass, are rapidly being developed as alternative to plants for production of biofuels and other biotechnology products. Although many biotechnological applications focus on axenic strains, it is becoming increasingly obvious that utilization of monocultures presents significant physiological and engineering challenges. These not only the cultures are nearly impossible to maintain in an axenic state in open systems, monocultures are substantially less resilient to environmental perturbations, stress, and predation. Other important drawbacks include mass-transfer limitations, substrate delivery as well as precise coordination and balancing of energy generating with the downstream biosynthetic pathways. To that end, co-culturing of microalgae and cyanobacteria with heterotrophic microorganisms may offer an alternative means to optimally engineer the photosynthetic production of biofuels and other-value added products. By using photosynthetic strains that excrete organic carbon compounds (organic acids or sugars) that can be utilized by a heterotrophic partner growing in co-culture, one can physically separate the processes of carbon fixation and photosynthate conversion and allow utilization of readily-engineered heterotrophic strains for major biotechnology products using CO2 and light instead of commodities such as glucose, sucrose, and agricultural feedstocks. Moreover, O2 as well as carbon and energy source(s) for the heterotrophic organism will be uniformly produced throughout the cultivation system, ensuring absence of shock by periodic excess or deficiency of nutrients and oxidants that conventional types of cultivation usually suffer. The heterotrophic organism will consume O2 produced as the result of photosynthesis, thus dramatically decreasing mass transfer energy expenditure and simplifying the design and operation of any cultivation system. The co-culture approach also allows the utilization of various carbon sources ranging from CO2 from power plants to municipal wastes. The co-cultivation of oxygenic photoautotrophs and aerobic heterotrophs also eliminates engineering problems associated with oxygen-sensitivity and substrate delivery by creating favorable micro-oxic CO2-enriched environments. This presentation describes the most current technological innovation used for production of biofuel precursors and other value-added products.

Doug DiLillo Abstract: Progress made in Commercializing the use of Membranes in Algal BioMass Processing Pall Corporation is commercializing Algal Separation & Concentration Filter (ASCF) systems using membranes operating in crossflow mode to harvest and concentrate micro algae from growth mediums. Integrating the ASCF into the process requires the consideration of downstream algae biomass processing requirements. The optimal system design will depend on the biomass consistency and degree of dewatering required. The effectiveness of the separation method used to dewater the biomass depends on properties specific to the algae suspension. Filtration works by separating materials of different sizes. In Crossflow filtration, the flow is parallel to the filter medium allowing 100 % separation and concentration of the algae while the growth medium passes through the filter medium. This provides the opportunity for optimal water/growth medium and

nutrient management. With 100% of the algal biomass harvested with the membrane system, the effluent is free of solids that may otherwise require treatment for discharge. More important, the growth medium, free of biomass may be recycled back within the process. This enables water and nutrient management while achieving maximum biomass harvesting yields. The effectiveness of cross- flow filtration to separate algae depends on the size of the algae in the nutrient broth, the membrane pore size and the system design. Costs for the equipment will depend on feed stream sizes and the final biomass concentration desired. Crossflow filtration is a low energy means for concentrating algae biomass. Determining the energy consumption at scale is one of the variables that Pall will be presenting from the data collected during its six plus years working on optimizing the use of membranes for commercial scale algae production. Multiple membrane based integration strategies for algae processes will be presented along with the current status of commercialization and topics under further refinement.

Paul Kadotam Metro Vancouver is continually seeking opportunities to implement integrated resource recovery opportunities as part of its operations and is exploring the future possibility of co-locating a facility for wastewater treatment and microalgae cultivation. Treated municipal wastewater effluent is a readily available source of nitrogen and phosphorus which can be used for cultivating microalgae for the production of bioenergy and other bioproducts. As a first step, the National Research Council of Canada and Metro Vancouver collaborated to test the suitability of wastewater from Metro Vancouver as a growing media for microalgae. Samples of treated effluent and centrate from solids dewatering obtained from the Annacis Island Wastewater Treatment Plant were evaluated at National Research Council facilities in Halifax as nutrient sources for two photosynthetic green algae. Scenedesmus sp. AMDD and Chlorella sorokiniana were grown in duplicate, 250 mL batch-flasks in controlled environment chambers at 22°C. The concentrations of dissolved NH3-N and PO43--P averaged 14 and 0.82 mg/L, respectively, in effluent and 1280 and 230 mg/L, respectively, in centrate samples. Carbon dioxide was added to the air stream to a final concentration of 2% (v/v). Effluent samples supported specific growth rates of approximately 1.0 d-1 in both species and average biomass yields of 0.59 g/L and 0.41 g/L in Scenedesmus and Chlorella, respectively. Blending the effluent with up to 5% centrate enhanced biomass yields significantly in both species and resulted in 100% N and P removal from the wastewater through algal growth. Given the successful bench-scale cultivation of the selected microalgae species, Metro Vancouver is seeking additional collaborators to pilot technologies at its recently established Sustainability Academy – Annacis Wastewater Centre as the next step.

Renewable Chemical Production

Wednesday, October 10, 2012 - 8:30am-10:00am Renewable Chemicals: Next Generation Monomers and Polymers Moderator: William Baum, Genomatica

TBD Nelson Barton, Genomatica

Direct Gaseous Fermentation to Isobutene, Butadiene, Propylene and Other Light Olefins. David Gogerty, Global Bioenergies S.A

Biobased Chemicals: The Path to Commercialization Max Senechal, Metabolix

Polymers for the 21st Century: New Planet and Human Friendly Materials with Exceptional Performance. Olga Selifonova, Reluceo, Inc.

Abstracts

David Gogerty As of today, most bioproduction processes are based on enhancing metabolic pathways naturally existing in microorganisms. Light olefins (ethylene, propylene, linear butylene, isobutylene, butadiene..) are currently obtained from fossil oil. They are the principal building blocks of the chemical industry, representing each an existing multi-billion dollar market. Light olefins are not produced by microorganisms and no direct bioprocess to produce these molecules industrially from natural resources has been developed so far. Global Bioenergies has been founded in 2008 to develop new metabolic pathways for the direct biological production of light olefins from renewable resources. ―Direct‖ refers to the fact that the product secreted by the micro-organism is the light olefin itself, and not an alcohol such as ethanol, isobutanol or butanediol which would then need to be dehydrated chemically and possibly undergo further chemical reaction steps (e.g. metathesis). At room temperature and hence in the production reactor, light olefins are gaseous and therefore spontaneously volatilize from the fermentation medium, entailing two major advantages compared to liquid fermentation products: - The gas does not accumulate in the reaction chamber and thereby does not reach concentrations slowing down or inhibiting the microorganism‘s production. Ethanol for example becomes toxic for yeast at concentrations slightly over 10%. - The purification process is considerably easier and cheaper since no energy consuming methods such as distillation or phase separation are necessary to purify the end product. Proof of principle has been obtained through successful bacterial production of Isobutene, a key building block for tires, organic glass, plastics and various polymers. It can also be converted into Gasoline (IsoOctane), JetFuel and Diesel. In June 2011, the company has carried out an IPO to finance the

industrialization of the isobutene process as well as to bring forward its programs on other light olefins. Global Bioenergies has established partnerships with Synthos, LanzaTech, a German car manufacturer and one of the 50 biggest US companies on bio-butadiene, SynGas, drop-in biofuels and isobutene.

Max Senechal Metabolix is a leading industrial biotechnology company and pioneer in the development of biobased plastics and chemicals, combining its expertise in bioscience with innovative engineering excellence to address global markets. The Metabolix scientific foundation and core science is the metabolic pathways for the production of a class of microbial biopolymers – polyhydroxyalkanoates (PHAs) – from renewable resources. PHAs can be tailored through metabolic pathway engineering and have a wide range of applications in industry, including use in polymer form as biobased plastics or in monomer form as chemical intermediates. The first commercial product using the fermentation route is Mirel biopolymers. Metabolix is also rapidly developing PHA fermentation routes to produce biobased replacements for gamma butyrolactone (GBL) and 1,4-butanediol (BDO) (C4) and acrylic acid (C3) chemical intermediates with a current addressable market over $10 billion, using its proprietary FAST recovery technology. We believe that volatility in the price of petroleum derivatives, rapid advances in biotechnology at all stages of the value chain, a growing commitment among brand owners and consumers to produce and use ―green‖ products, and an increase in the renewable content of products will accelerate the pace of adoption for chemicals made from renewable sources. In this presentation, Metabolix‘s Vice President of Biobased Chemicals Max Senechal will outline the Company‘s experience developing biobased ―drop-in‖ replacements for petroleum-based industrial chemicals, and the path to commercialization.

