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.

Anaerobic detoxification of acetic acid in a thermophilic ethanologen.

BACKGROUND: The liberation of acetate from hemicellulose negatively impacts fermentations of cellulosic biomass, limiting the concentrations of substrate that can be effectively processed. Solvent-producing bacteria have the capacity to convert acetate to the less toxic product acetone, but to the best of our knowledge, this trait has not been transferred to an organism that produces ethanol at high yield. RESULTS: We have engineered a five-step metabolic pathway to convert acetic acid to acetone in the thermophilic anaerobe Thermoanaerobacterium saccharolyticum. The first steps of the pathway, a reversible conversion of acetate to acetyl-CoA, are catalyzed by the native T. saccharolyticum enzymes acetate kinase and phosphotransacetylase. ack and pta normally divert 30% of catabolic carbon flux to acetic acid; however, their re-introduction in evolved ethanologen strains resulted in virtually no acetic acid production. Conversion between acetic acid and acetyl-CoA remained active, as evidenced by rapid (13)C label transfer from exogenous acetate to ethanol. Genomic re-sequencing of six independently evolved ethanologen strains showed convergent mutations in the hfs hydrogenase gene cluster, which when transferred to wildtype T. saccharolyticum conferred a low acid production phenotype. Thus, the mutated hfs genes effectively separate acetic acid production and consumption from central metabolism, despite their intersecting at the common intermediate acetyl-CoA. To drive acetic acid conversion to a less inhibitory product, the enzymes thiolase, acetoacetate:acetate CoA-transferase, and acetoacetate decarboxylase were assembled in T. saccharolyticum with genes from thermophilic donor organisms that do not natively produce acetone. The resultant strain converted acetic acid to acetone and ethanol while maintaining a metabolic yield of 0.50 g ethanol per gram carbohydrate. CONCLUSIONS: Conversion of acetic acid to acetone results in improved ethanol productivity and titer and is an attractive low-cost solution to acetic acid inhibition.

Substrate-level phosphorylation is the primary source of energy conservation during anaerobic respiration of Shewanella oneidensis strain MR-1.

It is well established that respiratory organisms use proton motive force to produce ATP via F-type ATP synthase aerobically and that this process may reverse during anaerobiosis to produce proton motive force. Here, we show that Shewanella oneidensis strain MR-1, a nonfermentative, facultative anaerobe known to respire exogenous electron acceptors, generates ATP primarily from substrate-level phosphorylation under anaerobic conditions. Mutant strains lacking ackA (SO2915) and pta (SO2916), genes required for acetate production and a significant portion of substrate-level ATP produced anaerobically, were tested for growth. These mutant strains were unable to grow anaerobically with lactate and fumarate as the electron acceptor, consistent with substrate-level phosphorylation yielding a significant amount of ATP. Mutant strains lacking ackA and pta were also shown to grow slowly using N-acetylglucosamine as the carbon source and fumarate as the electron acceptor, consistent with some ATP generation deriving from the Entner-Doudoroff pathway with this substrate. A deletion strain lacking the sole F-type ATP synthase (SO4746 to SO4754) demonstrated enhanced growth on N-acetylglucosamine and a minor defect with lactate under anaerobic conditions. ATP synthase mutants grown anaerobically on lactate while expressing proteorhodopsin, a light-dependent proton pump, exhibited restored growth when exposed to light, consistent with a proton-pumping role for ATP synthase under anaerobic conditions. Although S. oneidensis requires external electron acceptors to balance redox reactions and is not fermentative, we find that substrate-level phosphorylation is its primary anaerobic energy conservation strategy. Phenotypic characterization of an ackA deletion in Shewanella sp. strain MR-4 and genomic analysis of other sequenced strains suggest that this strategy is a common feature of Shewanella.

Shewanella secretes flavins that mediate extracellular electron transfer.

Bacteria able to transfer electrons to metals are key agents in biogeochemical metal cycling, subsurface bioremediation, and corrosion processes. More recently, these bacteria have gained attention as the transfer of electrons from the cell surface to conductive materials can be used in multiple applications. In this work, we adapted electrochemical techniques to probe intact biofilms of Shewanella oneidensis MR-1 and Shewanella sp. MR-4 grown by using a poised electrode as an electron acceptor. This approach detected redox-active molecules within biofilms, which were involved in electron transfer to the electrode. A combination of methods identified a mixture of riboflavin and riboflavin-5'-phosphate in supernatants from biofilm reactors, with riboflavin representing the dominant component during sustained incubations (>72 h). Removal of riboflavin from biofilms reduced the rate of electron transfer to electrodes by >70%, consistent with a role as a soluble redox shuttle carrying electrons from the cell surface to external acceptors. Differential pulse voltammetry and cyclic voltammetry revealed a layer of flavins adsorbed to electrodes, even after soluble components were removed, especially in older biofilms. Riboflavin adsorbed quickly to other surfaces of geochemical interest, such as Fe(III) and Mn(IV) oxy(hydr)oxides. This in situ demonstration of flavin production, and sequestration at surfaces, requires the paradigm of soluble redox shuttles in geochemistry to be adjusted to include binding and modification of surfaces. Moreover, the known ability of isoalloxazine rings to act as metal chelators, along with their electron shuttling capacity, suggests that extracellular respiration of minerals by Shewanella is more complex than originally conceived.