358 Cellulase for commodity products from cellulosic biomass Michael E Himmel*?, Mark F Ruth*1 and Charles E Wymans A vital objective for second millennium biotechnology will be the enzymatic conversion of renewable cellulosic biomass to inexpensive fermentable sugars; new and more efficient fermentation processes will convert this biological ‘currency’ to a variety of commodity products. Although early strides will be made using process development and engineering disciplines, mid-term and longer advances must rely heavily on insight gained through protein and metabolic engineering technologies. These challenging goals can be met most effectively by the full integration of academic, federal, and industrial efforts in teams that develop and apply new fundamental knowledge to key cost drivers. Addresses *National Renewable Energy Laboratory, 1617 Cole Boulevard, Golden, CO 80401, USA fe-mail: Michael-Himmel@nrel.gov te-mail: Mark-Ruth@nrel.gov OThayer School of Engineering, Dartmouth College, 8000 Cummings Hall, Hanover, NH 03755, USA; e-mail: Charles.E.Wyman@Dartmouth.edu Current Opinion in Biotechnology 1999, 10:358-364 http://biomednet.com/elecref/0958166901000358 0 Elsevier Science Ltd ISSN 0958-l 669 Abbreviations CBH cellobiohydrolase FPU filter paper units NREL National Renewable Energy Laboratory SDM site-directed mutagenesis SSCF simultaneous saccharification and co-fermentation Introduction Plant biomass, which represents the cellulosic materials that compose the cell walls of all higher plants, is the most abundant source of fermentable carbohydrates in the world. When biologically converted to fuels, such as ethanol and various other low-value high-volume com- modity products, this vast resource can provide environmental, economic, and strategic benefits on a large scale, with some, such as reduced release of greenhouse gases, unparalleled by any other sustainable resource [l-3]. As an example, the cost of biomass ethanol production has been reduced dramatically over the past two decades, to the point where the fuel is now competitive for blending with gasoline to reduce greenhouse gas emissions, enhance octane, extend the gasoline supply, and promote more complete combustion [4,5], but further processing cost reduction opportunities have also been identified that would make it competitive as a pure fuel without subsidies [6]. Cellulase enzymes provide a key opportunity for achieving the tremendous benefits of biomass utilization in the long term because of the high glucose yields possi- ble and the opportunity to apply the modern tools of biotechnology to reduce costs. In this review, we present an estimate of the current cost of making cellulase to pro- duce ethanol and other commodity products from cellulosic biomass on a large scale, review recent develop- ments in cellulase technology in this context, and suggest opportunities to improve cellulases further. Cellulase production costs Overall process description A new analysisof enzymatic hydrolysis of cellulose to glu- cose for fermentation to ethanol by the National Renewable Energy Laboratory (NREL) provides a useful benchmark of the estimatedcost of cellulase enzymes in a large commodi- ty plant [7]. As pictured in Figure 1, the process revolves around dilute acid hydrolysis of hemicellulose in hardwood chips followed by enzymatic hydrolysis of the exposedcellu- lose to release glucoseat high yield. The latter operation is performed in the same vessel used to ferment the sugars from both cellulose and hemicelluloseto ethanol to reduce inhibition of enzymes by the sugars released. This combined process step, known assimultaneous saccharification and co- fermentation (SSCF), is carried out in a series of continuous anaerobic fermentors. Before fermentation, the hydrolyzate isconditioned to remove compounds formed (e.g. furfural) or released (e.g. acetic acid) during the hemicellulosepretreat- ment step that are inhibitory to the fermenting organism and cellulase. After a seven-day residence time, the fermentation broth is transferred to a distillation and dehydration unit for recovery of ethanol, while the residual solidsare burned to provide heat and electricity for the process; excess electricity is sold to the grid. A portion of the water from the beer col- umn (distillation) bottoms is recycled, and the methane released during cleanup of the water before recycling or dis- chargeis burned with the residualsolids. Cellulase production Cellulase is produced by a microorganism fed on a small portion (3-S%) of the conditioned hydrolyzate slurry in eleven l,OOO,OOO L (264,000 gallon) batch aerobic bioreac- tars. At any one time, eight bioreactors are in operation, another is being drained, one is being filled, and one is being cleaned and sterilized. Whole corn steep liquor and other trace nutrients are also added to the bioreactors and ammonia is used to control pH and provide fixed nitrogen to the organisms. Three parallel seedtrains, of three batch fermentors each, produce the 5% inocula for the process using the carbon source and nutrients described above. All main and inoculum vessels are aerated at 0.577 VVM (vol- ume of gas/volume of reactor/min) without oxygen supplementation, and the temperature is controlled by chilled water running through internal coils. Corn oil pre- vents excessive foaming in the fermentors. Cellulase yields above 1.50 filter paper units (FPU)/g cellu- lose and productivities above 55 FPU/L-hr were achieved