Olga Selifonova Development of the petrochemical industry in the 20th century resulted in creation of remarkable new polymer materials and plastics that became inseparable parts of human life. However, today, the majority of fossil-based polymers and plastics are not degradable hence posing a multitude of pollution problems with gargantuan mountains of plastic trash on the land and in the oceans. Challenging such a socially and environmentally unacceptable status quo, we embark on a quest to develop and industrialize sustainable and responsible alternatives to those 20th century polymer technologies that are at the heart of the Trash Planet problem. Introduction of new degradable plastics from renewable sources faced and will continue to face enormous challenges. Largely, these challenges arise from limitations of the fundamental properties of PLA, PHB(A)s, cellulose acetate and starch-based alternatives developed and commercialized during last decades. We will discuss how we discover and develop the next generation of planet and human friendly polymers and polymer systems that are devoid of toxins, have exceptional performance, made from abundant renewable sources, beautifully designed to re- enter the carbon cycle when they are no longer needed, and are competitive with the best fossil carbon polymers. The presentation will highlight further development of a high performance renewable bioplastic PXLK derived from C5 carbohydrates

and Reluceo‘s degradable high performance non-acrylic superabsorbent polymers (SAP).

Wednesday, October 10, 2012 - 10:30am-12:00pm Commercial Applications for Biopolymers and Bioplastics Moderator: Murray McLaughlin, Sustainable Chemistry Alliance and BioIndustrial Innovation Centre

Wadood Hamad, Celluforce Inc. Toby Ried, Solegear John Shaw, Itaconix Amar Mohanty, University of Geulph

Abstract

Bio-based materials can provide a competitive alternative and provide new functionality to non-renewable based options. Market demands on access to renewable materials in durable and non-durable products continue to open new market opportunities and spark new innovation. This panel will highlight technology, market trends and opportunities in developing applications for bio- based polymers and plastics.

Wednesday, October 10, 2012 - 2:30pm-4:00pm Sugars: The Key to Renewable Chemicals Moderator: Bhima Vijayendra, Battelle

Sugars: Key to Unlocking Cellulose as a Petroleum Replacement in Fuels and Products Mike Hamilton, Renmatix

„Getting the World‟s First Industrial-Scale Cellulosic Ethanol Plant Up and Running in 2012‟ Dennis Leong, Beta Renewables

Production of Chemicals and Low Cost Sugar from Lignocellulosic Feedstocks Ali Manesh, American Science and Technology Corporation

Teaching Yeast to Convert Plant Cell Wall Sugars to Renewable Fuels and Chemicals Na Wei, University of Illinois – Urbaba Champaign

Abstracts

Mike Hamilton

High energy and commodity prices, as observed in recent years, have increased demand for integration of renewable resources in energy production and biomaterial applications. Biochemical products offer a natural and sustainable solution to the price volatility and environmental impact associated with petrochemicals. There is great potential to tap sugar from cheap, abundant waste biomass to satisfy the ever-growing demand for fuels and products in place of petroleum feedstocks. Drawing insight from a Deutsche Bank report, Renmatix projects the total global chemicals market in 2025 will be roughly $5.1 to $7TN. Currently, the cost of producing cellulosic sugar is one of the biggest factors inhibiting the biochemical industry‘s ability to occupy a large portion of this huge market. The challenge is to unlock the sugars inherent in biomass at a cost point that will enable the industry to compete on an economic basis with petroleum derived products. These sugars can then be used in intermediate chemicals to make everything from plastic bottles, paints, tennis shoes, and tires. Current methods of breaking down biomass—enzymatic or acid hydrolysis—require expensive enzymes or harsh chemicals, and can take up to three days to yield sugars. Renmatix, however has pioneered a new process. Using supercritical water—water at elevated pressures and temperatures—Renmatix is able to deconstruct a wide range of non- food biomass in seconds. The water-based process uses no significant consumables and produces much of its own process energy. With significant advantages in cost and speed, Renmatix is able to provide cellulosic sugar affordably and on large- scale. The process is also biomass agnostic and can be located near the source of biomass, reducing transportation and logistics costs. While supercritical liquids have an established history in industrial processes, such as coffee decaffeination and pharmaceutical applications, Renmatix is the first company to successfully yield sugar from biomass at significant scale. In this panel presentation, Renmatix CEO Mike Hamilton will discuss how supercritical hydrolysis provides a low cost alternative to traditional biomass-to-sugar conversion methods and can enable the transition from petroleum-based products to biobased alternatives.

Dennis Leong The plant in Crescentino, Italy is expected to be the world‘s first commercial-scale cellulosic ethanol plant when it starts operations this year. The plant, built by Chemtex, with a design capacity of 20 million gallons per year, represents a major commercialization milestone for the industry after over $200 million invested in R&D by Gruppo Mossi & Ghisolfi (M&G). This presentation will discuss the underlying PROESA technology from Beta Renewables, plant status, economics, and recent developments in licensing the technology to additional producers worldwide.

Ali Manesh In the last few years, sugar is emerged as the main feedstock to derive several potential chemicals and bio-fuels. The cost availability of these fuels/chemicals are vastly depends on the source of sugar. Currently most of the processes are based on sugar from corn, sugar cane and sugar beets where use of such food-derived raw materials is not sustainable. Lignocellulosic sugar is sustainable however so far has not economical been compared to other food-based feedstocks. American Science and Technology has developed technologies that can produce low cost sugar from lignocellulosic biomass feedstocks which are not part of nation‘s food

supply and are readily available. AST process produces bio-chemicals and its byproducts are short fibers and high quality lignin. Based on the results obtained from pilot production plant, we now have cellulosic sugar that economically can compete with the 1st generation food based sugars. Keywords: lignocellulosic feedstock, sugar, biofuel

Na Wei Sugars derived from plant cell walls are highly abundant in nature and hold promise to provide an abundance source for production of renewable fuels and chemicals. However, the cost and logistics of releasing these sugars from plant biomass remains prohibitive on commercial scales. The yeast Saccharomyces cerevisiae has a proven track record as a research platform and industrial host for production of biofuels and chemicals. Yet, S. cerevisiae is unable to utilize sugars released from the plant cell wall–cellodextrins and xylose. In collaboration with scientists in the Energy Biosciences Institute (EBI), we have used synthetic biology methods to engineer S. cerevisiae to convert cellodextrins and xylose simultaneously into biofuels with high yield and productivity. Having never used these sugars in their natural state, these yeast serve as a ―blank slate‖ to understand the key bottlenecks in cellodextrin and xylose utilization. I will present recent work that uses synthetic and systems biological methods to further improve the performance of cellodextrin- and xylose-utilizing S. cerevisiae in the production of renewable fuels and chemicals.

Thrusday, October 11, 2012 - 2:00pm-3:30pm Advances in the Commercial Production of Biobased Products Moderator: Chas R. Eggert, OPX Biotechnologies

Advances in the Successful Production of Biobased Products, a Strong Commercial Approach Stephen J. Gatto, Myriant Corporation

The Production of Green Nylon E. William Radany, Verdezyne, Inc

BioIsoprene™: Development of a Bio-based Process for Production of Isoprene Monomer from Renewable Resources Richard LaDuca, DuPont Industrial Biosciences Division

Abstract

Stephen J. Gatto Myriant is a leading biochemical company developing technologies for the production and manufacturing of biobased chemicals, including succinic and lactic acid. These chemicals are derived from sugars and not from conventional petroleum derived feedstocks. Myriant‘s first succinic acid commercial facility located in the

Port of Lake Providence, Louisiana is expected to be producing 30 million pounds of succinic acid in early 2013. Myriant leaders will communicate the challenges and opportunities faced from development to commercialization of biobased chemicals. Also, the presentation will discuss new trends on the development and commercialization of biobased chemicals.

