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Figure 12.4 Cellulose, a polymer of 4 linked glucose.

that cellulose exists primarily in the crystalline state (Figure 12.5) and is impermeable to water and it is, therefore, generally resistant to microbial attack.

Since cellulose is insoluble, the utilization of cellulose by fungi is viewed as a problem of converting the insoluble material into soluble sugars for transport within the mycelium and one that could be translated for converting cellulose into glucose. Scientists at the US Army Natick Laboratory in Natick, Massachusetts, envisaged a practical process for the production of glucose from cellulosic material (wood waste and wastepaper) and its fermentation into ethanol for use as biofuel (Mandels, 1975). In the 1970s, when the energy crunch became acute, a world-wide program started for screening and selecting fungi that secreted mixtures of exo- and endocellulase and (3-glucosidase in culture medium. Strains of Trichoderma were developed through mutagenesis and selection that, under optimized conditions, secreted up to around 30 grams of cellulase enzyme per liter of the

Figure 12.5 Diagram of cellulose showing crystalline and amorphous parts due to tight or loose packing of glucose chains. (From Meyer, B.S. and Anderson, J.B. (1950), An Introduction to Plant Physiology, Van Nostrand.)

culture medium, generating much euphoria over the possibility of developing an industrial process for converting cellulosic material into glucose and ethanol.

A few species of fungi were found that degraded cellulose completely under the growth conditions (Bhat and Maheshwari, 1987). Commonly, three types of hydrolytic enzymes are found in culture filtrates of cellulolytic fungi: (i) endoglucanase, or 1,4-p-D-glucan glucanohydrolase (molecular weight 25 to 50 kDa), which cleaves p-linkages at random, commonly in the amorphous part of cellulose; (ii) the exoglucanase or exo(1,4)p-glucanase (molecular weight 40 to 60 kDa), which releases cellobiose from the crystalline parts of cellulose; and (iii) the p-glucosidase (molecular weight 165 to 182 kDa), which releases cellobiose and short chain cellooligosaccharides. One of the most active fungi capable of utilizing cellulose rapidly and completely is a thermophilic fungus Sporotrichum thermophile (Figure 12.6). Its rate of cellulose utilization in shake-flask cultures is even faster than of Trichoderma viride, even though its secreted levels of endoglucanase and exoglucanase enzymes are lower. This raised the question whether endo- and exocellulases and p-glucosidase are the primary enzymes required for extracellular solubilization and utilization cellulose. In experiment, the growth of Sporotrichum thermophile in shake-flask cultures was stopped by the addition of cycloheximide, a protein synthesis inhibitor. Even though the endoglucanase, exoglucanase and p-glucosidase secreted prior to the inhibitor treatment were in the growth medium, the utilization (solubilization) of cellulose in the culture medium was interrupted. It was hypothesized that some crucial factor(s), replenished by growing of the mycelium, are involved in the utilization of cellulose, implying that cellulose degradation is a growth-associated process. The cellulose-grown culture filtrates of Sporotrichum thermophile had limited action under the in vitro conditions. Light microscopy showed that action of culture filtrate enzymes on Whatman filter paper (a substrate made from cotton, commonly used in experiments with cellulose enzymes) was initiated at naturally existing "disjointed regions" in the cell wall of

Figure 12.6 Photomicrograph of cellulose (filter paper) fiber treated in vitro with culture filtrate of a cellulolytic fungus Sporotrichum thermophile. Courtesy of M.K. Bhat.

elongated cells. The concentrated cell-free culture filtrates shortened the cellulose fibers and caused the splaying of the cell wall (Figure 12.7), although solubilization (i.e., production of reducing sugars or glucose) was limited.

