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Toxic Response of Clostridium Beijerinckii and Escherichia Coli to Lignin Hydrolysate-Related Compounds

Robert J. Mitchell1, Yun-Jie Lee1, Sun-Mi Lee1, Joo-Myung Ahn2, Dae Haeng Cho3, Youngsoon Um1, Yong Hwan Kim3, Man Bock Gu4, and Byoung-In Sang1. (1) Center for Environmental Technology Research, Korea Institute of Science and Technology (KIST), Seoul, South Korea, (2) Dept of Biotechnology, Korea University, Seoul, South Korea, (3) Dept. of Chemical Engineering, Kwangwoon University, Seoul, 139-701, South Korea, (4) Department of Biotechnology, Korea University, Seoul, South Korea

The toxicity of several compounds commonly found in lignin hydrolysates were evaluated individually by monitoring the production of acetone, butanol and ethanol (ABE) from Clostridium beijerinckii. The compounds selected for analysis included coumaric acid, syringaldehyde, furfural, ferulic acid and glucuronic acid. Assays with C. beijerinckii were performed using batch cultures while the growth and production of each of the ABE compounds were assessed over a 72-hour period. Very significant losses in butanol and acetone production were evident when the cultures were exposed to 0.5 mg/ml coumaric or ferulic acid, with a concurrent increase in the amount of acetic acid and butyric acid being produced. Likewise, exposure to syringaldehyde led a reduced butanol production while the addition of up to 1mg/ml of furfural or glucuronic acid had little or no effect on the metabolic output of this strain. To further evaluate, and potentially identify, the stresses C. beijerinckii experience when exposed to each of these compounds, tests were performed using a panel of bioluminescent strains of E. coli, each of which carry a plasmid harboring a specific promoter fusion with the luxCDABE genes. The strains included in this study are specific for and respond to oxidative damage (strains DK1 (katG) and DS1 (sodB)), membrane damage (strain DPD2540 (fabA)), protein damage (TV1061 (grpE) and DC1 (clpB)) and DNA damage (DPD2794 (recA)). Two other strains that were included were GC2 (lacZ) and DNT5 (nagR-nagAa), which are used to evaluate for the overall cytotoxicity and the presence of salicylic acid derivatives, respectively. Therefore, the stresses experienced when bacteria are exposed to each compound were characterized based upon the bioluminescent responses from these strains. Syringaldehyde clearly leads to a significant amount of membrane damage, with the bioluminescent response from strain DPD2540 being induced more than 400-fold when syringaldehyde was added to a concentration of 1mg/ml. Interestingly, glucuronic acid had little effect on C. beijerinckii but led to a significant response (about 200-fold with a concentration of 1.56 mg/ml) from DPD2540, suggesting that it does lead to some membrane damage. Furthermore, a slight but significant response (2.6-fold) was seen from strain DK1 when glucuronic acid was added at a concentration of 0.78 mg/ml, while higher concentrations led to a reduced response.