E. William Radany As consumer demand for sustainable products continues to gain momentum, Verdezyne provides an in-depth look at the development of its bio-processes for cost-advantaged production of renewable chemicals including adipic acid and dodecanedioic acid (DDDA). Adipic acid is used to manufacture nylon 6,6 and thermoplastic polyurethane. DDDA is used to manufacture nylon 6,12 for engineered plastics requiring special properties such as heat and abrasion resistance. Verdezyne‘s proprietary fermentation technologies offer feedstock flexibility and other noteworthy advantages. The introduction of these bio-based processes delivers a positive impact on the world by replacing environmentally unsustainable methods with cost-reducing cleaner and greener alternatives.

Richard LaDuca Abstract: A bio-based production system for isoprene production based on microbial fermentation is in development. Isoprene is a major chemical intermediate used in a wide variety of industrial applications including the production of synthetic rubber for tires and coatings, use in adhesives and development of specialty elastomers. All of the current production of isoprene is derived from petrochemical sources and with an increasing global need for more capacity with improved environmental attributes an opportunity presents itself for a new way to produce isoprene. This presentation describes the engineering of a microbial cell factory to convert renewable carbon feedstocks into isoprene using metabolic pathway engineering.

Thrusday, October 11, 2012 - 4:00pm-5:30pm Bio-Based Polyols – Challenges and Opportunities Moderator: Jack Grushcow, Linnaeus Plant Sciences Inc.

Bio-Based Polyols – Challenges and Opportunities Hamdy Kahlil, Woodbridge Foam Jeff Uhrig, Elevance Jack Grushcow, Linnaeus Plant Sciences Inc.

Opportunities for Bio-based Polyols in Engineering New Green Materials for Value-added Industrial Uses Manju Misra, University of Guelph

Abstracts

Hamdy Kahlil; Jeff Uhrig; Jack Grushcow The panel will seek to present the latest research into bio-based polyols. The organizations participating in the panel have recognized the value of a bio-based platform for their products/programs and will speak to their success and planned developments. This panel will make a unique connection between the end user, feed stock provider and oil seed developer. The group will explore how end user needs can be reflected in process design and how this in turn makes specific demands on oil seed profiles. It is a detailed look at how rational design can be used to guide molecular biology to deliver optimized industrial feedstocks.

Manju Misra Sustainability, industrial ecology, and green chemistry are guiding the development of the next generation of materials, products, and processes. New materials based on annually renewable agricultural and biomass feedstocks can form the basis for a range of sustainable, eco-efficient products which can substitute petroleum-based industrial products. The incorporation of bio-resources such as crop-derived green plastics and plant-derived biofibres (agro-fibres / agro residues) into composite materials are gaining prime importance in the design and engineering of green composites. Renewable vegetable oil based polyols as used in the manufacturing of bio- polyurethanes are sustainable green materials; widely used in the production of foams, elastomers, adhesives and sealants, and composite plastic parts. New biocomposites and nano-biocomposites have been developed and engineered in our laboratories from vegetable oil based polyols. These newly developed biomaterials show tremendous promise in industrial applications such as automotive, housing, aeronautical and other transportation systems, marine industries, recreation equipment, and farm equipment. These advanced biomaterials are processed from hybrid biobased polyurethane (PUR) matrices which are reinforced with natural fibers, cellulose, lignin, or multi-walled carbon nanotubes (MWCNT). Different processing techniques, such as compression molding

and vacuum assisted resin transfer molding (VARTM) are being used to engineer a wide range of biopolyol-based biomaterials. This presentation highlights the innovative value-added industrial applications of functionalized vegetable or plant derived oils. This research is financially supported by the Ontario Ministry of Agriculture, Food, and Rural Affairs (OMAFRA)-New Directions Research Programs; University of Guelph-OMAFRA (Bioeconomy for Industrial Uses Program); the Ontario Research Fund (ORF) Research Excellence (RE) Round-4 from the Ontario Ministry of Economic Development and Innovation (MEDI); Natural Sciences and Engineering Research Council of Canada (NSERC)-CRD grant; Grain Farmers of Ontario (GFO); and Manitoba Pulse Growers Association (MPGA).

Friday, October 12, 2012 - 8:30am-10:00am Integration of Bio-Based and Fossile-based Chemical Manufacturing Moderator: Uwe Welteroth, Linde Engineering Dresden

Integration of Bio-Based and Fossile-Based Chemical Manufacturing Tom Browne, FPInnovations Hassan Taheri, Petron Scienctech, Inc. Markus Wolperdinger, Linde Engineering Dresden GmbH

Lignin, A Renewable Chemical Feedstock for the Future Michael Rushton, Lignol Innovations Ltd.

Abstracts

Tom Browne; Hassan Taheri; Markus Wolperdinger The panel focuses on the integration of bio-based and fossil-based chemical manufacturing approaches in three distinctive, yet interrelated fields: (i) Forestry- based biorefinery systems and chemicals from wood, (ii) Value-added chemical intermediates from bio-based ethylene, and (iii) Pathways to bio-based gases for chemical manufacturing. The approaches will be discussed from a technical and economic perspective. Examples for applications of forest-based chemicals, intermediates from bio-ethylene, and biogenic gases in the chemical manufacturing environment will be given. In Talk 1, FP Innovations will focus on the value-chain of wood-based chemicals. Talk 2 by Petron focuses on aspects associate with the manufacture of intermediates and chemicals via bio-based Ethylene. Talk 3 by Linde will discuss technical approaches for the generation of bio-based gases, e.g., hydrogen, as well as industrial applications for such gases, including scale-up activities and the utilization of bio-based hydrogen in chemical manufacturing environments.

Michael Rushton Increasingly, lignin is being recognized as a system of chemically discrete entities as opposed to an amorphous undefined waste product from the pulp and paper and biorefining industries. This recognition is essential in justifying and defining a path

forward to commercialization, a path which begins with sources of lignin and will end with broad and successful commercialization of lignin-based products with sales volumes of the same order as some of today‘s ubiquitous specialty chemicals. The near end of the commercialization path relies on the knowledge that the very versatile and controllable chemistry of lignin will be heavily determined by its biomass source, and of equal importance, by the process by which the lignin is extracted from biomass and subsequently isolated, purified and modified. The far end of the commercialization path remains presently, to a large degree, within the realm of ―blue sky thinking‖. A careful examination of the literature and reports from industry do, however, clearly show that the mid-term segments of the commercialization path contain many reports of R&D-driven discoveries and innovations which do constitute both milestones and essential developments in the creation of new and useful applications of lignin. These new applications range from extensions of lignin use in resins to coatings, adhesives/binders, antioxidants/anti- ozonants, and foams, further on to low cost carbon fiber, fusible binders, activated carbon nano- structures, and eventually into the arena of human and animal health and nutrition. The purpose of this presentation is to explore some commercialization scenarios for lignin in the context of some of these newly emerging applications and highlight some of the key developments which validate the commercialization efforts.

Technical and Research Presentations

Wednesday, October 10, 2012-8:30am - 10:00am Economical Fermentable-Based Processes Moderator: John Bhatt, Novasep

Novel Yeasts for a Microbiological Biorefining Platform (BEACON) Sreenivas Rao Ravella, Institute of Biological Environmental and Rural Sciences (IBERS)

Ethanol Production by Recombinant Flocculent Saccharomyces Cerevisiae that can Effectively Co-Ferment Glucose and Xylose Akinori Matsushika, Biomass Refinery Research Center (BRCC), National Institute of Advanced Industrial Science and Technology (AIST)

Progress of 1,3-Propanediol Production with Genetic Modified Klebsiella Pneumonia Challenges in Developing Microbes for Industrial Biotechnology Rishi Jain, Praj Matrix – The Innovation Center

Metabolic Regulation of L-lactate Production without Neutralization Using Fission Yeast, Schizosaccharomyces Pombe Futoshi Hara, Asahi Glass Co., Ltd.

