Enteric Bacterial Catalysts for Fuel Ethanol Production

Abstract

The technology is available to produce fuel ethanol from renewable lignocellulosic biomass. The current challenge is to assemble the various process options into a commercial venture and begin the task of incremental improvement. Current process designs for lignocellulose are far more complex than grain to ethanol processes. This complexity results in part from the complexity of the substrate and the biological limitations of the catalyst. Our work at the University of Florida has focused primarily on the genetic engineering of Enteric bacteria using genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase. These two genes have been assembled into a portable ethanol production cassette, the PET operon, and integrated into the chromosome of Escherichia coli B for use with hemicellulose-derived syrups. The resulting strain, KO11, produces ethanol efficiently from all hexose and pentose sugars present in the polymers of hemicellulose. By using the same approach, we integrated the PET operon into the chromosome of Klebsiella oxytoca to produce strain P2 for use in the simultaneous saccharification and fermentation (SSF) process for cellulose. Strain P2 has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. Recently, the ability to produce and secrete high levels of endoglucanase has also been added to strain P2, further reducing the requirement for fungal cellulase. The general approach for the genetic engineering of new biocatalysts using the PET operon has been most successful with Enteric bacteria but was also extended to Gram positive bacteria, which have other useful traits for lignocellulose conversion. Many opportunities remain for further improvements in these biocatalysts as we proceed toward the development of single organisms that can be used for the efficient fermentation of both hemicellulosic and cellulosic substrates.

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status

“…The nature and availability of lignocellulosic feedstocks in different parts of the world depends on climate and other environmental factors, agricultural practice and technological development (Claassen et al 1999). Lignocellulose consists of an intermeshed and chemically bonded complex of three main polymers (Table 1): cellulose, hemicellulose, lignin and depending on the feedstock, pectin (Hahn-Hä gerdal et al 1991;Ingram et al 1999;Perez et al 2002;Zaldivar et al 2001). …”

“…Lignin, constituting 10-20% of biomass dry weight (Table 1), is an aromatic polymer containing phenolic residues such as trans-qcoumaryl alcohol, trans-q-coniferyl alcohol and trans-q-sinapyl alcohol (Hahn-Hä gerdal et al 1991;Ingram et al 1999;Klinke et al 2004;Perez et al 2002;Zaldivar et al 2001). While the lignin fraction does not contribute fermentable carbon sources, it is relevant as a potential source of microbial inhibitors (see below).…”

“…During this pre-treatment, hemicellulose is readily chemically hydrolyzed, while cellulose is essentially inert (Galbe and Zacchi 2002). Pretreatment of hemicellulose results in the release of monomeric sugars and in the production of a complex mixture of compounds that inhibit the bioconversion of these sugars (Ingram et al 1999;Klinke et al 2004;Mussatto and Roberto 2004). It has been proposed that the lignin fraction, which can be separated after pretreatment, may be used as an ash-free, solid fuel for the generation of heat and/or electricity (Galbe and Zacchi 2002).…”

“…This can, for instance, be avoided by simultaneous hydrolysis and fermentation. For comparison of separate or simultaneous hydrolysis and fermentation we refer to excellent papers by Lynd (1996), Galbe and Zacchi (2002) and Ingram et al (1999). In practice, it may not be necessary to completely hydrolyse polymers to monosaccharides.…”

“…Of the many weak organic acids that occur in lignocellulose hydrolysates, acetic acid is most common to hemicellulose hydrolysates (Ingram et al 1999), while hydroxycarboxylic acids like glycolic acid and lactic acid are also present when alkaline hydrolysis is performed (Sjö strö m 1991). Furthermore, formic acid is generated by degradation of sugars or lignin (Klinke et al 2002) with levulinic acid commonly arising from 5-HMF degradation (Palmqvist et al 1999).…”