Cellulosic biofuels promise a sustainable, renewable path to replacing the ~200 billion gallons of fuel consumed in the United States in 2008, but the key hurdle is how to economically convert cellulosic biomass into liquid fuels. Cellulose, the most abundant natural polymer on earth, is made of sugar molecules connected by difficult-to-break bonds. To make fuels from cellulosic biomass, it must be broken down into fermentable sugars, which is done industrially with biochemical processes that use inefficient and thus expensive proteins. Increasing their efficiency presents the best opportunity to decrease the cost of this critical cellulosic biofuel processing step, making sustainable, cellulosic biofuels cost competitive with fossil fuels. The aim of this Phase I SBIR is to demonstrate an ultra-fast biochemical platform to double the efficiency of biomass conversion to glucose. The effectiveness of this type of approach depends on two factors: how many variations can be tested, and how accurate or relevant the testing conditions are. The more variations that can be tested-that is, the higher the throughput-the more likely a newer, more efficient protein can be found. However, current high-throughput approaches do not test against the actual, solid biomass that will be eventually used in the production of biofuels, instead relying on easy-to-use, water soluble proxies . Our technique meets both challenges, to test against solid biomass components with throughput millions of times higher than competing technologies. OBJECTIVES: The objectives of this USDA-funded SBIR grant are to prove the viability of our high-throughput by improving the efficiency of a key step in the conversion of sustainable, cellulosic biomass to renewable fuels. While current processes can be used for producing fuels such as ethanol from cellulosic feedstocks such as switchgrass, the process is inefficient and thus costly. Much of this inefficiency lies in the process of converting biomass into fermentable sugars for microbial fuel production. Allopartis has developed a high throughput method for improving the efficiency of this process, which can operate on solid feedstocks. The ultimate objective is to prove, using pure feedstock components, that our technique can double the efficiency of biomass conversion to fermentable sugars. To reach this objective, the individual components of our development platform must first be proven. This includes protein selection and validation in our system as well as a number of bioconjugation strategies. Once complete, we will begin modifications of the proteins and their efficiencies at small scale. Finally, the entire platform will be run at full scale, which should enable benchtop screening rivaling the speed of a large robotic platform without the attendant cost and complexity. Our full-scale system will then be employed iteratively to improve protein efficiencies against our prepared biomass components. APPROACH: Our core platform technology aims to reduce the cost of converting cellulosic biomass to fermentable sugars for the production of sustainable biofuels. This step in the production process for biofuels will be made more efficient by increasing the efficiency of the proteins that perform the conversion reactions. Our proprietary technology allows us to leverage the two most important, but least compatible aspects of current protein improvement strategies: high-throughput screening capacity and the ability to screen for improvements directly against solid biomass components instead of specialized, soluble proxy molecules. In screening campaigns, it is most often the case that "you get what you screen for", meaning that unless tests are made directly against solid, insoluble biomass components, the resultant output proteins will only display improved efficiency against the proxy molecules, not against the industrially relevant materials. However, screening against solid-phase targets is usually relegated to very labor-intensive, low-throughput methods that are only scalable through expensive robotic systems. Our technology allows us to appropriately compartmentalize biomass solids in high density, volumetric arrays to be able to assay for improved activity with more than a million-fold more throughput than other techniques. The high density of our screening directly on solid particles of biomass components is the central, unique keystone of our technology and is key to the successful completion of the goals for this SBIR award. PROGRESS: 2009/06 TO 2010/01 OUTPUTS: The primary outputs of the work conducted under this Phase I SBIR award were the validation of the technical approach put forward in the application, the development of quantitative QC methods, and the successful implementation of the technology for improving the activity of biomass degrading enzymes. Most significantly, the enzyme improvement platform that had been proposed has been demonstrated from beginning to end, allowing the facile, high-throughput screening of improved enzyme variants that make this approach so potentially powerful. Several technical innovations made during the period of performance of this award that allowed our enzyme platform technology to function at a much higher efficiency and helped to establish clear metrics to assess the quality of the data at each of the many steps in the process. Additional second and third generation improvements offered similar augmentation culminating in tangible improvements in enzyme performance. There was no dissemination of the outputs from this award. PARTICIPANTS: Robert Blazej - PI and scientist specializing in genetics. 160+ hours Charles Emrich - scientist specializing in chemistry and protein science Nicholas Toriello - scientist specializing in high throughput screening TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period. IMPACT: 2009/06 TO 2010/01 The outcomes of the work performed under this Phase I SBIR award included changes in knowledge, changes in actions, and changes in conditions. The aim of the proposed research was to prove a technological approach to high throughput enzyme optimization for the purpose of improving the efficiency of the conversion of sustainable biomass to fermentable sugars. The proposed technological approach required several developments to reach its full potential and required the development of rigorous quality metrics to gauge the success of individual steps during operation. The realization of this necessity of process improvements and the subsequent creation of improved processes constitute the bulk of the change in knowledge, with the adjustments and improvements to our process constituting a change in action. Leveraging the enzyme enhancement platform technologies developed and proved during the course of this Phase I SBIR award makes it possible to directly affect conditions as the commercial application of this technology can help speed the adoption of sustainable biomass as a feedstock for renewable fuels and chemicals