Abstracts

Sreenivas Rao Ravella A biorefinery is a facility that integrates biomass conversion processes and equipment to produce fuels, chemicals, value added products, feed, materials and energy from biomass. The BEACON biorefinery platform is an innovation lead research project focused on working with businesses to develop new products, processes and methodologies that can deliver on innovation and commercial impact. BEACON aims to: optimize the use of bio-based resources; minimize wastes and produce value added products from plant based biomass to maximizing benefits and profitability. This project also focuses on optimizing the efficiency of supply chains and undertakes life cycle assessment to identify processes that will have significant impact on the sustainability of bio-based products. Processing and conversion of plant biomass presents a variety of opportunities for optimization. Developments in microbial conversion technologies will be part of the challenge e.g. developing new microbial strains for the direct conversion of hemicellulose hydrolysates. At present, there is a need for more efficient and robust microorganisms that can withstand higher temperatures and that are resistant to inhibitors. Progress in these areas will enable efficient conversion/ fermentation of plant based substrates into higher value products. We are currently developing robust yeasts for xylose utilization.

Akinori Matsushika Bioethanol production from xylose is important for utilization of lignocellulosic biomass as raw materials. The research on yeast conversion of xylose to ethanol has been intensively studied especially for genetically engineered Saccharomyces cerevisiae during the last decade. However, processes utilizing these strains cannot economically produce ethanol from lignocellulosic biomass, mainly because these strains cannot yet efficiently convert xylose to ethanol with the high yields and fermentation rates possible with glucose. Recently, we have reported the development of a recombinant industrial S. cerevisiae strain MA-R4 that can simultaneously co-ferment glucose and xylose to ethanol and has high ethanol productivity (1). The MA-R4 strain was engineered by chromosomal integration to express genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) from Scheffersomyces (Pichia) stipitis along with S. cerevisiae xylulokinase (XK) gene under the control of PGK1 promoters using the alcohol-fermenting flocculent yeast strain IR-2. IR-2 has the highest xylulose-fermenting ability among many different industrial diploid strains, suggesting that IR-2 is a useful host strain for genetically engineering xylose-utilizing S. cerevisiae (2). To gain further insight into the metabolic engineering of IR-2 for efficient ethanol production from xylose, here we constructed a set of recombinant isogenic strains harboring different combinations of the genetic modifications present in MA-R4. The results showed that high activity of XK increased ethanol production from xylose at the expense of xylitol excretion, and that high activity of XR increased the rate of xylose consumption. These results suggest that fine-tuning of introduced enzyme activity in S. cerevisiae is important for improving xylose fermentation to ethanol, and therefore MA-R4 could be a suitable recombinant strain for ethanol production from xylose. Furthermore, in this study the effects of the initial cell concentration and various glucose:xylose ratios in fermentation media on glucose/xylose co- fermentation parameters using the MA-R4 strain. The initial cell concentration of MA-R4 greatly affects the rate of xylose fermentation and ethanol production, and ethanol selectivity is increased when a higher proportion of glucose is available. From these results, we conclude that inoculation at the appropriate initial cell concentration and control of the extracellular glucose concentration is important for effective glucose/xylose co-fermentation.

Rishi Jain Metabolic abilities of microbes belonging to all the three domains – archae, bacteria and eukarya – are being harvested for the commercial production of valuable chemicals. The availability of technology is helping us isolate even more exotic microorganisms from extreme environments. High throughput technologies are assisting us in characterization of microorganisms at an increasing pace that can now lead us into the industrialized production of biochemicals. One of the challenges that lie ahead is the ―economic‖ scale-up of these fermentation-based processes. Viable titers, yields and productivity of the chemical of interest are the first targets that have to be addressed in the development of such processes. Other major contributors to the cost stack are feedstock, nutrient requirements, down-

stream processing and sterility requirements. Application of waste streams from other industries/sectors is now being seen as a viable option since it adheres with the process economics. However, different kinds of inhibitors come along with these waste streams that need extra efforts in addressing them. This presentation will cover certain aspects of finding solutions to the challenges in developing microbes for industrial biotechnology. Addressing the above problems lies in trying to approach all the parts of the cost stack during a microbial development program. A systems biology approach needs to be taken that includes high throughput omics techniques amalgamated with bioinformatics. Classical mutagenesis also plays an important role in addressing some of the issues. In addition, modern synthetic biology is starting to play its role in industrial biotechnology. However, one needs to find a fine balance between the costs associated with an inter-disciplinary infrastructure/expertise and the path to commercialization. Collaboration between industries and academics will go a long way to take the leap into the era of industrial biotechnology.

Futoshi Hara, Asahi Glass Co., Ltd. The current main D-/L- lactic acid production method is fermentation by the lactic acid bacterium. However, neutralization was needed with lactic acid bacterium fermentation and low optics purity raise a refinement cost. Gene engineering of fission yeast Schizosaccharomyces pombe and adequately culture and ferment can produce the lactic acid that the optics purity was high with low cost. This study aimed at elucidating what kind of gene control is performed in the fermentation. Great improvement was seen in production speed and yield by letting division yeast S.pombe which changed a gene ferment by high cell density. In addition, fermentation by no neutralization was enabled by this. Transcription of the gene group that promoted for the stationary phase was seen at the repeated fermentation.

Wednesday, October 10, 2012 - 10:30am-12:00pm Efficacy of the De Novo Enzyme Technology Moderator: Eric Althoff, Arzeda Corp.

Novel Enzymes to Enable New Chemistries Eric Althoff, Arzeda Corp.

Efficient Enzymatic Conversion of Corn Fiber into C5/C6 Sugars David Mead, C56 Technologies

Yeast-Derived Synergists in Environmentally Friendly Materials for Cleaning, Bioremediation and Agriculture Carl W. Podella, Advanced BioCatalytics Corporation

Comparison of Alkali and Enzymatic Delignification of Wheat Flour Mill Waste for Bioethanol Production by Saccharomyces Cerevisiae Muhammad Asgher, Department of Chemistry & Biochemistry, University of Agriculture, Faisalabad, Pakistan

Abstracts

Eric Althoff Arzeda's technology applies the power of computation to solve enzyme design problems. Arzeda has demonstrated the efficacy of the de novo enzyme technology by introducing entirely new active sites and enzyme activities into protein scaffolds. Arzeda's proprietary enzyme design algorithms can be applied to a variety of enzyme and protein design problems, and represents a technological change of mindset and a new addition in the enzyme engineering toolkit, as demonstrated by our successful collaboration with Pioneer Hi-Bred. To complement this successful technique of de novo enzyme design, we have developed an innovative approach – Enzyme Identification - that leverages computational enzyme design to rapidly engineer enzymes with known catalytic mechanisms for unnatural substrates as well as increasingly demanding transformations and reaction environments. This new technique has been important in the recent design successes for Arzeda. This talk will focus on the computational techniques used and lessons learned in all of these design projects. Coupled with traditional enzyme optimization techniques such as directed evolution and gene shuffling, computational identification and design techniques open new avenues for enzyme engineering and discovery and their expansion into other applications of synthetic biology. In addition to the discussion of the technology, the presentation will also go Arzeda's work in applying its unique technology to the development of next generation bio-based chemicals.

David Mead Corn fiber is a byproduct of the corn milling industry. Approximately 43 million tons of DDGS, which is 50% fiber,was produced in 2012 and marketed as an animal feed ingredient of very low value. The corn fiber contains residual starch and a cell wall

fraction of the kernel, which are both rich in polymeric carbohydrates. Economical conversion of this sugar stream into ethanol could provide an additional $4B in fuel revenue. Corn fiber is very difficult to degrade and there has not been any progress in the field for 40 years. C5•6 Technologies has developed a potent enzyme product that degrades this previously unfermentable carbohydrate stream within corn ethanol plants into fermentable sugar. BranBuster™ enzyme technology improves sugar yield 10% and leapfrogs existing products by providing a high potency fiber- degrading enzyme in a form that is compatible with existing ethanol plant infrastructure, saving the corn ethanol industry either 10% on feedstock costs or generating an additional 10% ethanol. The conversion of the corn fiber into sugar has numerous competitive benefits for the ethanol plant, farmers and the environment. Fiber holds large amounts of water and converting it to sugar will save large quantities of water, gas and electricity otherwise needed for drying the low value fiber. A higher protein content animal feed will improve its value and marketability to more livestock, while dramatically decreasing the volume of manure and greenhouse gases such as methane created by high fiber feed. A typical 100MGY ethanol plant would realize a $31M/yr EBITA gain using BranBuster™ enzyme product. Beyond corn ethanol, BranBuster™ enzyme is compatible with cellulosic biofuels conversion technologies, an emerging market projected to reach $1B in enzyme sales by 2020.

