Microbial cellulose utilization: fundamentals and biotechnology

Abstract

Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

FIG. 1.

Schematic representation of the hydrolysis of amorphous and microcrystalline cellulose by noncomplexed (A) and complexed (B) cellulase systems. The solid squares represent reducing ends, and the open squares represent nonreducing ends. Amorphous and crystalline regions are indicated. Cellulose, enzymes, and hydrolytic products are not shown to scale.

FIG. 2.

General strategies of cellulose hydrolysis and utilization by aerobic (top) and anaerobic (bottom) bacteria. Cellulose, degradation products, and cellular features are not shown to scale. Some features of the alternate strategy type are utilized by one or more species. For example, the cellulase of the anaerobe Clostridium stercorarium is of the noncomplexed type, and members of the facultatively anaerobic Cellulomonas utilize an aerobic-type strategy for hydrolyzing cellulose but perform a mixed-acid fermentative catabolism of the hydrolytic products. Glycocalyces are the dominant means of adhesion among ruminal cellulolytic bacteria, but the importance of such structures in other anaerobic groups has not yet been systematically investigated. Refer to Fig. 1 for a more detailed comparison of complexed and noncomplexed systems at the enzymatic level.

FIG. 3.

Hypothesis for the role of oligomers during microbially and enzymatically mediated cellulose hydrolysis.

FIG. 4.

Effect of chain length (number of glucosyl units) on the ATP expenditure required per mole of glucosyl unit transported and on the potential ATP benefit per mole of glucosyl unit arising specifically from phosphorolytic activation via intracellular cellobiose phosphorylase and cellodextrin phosphorylase. The calculations assume a requirement for transport of 1 mol of ATP per mol of sugar regardless of chain length (see the text).

FIG. 5.

Relationship between growth temperature and maximum specific growth rate constant for aerobic (open circles) and anaerobic (solid circles) microorganisms grown on crystalline cellulose (r² = 0.90). Data are from references , , , , , , , and .

FIG. 6.

Summary of material flows and bioenergetics for process configurations featuring dedicated cellulase production and consolidated bioprocessing.

FIG. 7.

Catabolic reactions leading to the formation of various end products by ethanol-producing cellulolytic bacteria. 1, Lactate dehydrogenase; 2, pyruvate-ferredoxin oxidoreductase; 3, NADH-ferredoxin oxidoreductase; 4, hydrogenase; 5, phosphotransacetylase; 6, acetate kinase; 7, acetaldehyde dehydrogenase; 8, alcohol dehydrogenase; 9, glyceraldehyde-3-phosphate dehydrogenase. From references , , and .