Degradation and Conversion of Lignocelluloses : Chapter 11 · 2011-12-16 · DEGRADATION AND CONVERSION OF LIGNOCELLULOSES . 269 . glucose and mannose, with mannose dominating. They

CHAPTER 11

Degradation and Conversion of Lignocelluloses T. KENT KIRK

11.1 Introduction/page 266 11.2 Chemistry and Structure of Lignocelluloses/page 266 11.3 Microbes that Metabolize Lignocelluloses/page 271 11.4 Growth of Fungal Hyphae in Wood and Microscopy of

Degradation/page 273 11.5 Chemistry and Biochemistry of Degradation/page 276 11.6 Deterioration of Lignocelluloses/page 283 11.7 Lignocellulose Conversion/page 284 11.8 References/page 289

11.1 Introduction The lignocellulosic tissues of higher land plants are the major repository of photosynthetic energy and renewable organic matter. These vascular tissues have a complex molecular architecture that gives them the ability to support the largest living structures while performing the transport functions. The molecular structure of lignocelluloses also presents micro-organisms with some notable barriers to biodegradation, which evolution has breached primarily via the filamentous fungi.

Because of the domineering abundance of lignocelluloses, their decom-position is probably the single most important biodegradative event in the earth’s carbon cycle. From a more limited perspective, there are two contrasting economic aspects of lignocellulose degradation by fungi: destruction of wood products, standing timber and other crops; and bioconversion to useful products. This chapter presents an overview of the lignocellulose substrate, its fungal degradation, and the potentials and problems in using fungi for its conversion.

11.2 Chemistry and Structure of Lignocelluloses Chemical composition Lignocelluloses are made up of the structural polymers cellulose, lignin and hemicelluloses; cellulose fibrils are embedded in an amorphous matrix of lignin and hemicelluloses. In the native state lignocelluloses are also associated with various non-structural components.

The non-structural components make up a considerable percentage of the weight of certain lignocelluloses, such as agricultural residues and the wood of certain tropical trees. These components, particularly the organic

DEGRADATION AND CONVERSION OF LIGNOCELLULOSES 267

extractives (phenolics, terpenes, alkaloids, etc.) can significantly influence biodegradability (Scheffer & Cowling, 1966).

Of the structural components, cellulose is the most abundant, making up 35 to 45% of the dry weight of most woody tissues. The hemicelluloses and lignin usually make up 20-40% and 15-35% respectively (Table 11.1). Immature tissues may contain a considerable percentage of another structural component, pectin, which is diluted to quantitative insignificance during secondary thickening and lignification of the cell walls. Aquatic plants, including marine algae, and mature terrestrial plant tissues rich in non-lignified cells such as pith, leaf, and mesophyll contain proportionately more polysaccharides. These are largely digestible by polysaccharidases and are degraded by many more microbes - including anaerobic bacteria -than are fully lignified tissues (Akins & Burdick, 1981). Whereas a wider variety of potential biotechnological conversions therefore exists for such tissues, they make up a relatively small proportion of the terrestrial biomass. This chapter is concerned with lignocelluloses, which are not found in the lower plants, and which in the native state have limited polysaccharidase digestibility.

Table 11.1 Cellulose, hemicellulose, and lignin contents of representative lignocelluloses.

The chemical and physical structure of cellulose is discussed in Chapter 12, and is represented schematically in Fig. 11.1. Cellulose is a highly regular linear polymer of anhydro-D-glucopyranose units linked β(1→4). The long parallel cellulose chains in lignocellulosic tissues are strongly hydrogen-bonded and exist in aggregate as largely crystalline microfibrils; these are visible by electron microscopy (Côté, 1977).

Several properties of cellulose influence its microbial enzymatic degradation (Cowling & Brown, 1969; Cowling & Kirk, 1976): (i) the

268 DEGRADATION AND CONVERSION OF LIGNOCELLULOSES

capillary structure in relation to the size of cellulases; (ii) the degree of crystallinity; (iii) the dimensions of the crystalline portions of the microfibrils; and (iv) the nature of substances with which the cellulose is associated - particularly lignin.