Carl W. Podella Baker‘s yeast subjected to a mild, non-lethal stress, releases low molecular weight stress proteins that form tight complexes with a broad range of surfactants. These protein-surfactant complexes (PSC) display surface activity surpassing that of synthetic surfactants alone, as verified by measurements of surface and interfacial tension, critical micelle concentration, and contact angle. Applications include, but are not limited to enhanced efficiency in industrial cleaning, such as oil- contaminated chemical cargo tank cleaning, decontamination of oil-spill, contaminated water, soil and equipment; odor control in sewer lines and for water and soil bioremediation. Improved spreading, wetting, and penetration of these PSC materials provide multiple agricultural benefits for various surfaces, such as foliar uptake of nutrients and protectants, simulation of plant root growth, prevention of scale in irrigation lines and significant reduction in watering requirements. PSC activates indigenous microflora in waste water processing, resulting in a faster elimination of organic contaminants, and reduction in sludge production. These improvements are due to an uncoupling of bacterial electron transfer from the ATP synthesis process, by increasing proton leak across bacterial cell membranes (uncoupling of Oxidative Phosphorylation).More recently, the PSC technology has shown the ability to enhance the kinetics function of catalytic activity in cellulose and lipase enzymes.Specific products are certified by the National Sanitary Foundation (NSF) for application in potable water treatment, by the International Maritime Organization (IMO) for tanker fleet cleaning applications and have been included on the U.S. EPA National Contingency listing for ocean water dispersants and shoreline and wetlands cleaning. The products developed are based upon the PSC core technology and are protected by several patents issued and patent applications pending. Data on the physical and biochemical properties of the PSC

will be presented, together with the examples of their applications in diverse arenas.

Muhammad Asgher The research project was planned to evaluate the cost effective pretreatment of a lignocellulosic waste for the production of low cost bioethanol. Ligninolytic and cellulolytic crude enzyme extracts were indigenously produced using Trametes versicolor and Trichoderma resiae, respectively under pre-optimized culture conditions. Lignocellulosic waste ―Mumni‖ from wheat flour mill was subjected to enzymatic as well as alkali pretreatment for delignification. Crude lignolytic enzymes (MnP, LiP, Laccase) mixture produced by Trametes versicolor IBL-04 under pre-optimized solid state culture conditions was used for producing delignified feedstock and it was compared with 4% NaOH treatment. The delignified substrates were further hydrolyzed by cellulase mixture produced by Trichoderma resiae for sugar release. The feed stock thus produced were subjected to liquid state fermentation for ethanol production using Saccharomyces cerevisiae. Biomass obtained after ligninase pretreatment proved to be a better feedstock for ethanol production, as compared to alkali pretreatment. Cellulose content of the enzymatically delignified biomass was higher as compared to alkali pretreated one. Hydrolysis of the biomass with crude T. resiae cellulase mixture also produced higher glucose concentration, subsequently resulting in higher ethanol production in case of enzymatic delignification, as compared to alkali pretreatment. After optimizing different fermentation parameters, maximum ethanol productivity of 32.2 g/dL was achieved in case of enzymatic treatment after 72 hours of incubation with 8g/100 mL of substrate at pH 5 and 30°C temperature. In case of alkali pretreatment, maximum ethanol productivity of 28.1g/dL was noted after fermentation by yeast under similar conditions. Ligninase pretreatment for delignification of Mumni was found to be better delignification method as compared to alkali treatment for ethanol production.

Wednesday, October 10, 2012 - 2:30pm-4:00pm Techno-Economic Evaluation: Preprocessing and Pretreatment of Biomass Moderator: Johan van Groenestijn, TNO

Biomass Pretreatment by a Continuous Flow of Superheated Steam Johan van Groenestijn, TNO

Ambient Conditions Pretreatment for Plant Biomass Followed by Saccharification Rajai H. Atalla, Cellulose Sciences International

Optimization Study of the Ultrasonic Oil Extraction and Insitu Transesterification of Microalgae for Biodiesel Production Mel Maizirwan, International Islamic University- Malaysia

Abstracts

Johan van Groenestijn It is known that the lignocellulosic complex can be broken under acidic conditions at high temperatures. In case heating is carried out by steam injection, most of the time such steam is stagnant and saturated. The use of superheated steam that flows through the biomass is an alternative with several advantages. By passing steam through heaps of straw an efficient and uniform heat transfer is established. Tests were carried out with wheat straw and reaction conditions were varied. The straw could be successfully pretreated within a few minutes at dry matter concentrations between 30% and 65%, steam pressures of 6 bara and temperatures of 160-180°C. High dry matter concentrations reduce heating costs and required acid concentrations are rapidly reached because of the low amount of water present. In addition, high biomass concentrations are favorable for the economy of the fermentation process and downstream processing. A large part of the volatile compounds such as furfural was stripped from the biomass, thus reducing toxicity of the biomass. During superheated steam treatment hemicellulose was 90% hydrolyzed into its monomers. After cooling and neutralization the cellulose could be 90-95% converted into glucose by the use of enzymes. Such pretreatment can be carried out in conventional steam dryers, with some modifications. In such systems the continuous feed in and out of relatively dry pieces of biomass through the superheated steam process, against a pressure drop of 6 bara, is easy: the inlet and outlet systems are proven technology.

Rajai H. Atalla Cellulose Sciences International (CSI) has developed a novel inexpensive process for transforming lignocellulosic biomass into a nanoporous form that is more accessible to enzymes than biomass pretreated by alternative methods that rely on the use of elevated temperatures and pressures. In addition to transforming the biomass into nanoporous form, the pretreatment limits or eliminates the inhibition of the action of enzymes by lignin and related compounds. The pretreatment utilizes inexpensive, commonly available reagents and together with commercially available enzymes, transforms the polysaccharides into monosaccharides rapidly and efficiently. The monosaccharides can be readily fermented to ethanol or other biobased compounds because avoidance of elevated temperatures prevents formation of thermal degradation products that are toxic to microorganisms. The saccharides can also be used as feedstocks for chemical processes that catalytically transform them into hydrocarbons or other products. The CSI process began with discovery of a previously unknown form of cellulose that has a structure analogous to that of the transition state during mercerization, and has properties dramatically different from other celluloses. It is much more accessible to modifying reagents whether chemical or enzymatic. Its porosity is illustrated by its accessibility to the large polyiodide anions responsible for the blue color obtained with starch. Accessibility to enzymes is such that prerequisite enzyme dosages are an order of magnitude less than required for the cellulose from which it is prepared. Its preparation is very simple; it requires treatment with a solution of NaOH in a co- solvent made up of water and an alcohol at ambient temperature. The key to stability is removal of the NaOH under conditions that prevent mercerization. After

enhancement of susceptibility to cellulases was demonstrated, possible inhibition by lignin was considered. The treatment was tested on corn stover, the plant biomass most often suggested as a source of lignocellulosic ethanol. It was observed that pretreatment overcomes inhibition of polysaccharide hydrolases by lignin while simultaneously opening up the structure of cell wall polysaccharide constituents to enhance accessibility to the polysaccharide hydrolases. As noted, the pretreatment involves use of NaOH in a co-solvent system made up of an alcohol and water and its application is entirely at ambient conditions of temperature and pressure. In the first stage, the pretreatment solution removes a significant fraction of the lignin attached to the cell wall polysaccharides. When treatment is completed, addition of commercial grade polysaccharide hydrolases results in sacharification that is more rapid than that resulting from corn stover subjected to other pretreatment technologies. In one instance the CSI process was compared with steam explosion. Corn stover from the same source was treated by the CSI and the results of hydrolysis contrasted with hydrolysis of corn stover that had been subjected to steam explosion. Application of the same enzyme cocktails under identical conditions resulted in conversion of 67% of the CSI treated sample while conversion of its steam exploded counterpart was only 35%. These are percentages of the pretreated samples.

