Hydrolysis of Cellulose by Aerobic and Anaerobic Microorganisms Biological Aspects

Cellulose and lignin are the main structural compounds of plants. Both substances are the most abundant biopolymers on earth. Cellulose fibers are formed of linear chains of 100-1400 glucose units linked together by p-1,4-glycosidic bonds. Inter-and intramolecular hydrogen bonds and van der Waals interactions arrange the highly organized fibrous regions (crystalline region), which alternate with less organized amorphous regions in the cellulose fibers. The fibers are embedded in a matrix of hemicelluloses, pectin, or lignin. The hemicelluloses consist mainly of xylans or glu-comannans, which have sidechains of acetyl, gluconuryl, or arabinofuranosyl units. To make cellulose fibers accessible to microorganisms, the hemicellulose, pectin, or lignin matrix must be degraded microbiologically or solubilized chemically. Cellulose degradation in the presence of oxygen in soil or in the absence of oxygen in the rumen of ruminants, in swamps, or in anaerobic digesters is the most important step in mineralization of decaying plant material. Cellulolytic organisms are found among aerobic soil fungi, e.g., within the genera Trichoderma and Phanaerochaete and in anaerobic rumen fungi, e.g. Neocallimastix and Piromyces, and among bacteria, e.g., within the genera Cellulomonas, Pseudomonas, and Thermomonospora (aerobic cellulose degraders) and Clostridium, Fibrobacter, Bacteroides, and Ruminococcus (anaerobic cellulose degraders). For more details on cellulolytic bacteria and the mechanism of cellulose cleavage, please see Coughlan and Mayer (1991).

Glycosyl hydrolases are involved in cellulose and hemicellulose degradation by cleaving glycosidic bonds between different carbohydrates and between carbohydrates and noncarbohydrates. Endo- and exocellulases - in some organisms organized in cellulosomes - must be excreted into the medium. Cellulases are complex biocatalysts and contain a catalytic site and a substrate-binding site. The presence of a noncatalytic substrate binding site permits tight attachment to the different forms of cellulose substrate and keeps the enzyme close to its cleaving sites. Substrate binding is reversible, which allows the enzyme to 'hike' along the fibers and obtain total solubilization. Many aerobic fungi and some bacteria excrete endoglucanases that hydrolyze the amorphous region of cellulose (degradation within the chain), whereas exoglucanases hydrolyze cellulose from the ends of the glucose chains. Cel-lobiose is cleaved off by cellobiohydrolases from the nonreducing ends in the amorphous region, and finally the crystalline region is also hydrolyzed.

Cleavage of cellobiose to glucose units by p-glucosidases is necessary to prevent cellobiose accumulation, which inhibits cellobiohydrolases (Be'guin and Aubert, 1994).

Anaerobic bacteria such as Clostridium thermocellum form a stable enzyme complex, the cellulosome, at the cell surface (Lamed and Bayer, 1988). Cellulosomes are active in degrading crystalline cellulose. The catalytic subunits of a cellulosome, endoglucanases and xylanases, cleave the cellulose fiber into fragments, which are simultaneously degraded further by p-glucosidases. Cellulosome-like proteins are found also in Ruminococcus sp. and Fibrobacter sp. cultures. The cell-bound enzymes are associated with the capsule or the outer membrane. Other specific adhesions or ligand formations with the cellulose can be facilitated by fimbrial connections, glycosylated epitopes of carbohydrate binding proteins, or the glycocalyx and carbohydrate binding modules (Krause et al., 2003). Some bacteria have not developed a mechanism to adhere to cellulose fibers, but excrete cellulases into the medium. Adsorption of bacteria onto cellulose fibers via cellulosomes offers the advantage of close contact with the substrate, which is hydrolyzed mainly to glucose, which is then taken up and metabolized. Small amounts of cellobiose must be present initially to induce cellulase expression. The contact of bacteria with the solid substrate surface keeps them close to cellobiose and thus keeps cellulase activities high. However, accumulation of hydrolysis products such as glucose repress cellu-lase activity.

In nature most cellulose is degraded aerobically. Only about 5%-10% is thought to be degraded anaerobically, which may be an underestimate. Since most ecosystems are rich in carbonaceous substances but deficient in nitrogen compounds, many cellulolytic bacteria can also fix dinitrogen. This is advantageous to them and to syntrophic or symbiotic organisms (Leschine, 1995). Other examples of mutual interactions between organisms of an ecosystem are interspecies hydrogen transfer between anaerobes (anaerobic food chain), transfer of growth factors (mycorhizzae, Kefir), and production of fermentable substrates for the partner organisms (bacterial interactions in the rumen).

Hydrolysis of biological structural components such as cellulose, lignin, and other structural or storage polymers (Table 1.2) is difficult. The limiting step of hydrol ysis seems to be liberation of the cleavage products. In contrast to the slow hydrolysis of celluloses, mainly due to lignin encrustation of naturally occurring celluloses, starch can be easily hydrolyzed. The branching and helical structure of starch facilitates hydrolysis (Warren, 1996). Whereas cellulose forms fibers with a large surface covered with lignin, starch forms grains with an unfavorable surface-to-volume ratio for enzymatic cleavage. Thus, although amylases may be present in high concentrations, the hydrolysis rate is limited by the limited access of the enzymes to the substrate.

Whereas cellulose and starch are biodegradable, other carbohydrate-derived cellular compounds are not biodegradable and - after reaction with proteins - form hu-mic acid-like residues by the Maillard reaction.

Table 1.2 Polysaccharides and derivatives occurring in nature.

Compound

Bond

Unit

Occurrence

Cellulose

ß-1,4-

glucose

plant cell wall

Chitin

ß-1,4-

N-acetyl glucosamine

fungal cell and insect wall exoskeleton

Murein

ß-1,4-

N-acetyl-glucosamine N-acetyl-muramic acid

bacterial cell wall

Chitosan

ß-1,4-

N-glucosamine, substituted with acetyl residues

fungal and insect materials

Mannans

ß-1,4-

mannose

plant material

Xylans

ß-1,4-

xylopyranose, substituted with acetyl-arabinofuranoside residues

plant material

Starch

a-1,4-

glucose amylose (contains few a-1,6-branches)

amylopectin (contains many a-1,6-branches)

storage material in plant

Glycogen

a-1,4-

highly branched glucose with a-1,6 bonds

storage material in bacteria

Dextran

a-1,2-

glucose

storage material in yeasts

a-1,3-

glucose

exopolymer of bacteria

a-1,4-

glucose

a-1,6-

glucose

Laminarin

ß-1,3-

glucose

reserve material in algae

12 | 1 Bacterial Metabolism in Wastewater Treatment Systems 1.2.5

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