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United States Department of Agriculture

Agricultural Research Service

Research Project: OPTIMIZING THE BIOLOGY OF THE ANIMAL-PLANT INTERFACE FOR IMPROVED SUSTAINABILITY OF FORAGE-BASED ANIMAL ENTERPRISES

Location: Forage-Animal Production Research

Title: Investigtion of the metabolic inhibition observed in solid-substrate cultivation of Clostidium thermocellum on avicel

Authors
item Dharmagadda, Vidya -
item Nokes, Sue -
item Strobel, Herbert -
item FLYTHE, MICHAEL

Submitted to: Bioresource Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 23, 2010
Publication Date: April 1, 2010
Citation: Dharmagadda, V.S., Nokes, S.E., Strobel, H.J., Flythe, M.D. 2010. Investigtion of the metabolic inhibition observed in solid-substrate cultivation of Clostidium thermocellum on avicel. Bioresource Technology. 101:6039-6044.

Interpretive Summary: Clostridium thermocellum is a thermophilic, anaerobic bacterium with an efficient cellulase system. It has been proposed that C. thermocellum could be used commercially for direct microbial conversion of cellulosic biomass to ethanol by combining cellulase production, hydrolysis and fermentation in a single step. Traditionally, similar fermentations are conducted in liquid media, but solid substrate cultivation (SSC) is an alternative method with a number of advantages. When C. thermocellum was grown in SSC, the rates of acetic acid and ethanol production were exceptional. However, acetic acid accumulated, the pH declined, and metabolism was inhibited more rapidly than liquid fermentations. Metabolism and ethanol production were improved by periodically flushing the SSC with liquid media (FSSC). Flushing removed acetic acid, increased pH, and permitted greater conversion of cellulose to ethanol. The intracellular water of C. thermocellum decreased at low pH when acetic acid was present. SSC is a very hypotonic environment, and bacteria generally attempt to maintain intracellular water under such conditions. It appears that the responses to fermentation acids and low water content are incompatible, and FSSC is a promising technology to mitigate the problem.

Technical Abstract: Clostridium thermocellum exhibits premature metabolic inhibition during solid-substrate cultivation on Avicel, but the reason for the inhibition is not well understood. Inhibition was investigated by comparing three different cultivation techniques; submerged fermentation (SmF), solid substrate cultivation (SSC) and solid substrate cultivation with media replacement by periodic flushing (FSSC). Cellulose conversion extent and end product concentrations were measured for each cultivation method. The SmF converted approximately 65% of the Avicel in 240 hours (10 days), whereas conversion in SSC was less than 8% in the same period. However FSSC converted approximately 25 % and 47% of initial substrate after 240 hours; 45% and 71% of initial substrate after 25 days, with media replacement every 24 and 12 hours, respectively. The SSC experienced higher initial production rates for all fermentation products, but differences between culture techniques were less pronounced with prolonged incubation times. Relatively constant amounts of ethanol, acetate and lactate were produced during each flush of the FSSC. The results indicate that flushed solid substrate cultivation maintained favorable growth conditions for C. thermocellum even up to 25 days, most likely because fermentation acids were removed and the pH was closer to neutral. This conclusion was supported by the observation that acetate decreased the intracellular volume of C.thermocellum cell suspensions at pH values similar to those observed in SSC. It appears that the acids produced by fermentation exacerbate the already unfavorable osmotic condition of SSC.

Last Modified: 9/10/2014
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