Switchgrass Leaching Requirements for Solid-State Fermentation by Acidothermus cellulolyticus Jean S. VanderGheynst, Farzaneh Rezaei, and Todd M. Dooley Dept. of Biological and Agricultural Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616 Alison M. Berry Dept. of Plant Sciences, University of California, Davis, One Shields Avenue, Davis, CA 95616 DOI 10.1002/btpr.366 Published online December 28, 2009 in Wiley InterScience (www.interscience.wiley.com). Growth of Acidothermus cellulolyticus in solid-state fermentation and its required growth conditions were investigated in this study. Extraction of switchgrass was required for growth. Under the experimental conditions, extraction ratio had the most significant effect on the growth of A. cellulolyticus. Heat treatment (in the form of autoclaving) of switch- grass did not have a significant effect on the growth rate; however, longer heat treatment times had a negative effect on the total growth. Moisture content adjustment had no effect on the release of inhibitors into extracts. Our results showed that leaching at a minimum 40:1 (gram water: gram dry biomass) removed inhibitory compound(s) from switchgrass. Upon extraction A. cellulolyticus colonized switchgrass in solid fermentation without exoge- nous addition of carbon and nitrogen sources. It is the first demonstration of growth of A. cellulolyticus in solid fermentation. V V C 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 622–626, 2010 Keywords: Acidothermus cellulolyticus, solid fermentation, bioconversion, leaching Introduction To use environmentally benign plant biomass for the pro- duction of biofuels and bio-based industrial organic chemi- cals, there is a need to pretreat the plant material. There are several different pretreatment approaches such as biological, chemical, physical and combinations of all three. 1,2 Acido- thermus cellulolyticus is an organism that has been investi- gated as a source of enzymes for biomass hydrolysis. Aerobic, thermophilic decomposition of compounds in ligno- cellulose including cellulose and hemicellulose by A. cellulo- lyticus has been confirmed experimentally. 3,4 Moreover, analysis of the recently sequenced genome of A. cellulolyti- cus demonstrates a much more extensive suite of lignocellu- lose-degrading enzymes than previously reported. 5 These features combined with the thermotolerance of A. cellulolyti- cus and its wide range of secreted enzymes make this orga- nism a promising candidate for industrial biotechnology. 6 Despite this potential, very little research has been done to examine the growth of Acidothermus cellulolyticus on com- plex lignocellulosic substrates such as those being considered as dedicated crops for liquid fuel production. One of the first steps toward the broad use of A. cellulolyticus in biological pretreatment is to understand the growth conditions for this bacterium on solid substrates. Here we identified conditions that support the growth of A. cellulolyticus on switchgrass in solid-state fermentation. Solid fermentation is fermentation in solid substrates in the absence of free water. It involves the growth of cells in a liquid film surrounding solid particles that comprise the solid fermentation matrix. In environments involving colonization of plant biomass such as switchgrass, compounds from bio- mass are leached into this liquid film. These compounds may support or inhibit the growth of cells. For this reason our experimental approach included screening studies that examined growth of A. cellulolyticus on both extracts from solids and extracted switchgrass solids. Methods Cultivation of A. cellulolyticus A. cellulolyticus inoculum was prepared in medium con- sisting of the following compounds in 1 L: 1 g NH 4 Cl, 1 g KH 2 PO 4 , 0.1 g MgSO 4 , 0.2 g CaCl 2 -2H 2 O, 1 g yeast extract, 0.5 g Cellobiose and 125 mmol NaPi at pH ¼ 5.2 (LPBM). Cultures were maintained in an orbital shaker at 55  C and 150 rpm. Culture optical density was measured at 590 nm (OD590) periodically to assess growth. Feedstock source and preparation of switchgrass extracts and washed solids Switchgrass material was harvested from 2-year-old plants of the cultivar ‘‘Kanlow’’ grown in a greenhouse with a min- imum 12-hr day length and 24  C temperature in 15.2 cm di- ameter cubic pots with daily watering and monthly application of 1 g per pot fertilizer. The feedstock was Correspondence concerning this article should be addressed to J. S. VanderGheynst at jsvander@ucdavis.edu. 622 V V C 2009 American Institute of Chemical Engineers