Cellulase, clostridia, and ethanol

Abstract

Biomass conversion to ethanol as a liquid fuel by the thermophilic and anaerobic clostridia offers a potential partial solution to the problem of the world's dependence on petroleum for energy. Coculture of a cellulolytic strain and a saccharolytic strain of Clostridium on agricultural resources, as well as on urban and industrial cellulosic wastes, is a promising approach to an alternate energy source from an economic viewpoint. This review discusses the need for such a process, the cellulases of clostridia, their presence in extracellular complexes or organelles (the cellulosomes), the binding of the cellulosomes to cellulose and to the cell surface, cellulase genetics, regulation of their synthesis, cocultures, ethanol tolerance, and metabolic pathway engineering for maximizing ethanol yield.

FIG. 1.

The clostridial coculture process in which C. thermocellum serves as the cellulase and hemicellulase producer. The hemicellulose-derived pentoses can be utilized by C. thermosaccharolyticum but not C. thermocellum. C. thermosaccharolyticum uses cellobiose faster and is a better ethanol producer. In addition to cellobiose, cellodextrins are also produced from cellulose and can be utilized directly.

FIG. 2.

A schematic diagram of the C. thermocellum cellulosome. The type I dockerins mediate attachment of the catalytic subunits to the scaffoldin, which is comprised of nine cohesins, a CBD, a hydrophilic domain of unknown function (X) and a type II dockerin. The scaffoldin likewise binds through its type II dockerin domain to a type II cohesin-containing protein on the bacterial cell surface that is thought to anchor the complex through a series of three SLH domains (see Fig. 3 for details). Reproduced from reference with permission of John Wiley & Sons.

FIG. 3.

Schematic drawing depicting cellulosome attachment to the cell surface through the type II dockerin-cohesin interaction with SLH domain-containing cell surface proteins, SdbA, Orf2p, and OlpB (22). On the other hand, OlpA, which contains a type I cohesin, is presumed to anchor an enzyme or protein containing a type I dockerin. Adapted from reference with permission of John Wiley & Sons.

FIG. 4.

Cellulase gene clusters found in C. thermocellum. The arrow indicates the transcription direction and the approximate size of the gene. GH, glycosyl hydrolase family.

FIG. 5.

Cellulase gene clusters found in mesophilic clostridia. The arrow indicates the transcription direction and the approximate size of the gene. The sequence of the C. josui cluster is incomplete, and additional genes of the cluster may exist.

FIG. 6.

Ethanol, lactate, and acetate fermentation by C. thermocellum. 1, enzymes of Embden-Meyerhof pathway; 2, lactate dehydrogenase; 3, pyruvate-ferredoxin oxidoreductase; 4, acetaldehyde dehydrogenase; 5, alcohol dehydrogenase; 6, phosphotransacetylase; 7, acetate kinase.