The hemicelluloses, like cellulose, are polymers of anhydro-sugar units linked by glycosidic bonds. Unlike cellulose molecules, however, each hemicellulose molecule is comprised of more than one kind of sugar unit. Also, hemicellulose molecules are much shorter and are branched and substituted, and as a result they are usually non-crystalline. Substituted xylans (β(1→4)-linked xylopyranose units) are the main hemicelluoses of angiosperm lignocellulose; the major one is acetylated (up to ~ 35% of the xylose residues) and, in dicotyledonous plants, substituted with 4-0-methylglucuronic acid (Fig. 11.2a). Angiosperm lignocellulose also contains glucomannans, which are linear β(1→4)-linked co-polymers of


glucose and mannose, with mannose dominating. They usually make up less than 25% of the hemicellulose fraction. Related polysaccharides, acetylated galactoglucomannans (Fig. 11.2b), are the dominant hemicelluloses in gymnosperm tissues. The minor hemicelluloses are arabino-4-0-methyl-glucuronoxylans, which are similar to angiosperm xylan, but substituted with arabinose branches. For detailed treatment of hemicellulose structure the reader is referred to Timell (1967) and Towle & Whistler (1973).

The encrusting material of lignocelluloses, lignin, is distinct from cellulose and the hemicelluloses (Fig. 11.3). It is an aromatic polymer synthesized by the oxidative polymerization of three substituted cinnarnyl alcohols: p-coumaryl-, coniferyl- and sinapyl alcohols (4-hydroxy-, 4-hydroxy-3-methoxy-, and 4-hydroxy-3,5-dimethyoxycinnamyl alcohol, respectively). These are oxidized in the nascent cell wall by single electron abstraction to give free radical species that couple randomly with each other and, primarily, with the growing lignin polymer, which contains phenolic residues and is itself oxidized by single electron abstraction. The proportions of the three precursor alcohols differ between the major angiosperm and


gymnosperm lignocelluloses, and also among the various taxa, particularly within the angiosperms. Gymnosperm lignins are usually > 90% coniferyl alcohol-derived, whereas angiosperm lignins are frequently derived from nearly equal proportions of coniferyl and sinapyl alcohols. Most if not all lignins also contain small proportions of p-coumaryl alcohol-derived units. The polymerization process involves reactive intermediate structures (quinone methides) which can add nucleophiles, including hemicellulose hydroxyls and carboxyls. Thus, lignin-hemicellulose ether and ester linkages occur; a frequency of about one linkage per 36 phenylpropane units has been determined (Obst, 1982). Lignins of grasses and certain other angiosperms also contain ester-linked phenolic acid residues, which apparently are enzymatically connected to the precursor alcohols prior to polymerization. The reader is referred to Sarkanen & Ludwig (1971), Adler (1977), and Higuchi (1 982) for detailed treatment of lignin structure and formation.

Physical structure of lignocelluloses Figure 11.4 illustrates the ultrastructure of a mature wood cell. Lignified tissues of other lignocellulose cells have analogous structures. Approximately 80% of the weight of the cell substance, and the largest percentages of each component, are attributable to the secondary cell wall. The compound middle lamella is primarily lignin (70% to 90% of its weight), but this region contains only about 10-15% of the total lignin. In the secondary wall, the cellulose microfibrils are oriented nearly parallel to the long axis of the cell, and are probably arranged to form interrupted "sheets"or lamellae that are embedded in the inter-penetrating matrix of hemicelluloses and lignin.


11.3 Microbes that Metabolize Lignocelluloses Below 18-20% lignin content, lignocelluloses are degraded to increasing extents with decreasing lignin content by cellulases and hemicellulases, and consequently by the many bacteria and fungi that secrete these enzymes (Fig. 11.5). Thus fungi from each of the major classes and anaerobic as well as aerobic bacteria can partially degrade and metabolize such substrates. As the accessible polysaccharides are removed, the residual core, enriched in lignin, becomes progressively resistant to further degradation, unless the attacking microbe can also degrade the lignin. Forage fibres, for example, become resistant to further degradation on passage through the digestive system of ruminants, where anaerobic cellulose- and hemicellulose-degrading bacteria hydrolyse the accessible polysaccharides. ~ In ‘fully lignified’ tissues (lignin content > 20%) major degraders are the filamentous higher fungi, primarily the Basidiomycetes that cause the white-rot type of wood decay and the so-called litter-decomposing Basidiomycetes. These can metabolize lignin. Included are hundreds of species in diverse taxa. Gilbertson (1980) estimated that there are between 1600 and 1700 North American species of wood-rotting fungi, of which over 90% are white-rot fungi, these belonging to 36 families in three orders. On a world-wide basis there are certainly well over 2000 species. There is undoubtedly overlap between the litter-degraders and the true wood-decay fungi, but there are many species of the former that are poor wood degraders. The litter-degrading fungi have received only minimal research attention. In any event, the total number of Basidiomycetes that degrade lignin is quite substantial, reflecting the ecological importance of the group.


