Strain and bioprocess improvement of a thermophilic anaerobe for the production of ethanol from wood.
BACKGROUND
The thermophilic, anaerobic bacterium Thermoanaerobacterium saccharolyticum digests hemicellulose and utilizes the major sugars present in biomass. It was previously engineered to produce ethanol at yields equivalent to yeast. While saccharolytic anaerobes have been long studied as potential biomass-fermenting organisms, development efforts for commercial ethanol production have not been reported.


RESULTS
Here, we describe the highest ethanol titers achieved from T. saccharolyticum during a 4-year project to develop it for industrial production of ethanol from pre-treated hardwood at 51-55 °C. We describe organism and bioprocess development efforts undertaken to improve ethanol production. The final strain M2886 was generated by removing genes for exopolysaccharide synthesis, the regulator perR, and re-introduction of phosphotransacetylase and acetate kinase into the methyglyoxal synthase gene. It was also subject to multiple rounds of adaptation and selection, resulting in mutations later identified by resequencing. The highest ethanol titer achieved was 70 g/L in batch culture with a mixture of cellobiose and maltodextrin. In a "mock hydrolysate" Simultaneous Saccharification and Fermentation (SSF) with Sigmacell-20, glucose, xylose, and acetic acid, an ethanol titer of 61 g/L was achieved, at 92 % of theoretical yield. Fungal cellulases were rapidly inactivated under these conditions and had to be supplemented with cellulosomes from C. thermocellum. Ethanol titers of 31 g/L were reached in a 100 L SSF of pre-treated hardwood and 26 g/L in a fermentation of a hardwood hemicellulose extract.


CONCLUSIONS
This study demonstrates that thermophilic anaerobes are capable of producing ethanol at high yield and at titers greater than 60 g/L from purified substrates, but additional work is needed to produce the same ethanol titers from pre-treated hardwood.
Mascoma Corporation, 67 Etna Rd, Lebanon, NH 03766 USA ; Thayer School of Engineering, Dartmouth College, 14 Engineering Drive, Hanover, NH 03755 USA.
Christopher D
CD
Herring
Mascoma Corporation, 67 Etna Rd, Lebanon, NH 03766 USA ; Verdezyne, Carlsbad, CA USA.
William R
WR
Kenealy
Mascoma Corporation, 67 Etna Rd, Lebanon, NH 03766 USA ; Novogy Inc, Cambridge, MA 02138 USA.
A
A
Joe Shaw
Mascoma Corporation, 67 Etna Rd, Lebanon, NH 03766 USA.
Sean F
SF
Covalla
Thayer School of Engineering, Dartmouth College, 14 Engineering Drive, Hanover, NH 03755 USA ; Bioenergy Science Center, Oak Ridge, TN USA.
Daniel G
DG
Olson
Mascoma Corporation, 67 Etna Rd, Lebanon, NH 03766 USA ; Genzyme, Cambridge, MA USA.
Jiayi
J
Zhang
Mascoma Corporation, 67 Etna Rd, Lebanon, NH 03766 USA ; Myriant Corporation, Quincy, MA USA.
W
W
Ryan Sillers
Mascoma Corporation, 67 Etna Rd, Lebanon, NH 03766 USA ; Novogy Inc, Cambridge, MA 02138 USA.
Vasiliki
V
Tsakraklides
Mascoma Corporation, 67 Etna Rd, Lebanon, NH 03766 USA.
John S
JS
Bardsley
Mascoma Corporation, 67 Etna Rd, Lebanon, NH 03766 USA.
Stephen R
SR
Rogers
Mascoma Corporation, 67 Etna Rd, Lebanon, NH 03766 USA.
Philip G
PG
Thorne
Mascoma Corporation, 67 Etna Rd, Lebanon, NH 03766 USA ; Washington, DC, USA.
Jessica P
JP
Johnson
Mascoma Corporation, 67 Etna Rd, Lebanon, NH 03766 USA.
Abigail
A
Foster
Mascoma Corporation, 67 Etna Rd, Lebanon, NH 03766 USA ; Nalco Champion, Houston, TX USA.
Indraneel D
ID
Shikhare
Bioenergy Science Center, Oak Ridge, TN USA ; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN USA.
Dawn M
DM
Klingeman
Bioenergy Science Center, Oak Ridge, TN USA ; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN USA.
Steven D
SD
Brown
Bioenergy Science Center, Oak Ridge, TN USA ; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN USA.
Brian H
BH
Davison
Mascoma Corporation, 67 Etna Rd, Lebanon, NH 03766 USA ; Thayer School of Engineering, Dartmouth College, 14 Engineering Drive, Hanover, NH 03755 USA ; Bioenergy Science Center, Oak Ridge, TN USA.
Lee R
LR
Lynd
Mascoma Corporation, 67 Etna Rd, Lebanon, NH 03766 USA ; Novozymes Inc, Davis, CA USA.
David A
DA
Hogsett
Bioprocess development; Cellulosic ethanol; Consolidated bioprocessing; Metabolic engineering; Organism development; Thermophilic bacteria