A reason for the limited solubilization of cellulose under in vitro conditions is end-product inhibition of cellulase activity by glucose and cellobiose that, under the growth conditions, are constantly removed by the organism. The cell-free culture filtrates of some fungi, such as Trichoderma viride (Fungi Anamorphici), Humicola insolens (Fungi Anamorphici), Chaetomium thermophile (Ascomycotina) and Thermoascus aurantiacus (Ascomycotina), solubilize cellulose much more rapidly under aerobic conditions than under anaerobic conditions, indicating that an oxidative reaction is involved in breakdown of cellulose. An oxidative enzyme, cellobiose dehydrogenase, is present in some fungi, which in the presence of Fe2+ can generate reactive hydroxyl radicals, H2O2 + Fe2+ ^ HO^ + Fe3+. These radicals, in cooperation with cellulase, can depolymerize cellulose (Mansfield et al., 1997). H2O2 + Fe2+, known as the Fenton's reagent, is thought to loosen cell-wall structure and allow the diffusion of enzymes (Koenigs, 1974a,b; Kirk and Farrell, 1987). Although a fundamental process in nature, our knowledge of the mechanisms involved in cellulose decomposition is far from complete.

In culture conditions, approximately 50% of the carbon in the growth medium is used in the production of new biomass. Yet in nature, a vast quantity of litter is decomposed by fungi year after year without visible accumulation of mycelia. A distinctive feature of

Figure 12.7 Utilization of cellulose by Sporotrichum thermophile. Light microscopy of samples inform shake-flask cultures of Sporotrichum thermophile. (A) Initial appearance of fibers. (B) 16-hour culture showing germination of conidia and no perceptible changes in structure of cellulose fibers. (C) 30-hour culture showing extensive fragmentation of fibers. (D) Magnified view of fibers from 30-hour samples showing fragmentation at "weak spots" (î). (E) 48-hour culture showing extensive fungal growth and nearly complete utilization of cellulose. (F) 72-hour culture showing total utilization of cellulose fibers and beginning of sporulation (î). Courtesy of M.K. Bhat.

Figure 12.7 Utilization of cellulose by Sporotrichum thermophile. Light microscopy of samples inform shake-flask cultures of Sporotrichum thermophile. (A) Initial appearance of fibers. (B) 16-hour culture showing germination of conidia and no perceptible changes in structure of cellulose fibers. (C) 30-hour culture showing extensive fragmentation of fibers. (D) Magnified view of fibers from 30-hour samples showing fragmentation at "weak spots" (î). (E) 48-hour culture showing extensive fungal growth and nearly complete utilization of cellulose. (F) 72-hour culture showing total utilization of cellulose fibers and beginning of sporulation (î). Courtesy of M.K. Bhat.

Figure 12.8 Xylan. A polysaccharide composed of five carbon xylose sugar. C-l of one five-carbon sugar ring is linked to C-4 of another sugar ring by a P-glycosidic bond.

the development of litter fungi on cellulosic substrates is the precocious differentiation of hypha into spores and the autolysis of hyphal cells. This may explain how in nature vast quantities of cellulose is constantly recycled but without the accumulation of fungal mycelium. Cellulose has a strong effect on fungal reproduction and spores are incorporated into the soil—a significant content of "soil" is fungal spores. Polymeric substrate and its depolymerized forms affect the morphology of the fungus differently (Gaikwad and Maheshwari, 1994).

12.2.3 Hemicellulose Degradation

Hemicelluloses (non-cellulosic polysaccharides) are composed of (3,1-4 linked pentose with side chains consisting of sugars, sugar acids and acetyl esters that prevent the aggregation of chains as in cellulose. Hemicelluloses are hydrogen-bonded to cellulose and covalently-bonded to lignin. Commonly occurring hemicelluloses are pectin, xylan, ara-binan and rhamnogalactouranan. Next to cellulose, xylan (Figure 12.8) is the most abundant structural polysaccharide in wood cell walls. The complete hydrolysis of xylan requires cooperative action of the endoxylanases that randomly cleave the (-1,4-linked xylan backbone, the ( -xylosidases that hydrolyze xylooligomers and the different branchsplitting enzymes that remove the sugars attached to the backbone, e.g., glucuronic acid and arabinose. Xylanases of fungi show a multiplicity of forms with molecular mass ranging from 20 to 76 kDa. Analysis of the breakdown products of delignified cell walls using xylanase sequentially and simultaneously with cellulase show that a mixture of these enzymes is more effective in degrading cell wall than individual enzymes (Prabhu and Maheshwari, 1999).

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