Another group of wood-decaying Basidiomycetes, causing brown rot of wood, also degrades fully lignified tissues, but without substantially depleting the lignin. These fungi, of which Gilbertson (1980) counted 106 species in North America, use an apparently unique mechanism for circumventing the lignin barrier to enzymatic cellulose degradation (Section 11.5).

Various Ascomycetes and Fungi Imperfecti cause the ‘soft-rot’ type of wood decay, recognized during the past 25 years. Just as white-rot fungi are related to litter-decomposing Basidiomycetes, so are the soft-rot fungi related to soil-inhabiting Ascomycetes and Imperfecti. It is probably a safe assumption that various ‘soil fungi’ also degrade wood and other lignocellulosics in a soft-rot manner (Duncan, 1960).

A number of species of Ascomycetes in the family Xylariaceae have been reported to cause typical white-rot of wood, e.g., Xylaria polymorpha (Fr.) Grev. and Hypoxylon deustum. ((Hoffm. ex Fr.) Grev. (= Ustulina deusta Hoffm. ex Fr.) Petrak). Over 45 years ago Campbell & Weirtelak (1935) reported that the chemical changes in naturally decayed wood associated with U. vulgaris Tul. basidiocarps are typical for white-rot. Decay by the group has received only slight attention since then. Merrill, French & Wood (1964) showed that several species of Xylariaceae could cause weight loss of wood, but decay was much slower than by Basidiomycetes included for comparison. The decay was favoured by high moisture and was limited to the outer surfaces of the wood blocks, and probably was of the soft-rot type; chemical analyses were not performed.

Slow degradation of lignin by certain soil bacteria has been demonstrated recently (for reviews see Crawford & Crawford, 1980; Crawford, 1981). The inability of most bacteria to thrust through the lignocellulosic substrate


probably in part limits the rate and extent of their attack. In accord with this is the fact that the most effective lignin-degrading bacteria are filamentous forms (Strepfomyces, Nocardia). Even so, there are no recognized bacterial wood decays.

Table 11.2 provides an overview of the groups of lignin-degrading microbes. Although the capacity to degrade lignin has evolved in diverse taxa, it seems clear that the filamentous fungi play the dominant role, no doubt in part because of the penetrating capacity of their hyphae.

Table 11.2 Lignin-degrading micro-organisms.

11.4 Growth of Hyphae in Wood and Microscopy of Degradation Hyphae of white- and brown-rot fungi are found in the cell lumens, whereas those of soft-rot fungi occur both there and within the secondary cell walls. In establishing themselves in wood all of these fungi apparently take the paths of least resistance and with the most available food, colonizing first the


rays, resin canals and vessels. Hyphae penetrate from cell to cell first through pits (the pectin membrane-covered openings between cells), and later via direct penetration of the cell walls - ‘bore hole’ formation. Wilcox (1970) has reviewed in detail the microscopical features of wood decays.

Early in decay by white- and brown-rot fungi, hyphae can be observed in virtually every cell where micro-environmental conditions are favourable.


Electron microscopy shows dissolution of cell wall substance caused by the secretion of lignocellulose-wall-degrading enzymes along the lateral hyphal surfaces as well as near the growing tips (Schmid & Liese, 1964; Eriksson et al., 1980) (Fig. 11.6).

Microscopical studies have shown that white-rot fungi produce a progressive and frequently rather uniform thinning of the secondary cell wall, beginning with the lumen surfaces (Fig. 11.7). Brown-rotted cells tend to shrink and collapse due to cellulose degradation (Cowling, 1961). Both microscopical and chemical investigations have shown that decomposition by the white-rot fungi takes place on exposed surfaces, whereas in brown rot the degrading agents completely penetrate the lignocellulose matrix in early stages of decay (section 11.5).

The mode of degradation of wood by soft-rot fungi is different from that of white- and brown-rot fungi. It is generally limited to the outer surfaces of wood. Soft rot is commonly found in wood under special environmental conditions of extreme wetness or frequent wet-dry cycles; for example, it is a common problem in cooling towers (Duncan, 1960). It seems likely that O2 limitation is responsible for the surface mode of attack, even though it is apparently a tolerance for low O2 that permits soft-rot fungi to decay water-soaked wood (Duncan, 1961). Decay progresses inwardly only as the outer surfaces are destroyed. Soft rot also differs from the other decays in that characteristic catenate, spindle-, or diamond-shaped cavities, oriented with the cellulose microfibrils, are formed, both on lumen surfaces and, around a penetrating hypha, inside the secondary wall (Fig. 11.8). Nilsson (1974b) has discussed the possible reasons for this pattern of cell wall dissolution.