Mel Maizirwan The objectives of this research are to identify the dominant factors in the extraction and in situ transesterification processes; and to determine the optimum state of particular combination of various factors using the ultrasonic method. This is an experimental laboratory study that was run using ultrasonic homogenizer Omni Ruptor 4000 for examining the effect of solvent type, solvent concentration, alga- solvent ratio, ultrasonic power, ultrasonic time, ultrasonic pulse and mixing toward yield. Box-Behnken Design of Response Surface Methodology by a quadratic model was used to evaluate the correlation of the parameters for analysing certain factors and combination of factors. As a result obtained in this study, it shows that power, time and pulse are the most significant factors that influence the yield. In the extraction process, the combinations of pulse-time give better result than power- pulse combination. Meanwhile, in the insitu transesterification, the power-time combinations give better result than power-pulse combination. Even though the optimum point has not been reached yet, in general the combination of power-time is categorized as the most influential combination to increase the yield. The experimental values are also shown that the coefficient of correlation (R2) is 0.97977 (for extraction) and 0.98743 (for in situ). The density of Nannochloropsis sp is 0.924 g/ml, saponication number is 114, 269 KOH/1 g oil. The percentage of FFA is 19.67% consisting of monounsaturated and polyunsaturated Octadecenoic acid (C18:1) 43.49%, Dedecanoic acid (C12) 16.30%, Hexadecanic acid (C16:0) 12.51%, Tetradecanoic acid (C14) 11.43%, Octadecadinoic acid (C18:2) 5.85% and Octadecanoic acid (C18:0) 5.62%.

Thrusday, October 11, 2012 - 2:00pm-3:30pm Innovative Approaches in Biochemical and Thermochemical Biomass Conversion Pathways

Moderator: Houston Brown, University of Alberta

Process Intensification to Reduce Cost in Biofuel Production Patricia Irving, InnovaTek

Renewable Solar Biobutanol Production Utilizing Carbon Dioxide as the Sole Feedstock Bruce Dannenberg, Phytonix Corporation

Process Simulation and Economic Evaluation for Bioethanol Production Implementing the Enzyme Recycling by Re-Adsorption Oscar Rosales Calderon, University of British Columbia

Mixed-valent First-row Metal Clusters for Catalytic Hydrodeoxygenation of Lignocellulosic Biomass Houston Brown, University of Alberta

Abstracts

Patricia Irving America‘s transportation sector relies almost exclusively (94%) on refined petroleum products, accounting for over 70% of the oil used. Nearly 9 million barrels of oil are required every day to fuel the 247 million vehicles that constitute the U.S. light-duty transportation fleet. Biomass from agricultural residuals or non- food sources is a near-term alternative to oil that can be used for sustainable production of liquid fuels. InnovaTek is developing technology to help reduce capital and operating costs of biofuel production to facilitate the replacement of transportation fossil fuels with renewable fuels. Catalytic conversion of biomass to a liquid transportation fuel requires a significant amount of hydrogen input. Fossil hydrocarbons are currently the main source for hydrogen production which makes it an expensive and insecure feedstock for sustainable energy production. Identifying renewable sources of hydrogen for biomass conversion is of great interest. Hemicelluloses are heterogeneous polysaccharides which typically account for up to 30% of lignocellulosic biomass materials. Our goal is to develop and demonstrate an approach for process intensification in biomass conversion to transportation fuels using catalytic micro-channel reactor technology for production of hydrogen from the normally unused hemicellulosic fraction of biomass. Compared to cellulose and lignin, hemicelluloses are generally much less stable and are prone to degradation during chemical or thermal treatments. For this reason, hemicellulose is considered an underutilized biomass component in most biomass conversion processes. Wheat straw, a predominant agricultural waste in the Pacific Northwest, is being used as the biomass feedstock in this project. The wheat straw cellulosic fraction will be hydrolyzed by cellulase enzyme to glucose. The glucose will then be hydrotreated with the hydrogen produced from the hemicellulose and then reformed to produce a liquid hydrocarbon fuel. Our engineered micro-structured catalytic reactor design achieves maximum activity of the catalyst, reduced reformer size, and hydrogen- rich reformate – all significant in intensifying the conversion of cellulose to bio-fuel

in an integrated process design. Two reaction steps will be integrated: pre- reforming and steam reforming reactions with highly active catalysts developed by InnovaTek. These reactions take place within different temperature zones of the system that are produced using a series of cross-flow heat exchangers that handle heating and cooling of the liquid and gas streams in the system. This represents an innovative approach to integrate biochemical and thermochemical biomass conversion pathways to maximize the utilization of all biomass components for bioenergy production and minimize the needs of external energy and hydrogen supply. The approach also represents an integrated and intensified lignocellulosic biomass conversion process that is greener and more efficient. The process eliminates the need for an external hydrogen supply which is typically derived from petroleum sources. The application of micro-channel reactors as a core conversion unit will reduce the size and capital cost for biomass conversion infrastructure as well as increase the efficiency of the conversion process through improved heat and mass transfer.

Bruce Dannenberg The sun is the most important source of sustainable energy, and biofuels and bio- chemicals can be used as renewable energy carriers when they derive their stored chemical energy from the sun. Biologically produced 1-butanol may become an alternative biofuel. Fermentative production utilizing cellulosic feedstocks requires additional production steps resulting in bottlenecks and high cost. This can be overcome by engineering cyanobacteria to secrete biobutanol via photosynthesis. Cyanobacteria can use light as the sole source of energy and carbon dioxide as the sole carbon-based feedstock. In contrast to eukaryotic green algae, genetic modifications in prokaryotic cyanobacteria is more easily achieved. As cyanobacteria do not have the native capability for producing butanol, heterologous pathways for butanol production have to be engineered into the cells. Atsumi et al. (2008b, 2009,2010) introduced a non-fermentative pathway into Synechococcus elongatus, resulting in isobutyraldehyde and isobutanol production directly from atmospheric CO2 as the carbon source demonstrating proof of concept for sustainable production of biobutanol. The process still has to be improved and optimized to be able to be used at commercial scale. Optimization requires the fine tuning of the production pathway as several enzymatic steps within multiple pathways options are involved. If these steps are misbalanced, intermediates can accumulate and disturb the whole cell metabolism, resulting in low yields. Besides systems biology approaches for the analysis of metabolic pathways, synthetic biology techniques will be utilized to optimizing metabolic pathway design. Phytonix Corporation owns the exclusive global license for the photobiological/photosynthetic production of butanol via engineered microbial cell factories utilizing carbon dioxide as the sole feedstock. The rapid development of synthetic biology opens up new possibilities for the construction of efficient biofuel producing cyanobacterial strains. The principal behind synthetic biology is to design and construct a cell with a desired function and metabolism. It is a molecular approach using standardized artificially synthesized genetic building blocks in a multipurpose cloning vector. The chemical DNA-synthesis allows the production and use of DNA sequences not found in nature. A fine tuning of new metabolic pathways is done by characterizing the single enzymatic steps in their kinetics and modeling the whole complex pathway

under consideration of the underlying microbial chassis on the computer. Phytonix‘s fuel organism development initiative focuses on the heterologous expression of codon optimised metabolic pathways for butanol synthesis in biosafe photoautotrophic cyanobacterial host organisms. The result will be novel, engineered, biosafety-guarded photosynthetic cyanobacterial cells producing biobutanol directly from solar energy, carbon dioxide and water. This is the objective of Phytonix‘s organism development team leader, Dr. Peter Linblad, at The Angstrom Laboratory of Uppsala University in Sweden.

Oscar Rosales Calderon In response to the increasing energy demand, research on alternative energy sources has been undertaken. Among the renewable sources of energy, lignocellulose is one of the most promising resources. As the technology to produce bioethanol from lignocelluloses is still under development, it is necessary to optimize the production process to decrease its cost. One of the highest operational costs in the process is due to the enzyme usage for the enzymatic hydrolysis of lignocellulose. As a result, different approaches have been developed to decrease its cost. Enzyme recycle in hydrolysis is one of these approaches, two methods have been proposed: recycle of enzymes by ultrafiltration and re-adsorption of enzymes into fresh substrate. An economic analysis on the ultrafiltration recycling method has been reported a few years ago. However, the economic analysis on the re-adsorption method has never been reported. As ultrafiltration is considered an expensive process due to its membrane cost, it is imperative to evaluate both methods and select the most advantageous. My research is focused on providing an economical analysis for the re-adsorption method. I monitored the concentration of each main compound and enzyme in the process. As a commercial enzyme preparation will be, pure enzyme preparations were used as standards to achieve a better approximation to the enzyme concentrations after hydrolysis. Recycling of the enzyme was done at different times for a range of enzymatic hydrolysis conditions. With the final mass balance, an improved hydrolysis continues process was designed and simulated. Finally, an economical analysis was presented and analyzed.

