Strain and bioprocess improvement of a thermophilic anaerobe for the production of ethanol from wood

Abstract

BackgroundThe 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.ResultsHere, 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.ConclusionsThis 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.

Development of a thermophilic coculture for corn fiber conversion to ethanol

“…As a potential coculture partner, Thermoanaerobacterium saccharolyticum can utilize xylan and other hemicellulose carbohydrates. It has also been engineered to produce ethanol at >90% of theoretical yield and up to 70 g/L titer 12 . However, its pH optimum is 6 while that of C. thermocellum is pH 7.…”

Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA , Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

“…Over the past decade, much work has been dedicated to engineering these organisms for increased ethanol production (1,2). In particular, Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at ϳ90% of the theoretical maximum yield and a titer of 70 g/liter (3,4). However, T. saccharolyticum cannot break down cellulose, which accounts for half to a third of plant biomass.…”

Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production

“…However, the maximum ethanol titer achieved by this engineered C. thermocellum strain (LL1319) was only 15 g/L, which is far short of the 70 g/L ethanol titer that engineered T. saccharolyticum (strain M1442) is capable of producing [5]. …”