Degradation from within the wall distinguishes soft rot from the white and brown rots microscopically. These cavities were not observed in wood decayed by members of the Xylariaceae studied by Merrill, French & Wood (1964), mentioned above.

11.5 Chemistry and Biochemistry of Degradation Changes in the gross chemical composition of wood during degradation by white-rot, brown-rot, and soft-rot fungi are illustrated in Fig. 11.9. Celluloses and hemicelluloses are metabolized by all three groups. Lignin is degraded only partially by brown-rot fungi, and more rapidly and extensively by white-rot than by soft-rot fungi.

Cellulose degradation Degradation of crystalline cellulose by white-rot fungi and various soil fungi and moulds results from the concerted, synergistic action of three types of


hydrolases: (i) endo- 1,4-β-glucanases, which cleave the cellulose chains randomly; (ii) exo-l,4-β-glucanases, which release cellobiose (together with glucose in some cases) from the non-reducing ends of cellulose; and (iii) β-glucosidases, which split cellobiose into two glucose units (see also Chapter 12).

Eriksson (1978) recently reviewed the mechanism of cellulose degradation by the white-rot fungus Sporotrichum pulverulentum Nov. (the anamorph of Phanerochaete chrysosporium Burds.). His extensive studies, as well as those by other workers with imperfect fungi (Reese, 1977; Gritzali & Brown, 1979; Wood & McCrae, 1979; Ryu & Mandels, 1980) support the hypothesis that endo- 1,4-β-glucanases (‘Cx’ enzymes) act randomly over the exposed surfaces of crystalline cellulose. The exposed non-reducing termini are then hydrolysed by exo-1,4-β-glucanases (cellobiohydrolases, ‘C1’ enzymes), with release of cellobiose. Cellobiose may be cleaved by a β-glucosidase, yielding glucose, or it may be oxidized to cellobionic acid and then cleaved. The endo-and exoglucanases actually act synergistically - perhaps as a loose complex (Wood & McCrae, 1979). The endo- and exoglucanases have molecular weights ranging up to about 75 000 (Cowling & Brown, 1969; Cowling, 1975), whereas the β-glucosidases are considerably larger (Ahlgren & Eriksson, 1967). The endoglucanases and probably the exoglucanases are repressed by high concentrations of monosaccharides (Eriksson, 1978).

Hydrolytic enzymes are probably not the only ones involved in cellulose degradation by white-rot fungi. Recent research has disclosed a hemoprotein, cellobiose oxidase, which oxidizes cellobiose to cellobiono-δ-lactone, with O2 serving as electron acceptor. The enzyme is responsible for the much more rapid hydrolysis of cellulose in O2 than in N2, presumably because it removes cellobiose, thus preventing the transglycosylation reactions and the inhibition of endoglucanase activity that occur when cellobiose builds up (Eriksson, 1978, 1981). Similar oxidizing activity is not found in the imperfect fungi examined (Wood & McCrae, 1979). Cello-biose is also oxidized to cellobiono-δ-lactone by cellobiose:quinone oxidoreductase; quinones serve as electron acceptors for this reaction (Westmark & Eriksson, 1973). A glucose oxidase also has been implicated in the overall process; it oxidizes glucose to gluconolactone with O2 as electron acceptor (Eriksson, 1978). These various oxidizing activities regulate the levels of glucose and cellobiose, and ultimately coordinate the rates of cellulose hydrolysis and metabolism of end products. Phenols also affect the levels of endoglucanase activity in white-rot fungi (Eriksson, 1978). Figure 11.10 summarizes the interconversions and regulatory interactions in cellulose hydrolysis by white-rot fungi as discussed by Eriksson (1978).