Houston Brown While the structural composition of lignin makes it an enticing potential source of liquid fuels and bulk chemicals, this biopolymer remains the most underutilized element of lignocellulosic biomass. Only about 2 % of lignin produced annually is used to source chemical products, while the vast majority burned on-site as a low- grade fuel for pulp and paper industries. Current refining technologies upgrade lignin to high-quality liquid fuels in two distinct steps. First, hydrogenolytic-lignin depolymerisation provides a mixture of functionalized phenols known as bio-oils. Second, bio-oils are catalytically refined to a mixture of etherified liquid fuels. The latter process deserves significant research investment, since catalytic reduction of such bio-oils provides a practical strategy for the commercial production of first- generation (unfunctionalized) bulk aromatics, including benzene, toluene, xylenes (BTX), along with various alkyl benzenes: building blocks of the petrochemical industry from an underappreciated renewable resource. Research and development related to the catalytic reduction of lignocellulose has focused on the ubiquitous

cobalt/nickel-promoted molybdenum hydrotreatment catalysts of the petroleum industry. The heterogeneous nature of these catalysts and the harsh reaction conditions required result in non-selective oxygen extrusion from these diversely functionalized biopolymers through competing mechanistic pathways, ultimately affording complicated mixtures of products at low conversions. Optimal catalysts for lignin refining will afford high conversions under mild conditions, thereby avoiding char formation and other competitive thermal processes. High selectivity for direct C-O bond hydrogenolysis is desired to suppress excessive hydrogen consumption as most heterogeneous catalysts hydrogenate aromatic rings prior to C-O bond scission. Moreover, the ideal biomass hydrotreatment catalyst will facilitate tandem depolymerisation and refining to convert biomass into higher value liquid fuels via non-destructive side-chain scission. Towards this goal, we have designed, synthesized, and fully-characterized discrete molecular catalysts that facilitate the hydrogenolysis of lignin-type C-O linkages under mild conditions. Our catalysts feature small clusters of cobalt centers (earth abundant, inexpensive, first-row transition metal) supported by thermally-robust anionic ligands, that can be tuned to optimize steric and electronic effects. We have shown these novel base metal hydrotreatment catalyst mediate selective C-O bond reduction under extremely mild conditions (110 oC, 1 atm H2). The catalytic hydrogenolysis of a range of aryl ethers and the ‗depolymerisation‘ of lignin model substrates have been investigated. Catalytic hydrodeoxygenation of refractory benzo- and dibenzofurans has also been explored.

Thrusday, October 11, 2012 - 4:00pm-5:30pm Advances in Enzymes used for Biomass Biorefining Moderator: Valdeir Arantes and Jack Saddler, University of British Columbia

Accessory Hemicellulases and Polysaccharide Oxidases for Hemicellulose Engineering Emma Master, University of British Columbia

Enzyme Synergy in Biomass Conversion as a Function of Pretreatment, Solids Loading, and Feedstock Stephen Decker, National Renewable Energy Laboratory (NREL)

Improving Cellulose Hydrolysis at High Substrate Concentrations and Low Protein Loadings to Achieve High Sugar Yields Jinguang Hu, University of British Columbia

Conceptualization of Biomass Sugar Platform Processes using a Flexible Technoeconomic Cost Model Brandon Emme, Novozymes

Abstracts Recent work on the biomass based, sugar platform, biorefinery process has shown how interrelated are: the source of biomass, pretreatment used and the nature of

the enzyme mixture required to achieve effective hydrolysis. This panel will describe various projects which have looked at the nature of the enzymes used to modify/hydrolyse the carbohydrate components of lignocellulosic substrates. The enzymes involved in both cellulose and hemicellulose will be discussed

Emma Master The aim of our research is to discover and engineer enzymes that can be used to synthesize high-value polymers and chemicals from hemicellulose, which remains an underutilized fraction of wood and agricultural fibre. Examples of bioproducts that can be synthesized from hemicellulose include packaging films, coatings for food preservation, and nutraceuticals. However, heterogeneity and loss in degree of polymerization are common limitations to broader application of this plant polymer. In an effort to overcome these limitations, we are isolating and engineering accessory hemicellulases that can reduce sample heterogeneity through selective removal of particular branching sugars. Glycoside hydrolases from families GH62 and GH115 are of particular interest, given the ability of enzymes from these families to function on polymeric substrates. In addition to glycoside hydrolases, we are engineering polysaccharide oxidases that oxidize specific hydroxyl groups on xylans and glucomannans to carboxylic acids. We are currently assessing how targeted introduction of carboxylic acids can facilitate site- specific chemical derivations, including co-polymerization. It is anticipated that biotechnologies that facilitate the synthesis of high-value bioproducts can improve the economics of biofuel production while generating new markets for forest and agricultural sectors.

Stephen Decker As the expanding biomass-to-fuels industry has branched out into new pretreatments and feedstocks, the role of activity-tailored enzyme hydrolysis has become even more critical for increasing yields and reducing costs. We have examined the synergism of xylanases and accessory enzymes with cellulases acting on different feedstocks treated by various pretreatment severities, chemistries, and solids loadings. Acetyl xylan esterase, ferulic acid esterase, and endoxylanase all enhanced Cel7A activity on pretreated corn stover, with a strong synergistic effect when added in combination. b-D-xylosidase addition increased both cellulose and xylan conversion of corn stover pretreated by various methods. Combined xylanase activities enhanced cellulase activity on alkaline peroxide pretreated corn stover, with the magnitude of the enhancement increasing with decreasing lignin content. Arabinofuranosidase activity also enhanced conversion of biomass in conjunction with cellulases and xylanases. These types of studies have demonstrated that tailoring the activities in enzyme cocktails to specific feedstocks and pretreatment conditions can reduce the production costs of lignocellulosic biofuels.

Jinguang Hu The rapidly evolving ―sugar platform‖ technologies are currently based on sugars derived from sugar cane/beet, and starch crops such as corn and wheat. Despite being the largest potential source of sugars in the world, the natural resistance of lignocellulosic substrates (such as agricultural and forestry residues) to biological deconstruction has make them remarkably difficult to cost-effectively process.

Significant advances in key unit operations in the overall biomass-to-sugars process, particularly the enzymatic hydrolysis step, are still needed to obtain good sugar yields while using low protein loadings. Our work on various types of lignocellulosic feedstocks has tried to optimizing the pretreatment step to better ―open up‖ the lignocellulosic materials. We have also tried to improve the enzyme performance by ―mixing and matching‖ enzymes with specific functionality (to create better enzyme mixtures that breakdown the polymeric sugars faster and more efficiently) and by assessing various enzyme recycling strategies to recover and reuse the enzymes for multiple rounds of hydrolysis. The possible role of key biomass-active proteins and their various interactions during hydrolysis will be described, as it relates to increasing sugar yields and reducing the overall enzyme loading required to achieve effective cellulose hydrolysis.

Brandon Emme Appropriate integration of process development steps in the biomass to sugars processing is a challenge. Building a fully flexible pilot plant to allow for multiple process configurations is cost prohibitive for most technology developers. For lack of fully integrated data, technoeconomic models can be employed to integrate smaller scale or individual unit operation data into the context of a full process to determine the best operational configuration and production cost. Furthermore, these models can be used to compare competing process concepts, both for state of technology as well as at n-th state conditions. This talk will present the Novozymes 2G flexible cost model and illustrate how it can be used to compare several possible sugar platform process concepts. The latest small and large scale hydrolysis data using the new commercial cellulase enzyme Cellic CTec3 on several different feedstocks will be presented, as well as some insights into what the models can tell us about the likely short and long term impacts of enzyme development on the future cost of biomass sugars.

Friday, October 12, 2012 - 8:30am-10:00am Natural Products Isolation, Purification, and Activity Moderator: Ronald Cascone, Nexant

Advanced Purification Technologies For Your Bio-Based Chemicals John Bhatt, Novasep, Inc.

