Bacteria are by far the most numerically abundant and taxonomically diverse microorgan­isms in bulk soil and are found both free-living and attached to the surface of soil particles. Many soil bacteria also interact with the roots of plants within the rhizosphere. Many of these bacteria have also been shown to possess hydrolytic enzymes that enable them to colonize and degrade plant biomass. They are widely distributed across phylogenetic groups that include diverse functional groupings. Many are native to soil, but have also been isolated from such diverse environments as hot springs, rumen contents, compost piles, termite guts as well as various other sites. The biochemical characteristics and physiology of biomass de­grading prokaryotes vary according to the environmental niche where they are found. Due, in part, to the diversity of prokaryotic microorganisms that produce cellulolytic enzymes and the promise of newer and better high-specific activity enzymes for industry, (Diversa is now called Verenium Corporation) these systems remain the focus of considerable study. Companies like Diversa Corporation in San Diego, California, collect DNA from hydrother­mal and other habitats worldwide, and then screen-extracted genomic DNA for the ability to produce useful enzymes for biomass conversion applications. Diversa’s business platform since 1995 has been to discover novel enzyme by creating gene libraries from bioprospecting globally diverse environments. Their ultimate goal is to develop enzymes capable of sur­viving at extreme process conditions for industry. Another company, Dyadic International, Inc, uses an integrated high-throughput technology platform targeted specifically at the discovery, expression, and modification of both prokaryotic and eukaryotic genes.

For microorganisms to degrade and metabolize the insoluble polysaccharides found in plant cell walls they must produce several extracellular hydrolytic enzymes. These enzymes can be secreted and act free in solution, or are cell associated. Adherence or colonization to insoluble substrates by microorganisms is common. Attachment can also be a factor in the control of enzyme expression and can be a precondition to the production of hydrolytic enzymes. Commonly, microorganisms act synergistically to enable efficient conversion of the substrate by the concerted action of several species. Synergism may involve the production of bacterial communication signals (quorum sensing) such as acylated homoserine lactones (AHLs) to establish these communities (20, 21). Quorum sensing has also been reported as a regulatory system for the control of extracellular enzyme synthesis in phytopathogenic bacteria, and in nitrogen-fixing rhizobia (22, 23). Bacterial communication signals may also allow the cells to regulate secretion of hydrolytic enzymes to reduce losses because of diffusion.

Because cellulose is a large insoluble polymer its use first requires binding of the enzymes either as a binary enzyme-substrate complex or as a ternary enzyme-substrate-microbe complex. Adhesion is most pronounced in the Gram-positive, thermophilic anaerobes such as Clostridium thermocellum or Clostridium cellulolyticum, which secretes an active and thermostable high molecular weight cellulase complexes (cellulosome) responsible for de­grading crystalline cellulose (24,25). Cellulosomes contain at least 30 polypeptides, most of the enzymes are endoglucanases (EC:3.2.1.4), but there are also some xylanases (EC:3.2.1.8), p-glucosidases (EC:3.2.1.21), and endo-p-1,3-1,4-glucanases (EC:3.2.1.73). Hydrolysis of cellulose by cellulosome producing organisms is dependent on adherence of the organism to the substrate through specific cellulose-binding proteins.

At least 46 unique bacterial producers of cellulases have been identified from many aerobic bacterial systems, including species within the genera Acidothermus, Bacillus, Cellulomonas, Cellvibrio, Cytophaga, Microbispora, Pseudomonas, and Thermobifida (26). Anaerobic bac­teria identified as biomass degraders include members of the genera Acetivibrio, Bacteroides, Clostridium, Micromonospora, and Ruminococcus. Bacteria that decay biomass have been isolated from temperature extremes that include psychrophilic, mesophilic, thermophilic, and hyperthermophilic conditions. The actinobacteria, for example, are widespread mi­crobial components of terrestrial and aquatic communities that have been demonstrated to play key roles in biomass turnover and nitrogen dynamics. Like fungi the actinobac­teria have the ability to penetrate lignocellulosic biomass which gives them the ability of secreting hydrolytic enzymes in confined cavities allowing for higher concentrations of free enzymes.

Recently sequenced genomes representative of the actinobacteria include organisms that are widespread in plant rhizospheres, plant tissues, and compost, including Streptomyces

spp., Thermobifida fusca, and Leifsonia xyli. A close relative to Acidothermus cellulolyticus and Frankia, Kineococcus radiotolerans is a drought-tolerant soil microbe that is also radiation tolerant, while Rubrobacter is a thermophilic genus containing radiotolerant species. The genomes of all these taxa contain cellulose — and other plant biomass-degrading metabolic pathways. The high-GC actinobacteria represent a large phylogenetic group and serve as a reservoir of organic carbon and nitrogen cycling capabilities. Many species of actinobacteria have adapted to diverse and often extreme environmental conditions. For example, Acti­nobacteria have been identified a the major group of cellulolytic organisms in ecosystems such as acid Sphagnum peat bogs (27).

The genomic sequence of A. cellulolyticus, a major cellulose degrader (see also Figure 15.2) originally isolated from the hot springs samples has recently been compiled and published by the Department of Energy Joint Genome Institute, accession NC-008578. A. cellulolyticus was originally isolated from submerged woody debris in a Yellowstone National Park hot spring, because of its efficient cellulose degradation (28). The organism was isolated using selective culturing methods of mud and decaying wood samples from thermal features in and around the Norris Geyser Basin region of the park. The microorganism was found to grow at 37-65°C with an optimum of 55°C. A type strain was selected and deposited in the American Type Culture Collection and is part of their National Park Service special collection (http://www. atcc. org/SpecialCollection/NPS. cfm). The genome contains a suite of glycosyl hydrolases responsible for cellulose degradation that have already been charac­terized as thermostable (29) as well as a xylanase and additional multifunctional hydrolytic activities.