Degradation of cellulose in wood by brown-rot fungi is an unusual process. Extracellular enzyme preparations from these fungi possess only endo-l,4-,β-glucanase activity (Johansson, 1966; King, 1966; Keilich, Bailey & Liese, 1969; Nilsson, 1974a; Highley, 1975). Thus, culture filtrates from brown-rot fungi will not degrade crystalline cellulose. Furthermore, endoglucanase production by many brown-rot fungi is not repressed by monosaccharides; the enzyme is produced during growth on monosaccharides (Highley, 1973). Wolter, Highley & Evans (1980) recently demonstrated a large enzyme complex in culture filtrates of Poria placenta (Fr.) Cke. that hydrolyses carboxymethyl cellulose, xylans, glucomannans


and glycosides. The initial attack by brown-rot fungi on cellulose in wood, however, is not by cellulases. Cowling (1961) discovered that in early stages of wood decay by Poria monticola (Murr.) ( = P. placenta) virtually all of the cellulose is severely depolymerized. This discovery explained the high destructive effects of these fungi on wood, but it presented a biochemical puzzle, because only a small percentage of the cellulose in wood is accessible to cellulolytic enzymes – namely that exposed to the cell lumens and contiguous openings. Thus, enzymatic degradation must cause a gradual loss in cellulose integrity, as is seen in white rot (Cowling, 1961).

The depolymerizing agent of brown-rot fungi not only reaches the cellulose within the cell walls, it completely penetrates the crystalline microfibrils, clearly indicating that only very small molecules can be responsible. In discussing this, Cowling & Brown (1969) noted with perspicacity that Halliwell (1965) had described experiments on the depolymerization of cellulose under physiological conditions with H2O2 and ferrous salts (‘Fenton’s reagent’). Subsequent studies by Koenigs (1974) demonstrated that brown-rot fungi secrete H2O2 and that wood contains enough iron for a possible involvement of an Fe+2/H2O2 system in cellulose degradation. Highley (1977) has since shown that cellulose is in fact oxidized


during attack by the brown-rot fungus Poria placenta. Schmidt, Whitten & Nicholas (1981) reported that oxalic acid, which is secreted by brown-rot fungi (Shimazono, 1955; Tsao, 1963; Takao, 1965), reduces the Fe+3

normally present in wood to Fe+2, the active form in Fenton’s reagent. They then proposed the mechanism depicted in Fig. 11.11 for the depolymerization of cellulose by brown-rot fungi. Recently Cobb, Whitten & Nicholas (1982 personal communication) demonstrated degradation of 14C-cellulose by Gloeophylium trabeum through a membrane with a nominal molecular exclusion limit of 1000.

This initial oxidative depolymerization of cellulose opens up the wood cell wall structure so that cellulolytic and hemicellulolytic enzymes can reach their substrates despite the presence of lignin.

Because white-rot fungi also secrete H2O2 (Koenigs, 1972), it is interesting that they do not also oxidatively depolymerize cellulose. One reason might be that they possess oxalate decarboxylase, which decomposes oxalate, whereas brown-rot fungi apparently do not (Shimazono, 1955).

The cellulolytic system of soft-rot fungi has received relatively little attention. It is probably similar to that of the closely-related Ascomycetes and Fungi Imperfecti, which have been studied in detail (Reese, 1977; Gritzali & Brown, 1979; Wood & McCrae, 1979; Ryu & Mandels, 1980).

Hemicellulose degradation Dekker & Richards (1976) have comprehensively reviewed microbial hemicellulases. Wood-rotting fungi produce enzymes capable of hydrolysing a variety of β-(1→4)-1inked glycan (mannan and xylan) substrates, as well as various glycosides (Lyr, 1960; King, 1966; Ahlgren & Eriksson, 1967; Keilich, Bailey & Liese, 1969; Highley, 1976). Corresponding enzymes from


the three groups of decay organisms appear to be similar, and also similar to those from other micro-organisms (Keilich et al., 1969; Dekker & Richards, 1976).

Endoglycanases from white-, brown- and soft-rot fungi all apparently act randomly, producing dimeric and higher oligomeric products (Sørensen, 1952; King & Fuller, 1968; Dekker & Richards, 1976; Ishihara & Shimizu, 1980). Uronic acid-substituted oligosaccharides are produced from glucuronoxylan substrates (Sørensen, 1952; King & Fuller, 1968; Ishihara, Shimizu & Ishihara, 1978; Highley, 1976); enzymes catalysing hydrolysis of the xylose-uronic acid linkage have not been reported. A mannanase purified from a brown-rot fungus hydrolysed both mannose-(1→4)-glucose and glucose-(1→4)-mannose linkages (Ishihara & Shimizu, 1980). An enzyme complex with multiple glycan and glycoside hydrolase activity in the brown-rot fungus Poria placenta has recently been reported (Wolter, Highley & Evans, 1980). With the exception of this complex, which had a molecular weight of 185 000, the hemicellulases (endoglycanases) of wood-rotting fungi have molecular weights of 3-6 × 104. All have acidic pH optima. Glycosidases active on hemiceilulose-derived disaccharides are also produced by the wood-rotting fungi (Ahlgren & Eriksson, 1967), but hemicellulose oligosaccharide exohydrolases have not been reported.

