The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading.
BACKGROUND
Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum.


RESULTS
Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel) initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids - predominantly L-valine and L-alanine - were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso- and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis.


CONCLUSIONS
C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.
Thayer School of Engineering, Dartmouth College, Hanover, NH 03755 USA ; BioEnergy Science Center, Oak Ridge, TN 37830 USA.
Evert K
EK
Holwerda
Mascoma Corporation, Lebanon, NH 03766 USA.
Philip G
PG
Thorne
Thayer School of Engineering, Dartmouth College, Hanover, NH 03755 USA ; BioEnergy Science Center, Oak Ridge, TN 37830 USA.
Daniel G
DG
Olson
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706 USA.
Daniel
D
Amador-Noguez
BioEnergy Science Center, Oak Ridge, TN 37830 USA ; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA.
Nancy L
NL
Engle
BioEnergy Science Center, Oak Ridge, TN 37830 USA ; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA.
Timothy J
TJ
Tschaplinski
Emeritus Industrial Biotechnology of Delft University of Technology, Delft, BC 2628 The Netherlands.
Johannes P
JP
van Dijken
Thayer School of Engineering, Dartmouth College, Hanover, NH 03755 USA ; BioEnergy Science Center, Oak Ridge, TN 37830 USA ; Mascoma Corporation, Lebanon, NH 03766 USA.
Lee R
LR
Lynd
2,3-butanediol; Amino acids; Cellulose fermentation; Clostridium thermocellum; Fusel alcohols; High solids; Isobutanol