Development of Processes for the Conversion of Xylose to Xylitol Using High Formic Acid Containing Hydrolysates Marilyn G. Wiebe, VTT Technical Research Centre of Finland

SqualaMatrix: Radiation Protection from Purified Concentrated Shark Liver Extract Lawrence Raithaus, ViiviBio Products

Production of Bioactive Mycelium and Polysaccharides by Liquid Fermentation of the Chinese Caterpillar Fungus Cordyceps Sinensis

Jian-Yong WU, The Hong Kong Polytechnic University

Abstracts

John Bhatt Biomass is of extremely diverse nature. Transforming biomass through chemical or biochemical catalysts leads to mixtures of impure products: fermentation broths, biomass hydrolysates, and chemical reaction mixtures. On the other hand, bio- based chemicals must be very pure to enter the value chain and be transformed into end products!! This is the challenge that every company developing a bio- based product is facing. Novasep‘s advanced purification technologies can fill this gap! Novasep is specialist in solving purification challenges, from process development to industrial installations, and from laboratory equipment to turnkey plants. We provide process development and engineering services, equipment and contract manufacturing services, for the biopharma and industrial biotech industries. Our unique know-how in separation and purification technologies includes membrane filtration, chromatography, ion exchange, adsorption, electrodialysis, evaporation and crystallisation. We use our proprietary computer modelling tools to speed up your process development and optimization. Success stories that we may develop in the presentation: • Organic acids purification by Continuous Ion Exchange: succinic acid, gluconic acid • Polyols desalting and purification by industrial SSMB chromatography • Cellulosic sugars separation and purification by industrial SSMB chromatography: C6 and C5 sugars, separation of xylose and arabinose • Hydrolysis enzymes recovery by ceramic membrane filtration: highly preserved enzymatic activity recovered from cellulosic biomass hydrolysates • Fermentation broth clarification with ceramic membrane filtration These examples will show how Novasep integrates the purification operations with the upstream process, to maximize efficiency, minimize waste and improve the overall process economics.

Marilyn G. Wiebe The five carbon polyol xylitol, derived from D-xylose, is currently used primarily as a sweetener, particularly in products which affect dental hygiene such as chewing gum and pastilles. Other applications (e.g. in co-polymers) for xylitol are under development. Xylitol is a natural product, being found in some plants and as a by- product of D-xylose metabolism in many microorganisms. Currently xylitol is produced chemically by hydrogenation of pure D-xylose, but environmentally friendly, biotechnological production routes are considered desirable and various studies have considered the potential yields and production rates which could be attained, particularly using various yeast. Since D-xylose is derived from lignocellulosic biomass, a process for efficient xylitol conversion directly from biomass hydrolysates would be suitable for incorporation into a biorefinery in which six carbon sugars and lignin would be used for other applications. To be economically viable, bio-production requires robust strains with high xylitol yields and production rates in these hydrolysates. Most investigations of xylitol production from plant biomass hydrolysates have focussed on the use of hydrolysates obtained by steam explosion or acid pre-treatments. Both 5 and 6 carbon sugars remain in the pre-treated material and further treatment is required to separate them. In

contrast, organosolv pre-treatments, such as that developed at CIMV, France, generate C5-enriched hydrolysate fractions directly during the process. Formic and acetic acid concentrations in such fractions may be considerably higher than from other pre-treatment methods. Here we explore the possibilities of using high formic acid C5 hydrolysates in the production of xylitol and demonstrate that xylitol concentrations over 100 g/l can be achieved with minimal detoxification of the hydrolysate.

Lawrence Raithaus The long term effects of repeated or continued exposure to radiation in its many varied forms has come into increased public awareness in recent years with reports of accidents such as Fukushima nuclear reactors, Chernobyl and Three Mile Island creating so-called ―safe‖ zones within a perimeter at a distance deemed to be safe, but still yielding low levels of radiation on a persistent basis. While the levels of radiation are barely measurable, little is known of the long term effects of these low levels on humans. BioStim, Inc, a Hawai‗i based marine bio products research and development company (that has a web presence under the name ViiviBio Products....www.viivibio.com), has developed and tested SqualaMatrix™ a nutraceutical dietary supplement that is safe and non-toxic,derived from the livers of a non-threatened species of dogfish shark. SqualaMatrix has undergone a number of controlled experimental studies that indicate important levels of bioactivity. In animal studies, mice treated with SqualaMatrix and then exposed to lethal doses of radiation had significantly higher survival rates than untreated mice. The possible protection of mammalian cells against radiation therapy suggested by the testing of our product in mice suggests that it could be used as an adjunct to radiation therapy, as well as in other situations where protection or attenuation of exposure to radiation is desirable. This product therefore has significant application in all situations where protection from the effects of long term exposure to low dose radiation, as in areas surrounding the Fukushima nuclear disaster, or patients undergoing repeated radiation studies, CT scans or treatments. Recent reports in the literature indicate a sharp rise in the use of CT over the past 23 years and a group of international researchers report children undergoing multiple CT scan to be 3 times more likely to develop brain cancer and 4 times more likely to be diagnosed with leukemia as a similar group who opted out of the scans. While physicians are being prompted to re-evaluate the apparent overuse of scans, a natural safe and edible shark-derived radiation protection product would have particular appeal in all cases where persistent exposure or increased risks are factors to be considered. This would not only include children undergoing CT scans, but anyone concerned about the accumulative effects of any form of low dose radiation, including diagnostic X-rays, airport scanning, prolonged high-altitude exposure, or any residents living within the "safe" zone of a nuclear disaster, such as Fukushima, Chernobyl, or Three Mile Island, or any of the hundreds of nuclear power plants currently operational. The cold extraction process used in production of SqualaMatrix was developed and patented in 1997. In this process, the oil-soluble and water-soluble fractions of shark livers are separated to produce a high-quality bioactive alkyglycerol rich shark liver oil and SqualaMatrix, which is the lyophilized water soluble fraction in an

encapsulated powdered shelf stable non-toxic form. BioStim has produced SqualaMatrix from Squalus acanthus dogfish sharks caught in British Columbia by a company that manages its shark fishery as a renewable resource with Marine Stewardship Council certification that assures environmental protection of a sustainable supply for years to come.

Jian-Yong Wu Edible and medicinal fungi (mushrooms) provide an abundant and reliable source of nutraceutical and pharmaceutical products. Polysaccharides (PS) represent a major class of bioactive constituents of medicinal fungi which have remarkable antitumor and immunomodulatory activities. Several polysaccharides or polysaccharide- protein originated from mushrooms have become commercial drugs for immunotherapy or as adjuvant for chemotherapy. The Chinese caterpillar fungus Cordyceps sinensis (Berk.) Sacc. (renamed as Ophiocordyceps sinensis (Berkeley) G.H. Sung et al. more recently), or Cordyceps in brief, is one of the most famous and highly valued medicinal fungi from China. It has been used in China for several centuries, mainly as a general tonic to strengthen and improve lung and kidney functions, to restore health after prolonged sickness and to maintain overall body health. It has also attracted world-wide attention in recent years. Modern scientific studies have demonstrated several health-promoting and pharmacological activities such as antitumor, antioxidant, antiaging, anti-inflammation, antiatherosclerosis and antifatigue. The limited supply and high value of natural Cordyceps plus the increasing demand in recent years have motivated enormous interest in development and application of Cordyceps fungal fermentation processes. Cs-HK1 is a fungus isolated from a wild Cordyceps fruiting body in our lab and has been applied to mycelial fermentation for production of fungal biomass and polysaccharides (PS). The Cs-HK1 mycelial fermentation has been successfully scaled up to 1.0-10 ton pilot and industrial fermentors, yielding 20-25 g/l biomass and 4-5 g/l exopolysaccharides (EPS) in 5-6 days. The Cs-HK1 mycelial biomass and PS have shown several health promoting effects through biochemical, cell culture and animal tests including antitumor, immumomodulation, antioxidant, and anti-fatigue. This paper will show the physical characteristics of Cs-HK1 mycelial liquid fermentation in stirred-tank fermentors, the engineering problems in product separation, and the medicinal properties of the mycelial extracts and polysaccharides.