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.

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Microbial cellulose utilization: fundamentals and biotechnology.

[This corrects the article on p. 100 in vol. 41.].

Energy conservation in chemotrophic anaerobic bacteria.

Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces. Despite the focus of modern microbiology research on pure culture, planktonic (free-swimming) bacteria, it is now widely recognized that most bacteria found in natural, clinical, and industrial settings persist in association with surfaces. Furthermore, these microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities. Numerous new experimental approaches and methodologies have been developed in order to explore metabolic interactions, phylogenetic groupings, and competition among members of the biofilm. To complement this broad view of biofilm ecology, individual organisms have been studied using molecular genetics in order to identify the genes required for biofilm development and to dissect the regulatory pathways that control the plankton-to-biofilm transition. These molecular genetic studies have led to the emergence of the concept of biofilm formation as a novel system for the study of bacterial development. The recent explosion in the field of biofilm research has led to exciting progress in the development of new technologies for studying these communities, advanced our understanding of the ecological significance of surface-attached bacteria, and provided new insights into the molecular genetic basis of biofilm development.

Microbial Biofilms: from Ecology to Molecular Genetics

Methanogens: reevaluation of a unique biological group.

Dissimilatory Fe(III) and Mn(IV) reduction.

The modification described utilizes a serum bottle closed with a butyl rubber stopper with a crimped metal seal for the growth of obligately anaerobic bacteria.

A serum bottle modification of the Hungate technique for cultivating obligate anaerobes

Two bacterial species were isolated from cultures of Methanobacillus omelianskii grown on media, containing ethanol as oxidizable substrate. One of these, the S organism, is a gram negative, motile, anaerobic rod which ferments ethanol with production of H2 and acetate but is inhibited by inclusion of 0.5 atm of H2 in the gas phase of the medium. The other organism is a gram variable, nonmotile, anaerobic rod which utilizes H2 but not ethanol for growth and methane formation. The results indicate that M. omelianskii maintained in ethanol media is actually a symbiotic association of the two species.

Methanobacillus omelianskii, a Symbiotic Association of Two Species of Bacteria*

A Theoretical Rumen Fermentation Balance

The influence of a H2-utilizing organism, Vibrio succinogenes, on the fermentation of limiting amounts of glucose by a carbohydrate-fermenting, H2-producing organism, Ruminococcus albus, was studied in continuous cultures. Growth of V. succinogenes depended on the production of H2 from glucose by R. albus. V. succinogenes used the H2 produced by R. albus to obtain energy for growth by reducing fumarate in the medium. Fumarate was not metabolized by R. albus alone. The only products detected in continuous cultures of R. albus alone were acetate, ethanol, and H2. CO2 was not measured. The only products detected in the mixed cultures were acetate and succinate. No free H2 was produced. No formate or any other volatile fatty acid, no succinate or other dicarboxylic acids, lactate, alcohols other than ethanol, pyruvate, or other keto-acids, acetoin, or diacetyl were detected in cultures of R. albus alone or in mixed cultures. The moles of product per 100 mol of glucose fermented were approximately 69 for ethanol, 74 for acetate, 237 for H2 for R. albus alone and 147 for acetate and 384 for succinate for the mixed culture. Each mole of succinate is equivalent to the production of 1 mol of H2 by R. albus. Thus, in the mixed cultures, ethanol production by R. albus is eliminated with a corresponding increase in acetate and H2 formation. The mixed-culture pattern is consistent with the hypothesis that nicotinamide adenine dinucleotide (reduced form), formed during glycolysis by R. albus, is reoxidized during ethanol formation when R. albus is grown alone and is reoxidized by conversion to nicotinamide adenine dinucleotide and H2 when R. albus is grown with V. succinogenes. The ecological significance of this interspecies transfer of H2 gas and the theoretical basis for its causing changes in fermentation patterns of R. albus are discussed.

Glucose Fermentation Products of Ruminococcus albus Grown in Continuous Culture with Vibrio succinogenes: Changes Caused by Interspecies Transfer of H2

Succinate is formed as an intermediate but not as a normal end product of the bovine rumen fermentation. However, numerous rumen bacteria are present, e.g., Bacteroides succinogenes, which produce succinate as a major product of carbohydrate fermentation. Selenomonas ruminantium, another rumen species, produces propionate via the succinate or randomizing pathway. These two organisms were co-cultured to determine if S. ruminantium could decarboxylate succinate produced by B. succinogenes. When energy sources used competitively by both species, i.e. glucose or cellobiose, were employed, no succinate was found in combined cultures, although a significant amount was expected from the numbers of Bacteroides present. The propionate production per S. ruminantium was significantly greater in combined than in single S. ruminantium cultures, which indicated that S. ruminantium was decarboxylating the succinate produced by B. succinogenes. S. ruminantium, which does not use cellulose, grew on cellulose when co-cultured with B. succinogenes. Succinate, but not propionate, was produced from cellulose by B. succinogenes alone. Propionate, but no succinate, accumulated when the combined cultures were grown on cellulose. These interspecies interactions are models for the rumen ecosystem interactions involved in the production of succinate by one species and its decarboxylation to propionate by a second species.

M. J. Wolin

Papers

A serum bottle modification of the Hungate technique for cultivating obligate anaerobes

Methanobacillus omelianskii, a Symbiotic Association of Two Species of Bacteria*

A Theoretical Rumen Fermentation Balance

Glucose Fermentation Products of Ruminococcus albus Grown in Continuous Culture with Vibrio succinogenes: Changes Caused by Interspecies Transfer of H2

Propionate Formation from Cellulose and Soluble Sugars by Combined Cultures of Bacteroides succinogenes and Selenomonas ruminantium