The genome of the closely related thermophile, Thermobifida fusca, has also been com­pleted. The T. fusca genome shares a high number of gene homologs with A. cellulolyticus. Also of interest are members of the genus Frankia, which are filamentous actinobacteria that form nitrogen-fixing root nodules on woody trees and shrubs in a symbiosis termed “actinorhizal.” Genome sequence data are now available for three strains of Frankia, an eco­logically important nitrogen-fixing root nodule symbiont which is the closest phylogenetic relative to A. cellulolyticus. The sequenced Frankia genomes contain homologs for cellulases, xylanases, peroxidases, and aromatic degradative enzymes, as well as genes for secondary metabolite biosynthesis. Similar metabolic capabilities have been identified in the genomes of Streptomyces spp., suggesting that actinobacteria maybe a rich source of enzymes for bio­conversion. Members of the Frankia have been found to inhabit root nodules, rhizosphere (termed actinorhizal), and the soil as a saprophyte. Frankia — actinorhizal plant symbio­sis has been reported within a range of actinorhizal plants scattered among eight families consisting of over 200 species of angiosperms (30, 31).

Thermobifida fusca is a moderate thermophilic soil actinomycete (growth temperature ranging from minimal 30°C to maximal 55°C) that is a major degrader of plant cell walls in heated organic materials such as compost heaps, rotting hay, manure piles, or mushroom growth medium. It produces spores that can be allergenic and causes a condition called farmer’s lung. Its extracellular enzymes, including cellulases, have been studied extensively because of their thermostability, broad pH range (32-37), and high-specific activity. It degrades all major plant cell wall polymers except lignin and pectin and can grow on most simple sugars and carboxylic acids. Its genome has been sequenced and closed by the JGI; it has a size of 3.4 Mb and encodes seven known cellulase genes, all which have been expressed and characterized. Four of the cellulases are endocellulases (Cel5A, Cel5B, Cel6A, Cel9B), one is an exocellulase attacking the nonreducing ends of cellulose molecules (Cel6B), one is an exocellulase attacking the reducing ends of cellulose molecules (Cel48A), and one is a new class of cellulase, a processive endocellulase, which have now been found in several cellulolytic bacteria. All the cellulases contain a family 2 cellulose-binding module (CBM) attached by a linker peptide. There are many hemicellulose genes in T. fusca and five of them have been expressed and characterized: Xyl11A, Xyl10A, Xyl10B, XG74A, and Gn81A.

There have also been extensive studies of the regulation of T. fusca cellulases that have shown that their synthesis is induced by cellobiose and repressed by any good carbon source. All the six-cellulase genes encoding cellulases that are regulated by cellobiose contain at least one copy of a 14-base inverted repeat sequence upstream of their start codon. This sequence is the binding site for a lacI family regulatory protein, CelR. CelR has been expressed and characterized in E. coli. Its binding to DNA containing the 14 base sequence is inhibited by cellobiose. There are a number of copies of this 14 base sequence in T. fusca DNA, including one copy upstream of an operon that contains a p-glucosidase gene and genes for a binding protein transport system. The two exocellulases make up about 75% of the total cellulase produced by induced T. fusca and both exocellulase genes contain a second copy of the CelR-binding site upstream of their start codon. It is not clear if this regulatory site is responsible for their higher transcription rate. It is also not known if CelR plays a role in carbon source repression.

The genome of Cytophaga hutchinsonii, another eubacterial cellulose degrader, has also recently been sequenced. C. hutchinsonii, an aerobic Gram-negative bacterium commonly found in soil that has been shown to rapidly digests crystalline cellulose (38). Molecular analysis of cellulose degradation by C. hutchinsonii is now feasible, since techniques for the genetic manipulation of this organism have recently been developed (39). C. hutchin­sonii belongs to the Cytophaga-Flavobacterium branch of the eubacterial phylogenetic tree. Members of this group are widely distributed in many environments and have the ability to move rapidly over surfaces by gliding motility (40, 41). Gliding motility is thought to be important in allowing C. hutchinsonii to colonize its insoluble growth substrate. The genome of C. hutchinsonii was sequenced by the JGI and has been shown to differ from most known cellulolytic microorganisms in that none of its cellulase genes encode processive cellulases and few of them encode a CBM. These results provide strong evidence that C. hutchinsonii does not use either of the two known mechanisms for cellulose degradation: secretion of a set of individual synergistic cellulases containing CBMs or production of cellulosomes, since processive cellulases and CBMs are key for both mechanisms. Determining the detailed mechanism of cellulose degradation by C. hutchinsonii is a major unsolved problem in plant cell wall degradation and this pathway might provide new proteins that improve the rate of cellulose degradation.

Recently, an interesting group of marine microorganisms, the marine complex polysac­charide (CP)-degrading bacteria from the Microbulbifer, Teredinibacter, and Saccharopha — gus group have been described. This group of organisms produces an array of enzymes to degrade complex polysaccharides including cellulose (42). The genome of the marine bac — terium, Saccharophagus degradans, has been completed and reportedly contains more than 180 open reading frames that encode carbohydrate-degrading proteins (43). S. degradans is a pleomorphic, Gram-negative, aerobic member of the у — proteobacterium isolated from decaying salt marsh cord grass.

C. phytofermentans is a recently discovered member of the order Clostridiales. Its genome is presently being sequenced by the JGI. It was found in forest soil and has a broad growth substrate range and is able to rapidly degrade and ferment several plant polymers, including cellulose, pectin, starch, and xylan (44). A remarkable property of cellulose-fermenting C. phytofermentans cultures is the production of high concentrations of ethanol, typically more than twice the concentration produced by other cellulolytic clostridia, and hydrogen.