(Hemi)cellulolytic enzymes from anaerobic fungi: production, characteristics and application potential

A sexual life cycle has not been descnded to date However, the fact that the aerobic chytndiomycete fungus Calhmastix cyclopsis has an alternation of generations, with an asexual stage in copepods and a sexual phase in mosquito larvae [125], suggests that the anaerobic fungi may also have a sexual life stage, perhaps even in an alternative host Survival and transferThe observation that anaerobic fungi could be isolated from air-dried faeces of ruminant as well as non-ruminant herbivores [21,22, 77, 115], even up to 10 months after the onset of drying [77, 115] suggested the existence of resistant structures like cysts or resistant zoosporangia This hypothesis was supported by the observation of a putative resistant zoosporangium in a NeocaUimastix sp which had a melanized wall and contained nuclei with four times the DNA content of nuclei in zoospores [143] A resistant stage (resting zoosporangium) is included in the life cycle shown in Fig 1 From the above results it was concluded that faeces may serve as a major route for transfer of anaerobic fungi between herbivores Transfer is likely to occur by accidental contact with fresh or dried faeces, since the host animals are not coprophagic Alternatively, anaerobic fungi may be transferred by saliva, either as aerosols or, more likely, by direct contact of mother and offspring during grooming or through shared food [68, 77] MetabolismAnaerobic fungi are able to grow on a large range of mono , oligo-and polysaccharides with mixed-acid fermentation profiles similar to those of enterobacteiia such as Escherichia coli Hexose is converted to formate, acetate, lactate, succinate, ethanol, C0 2 and hydrogen by most strains [10, 69, 109, 110] Research on carbon and electron How pathways has so tar only been performed for Neocallimastn species [72, 83, 147] A model for glucose catabohsm as proposed for Neocallimasttx sp L2 [72] is shown in Fig 2 Glycolysis to phospho enolpyruvate (PEP) proceeds via the Embden Meyerhof-Parnass pathway The pathway from PEP to the fermentation products is fairly similar to the pathways in N patnciarum [147] and N jrontalis EB188 [83] However, oxaloacetate is produced from PEP in NeocaUimastix sp L2 and N patnciarum but from pyruvate in N fiontahs Furthermore, acetaldehyde is formed from acetyl-CoA in NeocaUimastix sp L2 and from pyruvate in the two other Neocallimastu species in all fungi the malate, derived from oxaloacetate, is transported into the hydrogcnosomes where enzymes generate energy by coupling its oxidation (to pyruvate) to electron transfer via NADH ferredoxin oxidoreductase Proton reduction is then achieved via ferredoxin and hydrogenase with the concomitant generation of H 2 In NeocaUimastix sp L2, pyruvate (from malate) is converted to acetyl CoA, also yielding electrons for H, production via hydrogenase In turn, acetyl-CoA is converted to acetate, the second hydrogenosomal end product Digestive activitiesAnaerobic fungi exhibit giowth on a variety of structural carbohydrates present in forage fiber, such as cellulose and hemicellulose [10,37, 70, 90, 104, 111,112] For that purpose the fungi excrete a wide range of hydrolytic enzymes to degrade plant polymers to readily utihzable carbon sources These enzymes include cellulases, hemicellulases, proteases, amylases, amyloglycosidases, feruloyl and p-coumaroyl esterase, various di sacchandases and Rembrandt Dijkerman, Huub J.