Information regarding regulation of the synthesis of the hemicellulases is somewhat contradictory (Dekker & Richards, 1976). Regulation per se, however, has not been the subject of detailed study in lignocellulose-degrading fungi. Multiple hemicellulase activity is found in culture filtrates of both white- and brown-rot fungi after growth on a variety of substrates (Highley, 1976). Hemicellulase production by the two types of fungi is different, however, in that brown-rot fungi exhibit good hemicellulase activities during growth on simple sugars, whereas white-rot fungi do not (Eriksson & Goodell, 1974; Highley, 1976). Production of the enzymes on simple sugars, however, might be induced in response to hyphal wall constituents following substrate depletion, as is xylanase in Stereum sanguinolentum Fr. (Ahlgren & Eriksson, 1967). Regulation in Poria placenta of the enzyme complex having multiple activity (Wolter, Highley & Evans, 1980) will be interesting to elucidate. The hemicellulases of the soft-rot fungus Chaetomium globosum apparently are induced specifically by their substrates (Sørensen, 1952).

Lignin degradation Research on the fungal degradation of lignin has accelerated greatly in recent years. Some of the reactions comprising degradation have been elucidated, and the unusual biochemical and physiological features are beginning to be described. Several recent reviews (Ander & Eriksson, 1978; Amer & Drew, 1980; Crawford & Crawford, 1980; Kirk, 1981, 1982) and two books (Kirk, Higuchi & Chang, 1980; Crawford, 1981) provide comprehensive coverage and specific literature references.

Research with the white-rot fungi has shown that the process is oxidative, that the ligninolytic system is quite non-specific, that it is not induced by lignin, and that depolymerization is not an obligatory initial step. Lignin degradation, therefore, is distinct from cellulose and hemicellulose


degradation; indeed, it differs from the biodegradation of all other studied biopolymers. Prominent reactions of lignin polymer degradation are oxidations and oxidative cleavages in the propyl side chains, demethyl-ation of methoxyl groups, and even cleavages in aromatic rings (see reviews). Elucidation of the chemistry of degradation has been - and is being - pursued both by characterization of the partially degraded polymer and through studies of the metabolism of low molecular weight ‘dimeric’ model compounds representing substructures in the polymer. With the model compounds, the specific reactions that comprise degradation are rapidly being elucidated; Fig. 11.12 shows the fate of a phenylcoumaran substructure model in ligninolytic cultures of Phanerochaete chrysosporium (Nakatsubo et al., 1981). Models representing other important substructures have been similarly studied (e.g., Enoki, Goldsby & Gold, 1980). Unfortunately, cell-free activity has not yet been demonstrated, and this, of course, has been a serious impediment to progress on biochemical aspects.

The non-specificity of the ligninolytic system in the white-rot fungi is evidenced by the facts that: (i) lignin is degraded despite the heterogeneity of inter-unit linkages and the variety of neighbouring groups around those linkages; (ii) it is degraded even after substantial modification by chemical pulping and pulp-bleaching reactions; and (iii) ligninolytic cultures metabolize a wide variety of aromatic compounds (Kirk, 1981).

Physiological studies with P. chrysosporium have revealed several intriguing traits of lignin metabolism. The ligninolytic system (i.e. lignin→ CO2) is not induced by lignin, but appears constitutively as cultures enter the ‘secondary metabolic’ state. Its initiation is triggered by carbo-hydrate-, nitrogen- or sulphur limitation, but not by phosphorus limitation. Evidence suggests that regulation of secondary metabolism, including lignin degradation, is somehow connected with glutamate metabolism (Kirk, 1981).

Secondary metabolism, also referred to as ‘maintenance’ and ‘idiophasic’ metabolism, has been studied mainly because of the biosynthesis of ‘secondary’ metabolites, and not because of any previously recognized


special importance in biodegradative processes. The biosynthetic aspects have been discussed extensively in this series (Berry, 1975; Bu’Lock, 1975; Righelato, 1975; Turner, 1975; Towers, 1976; Wright & Vining, 1976; Martin & Demain, 1978). Why lignin degradation should be associated with the secondary state in P. chrysosporium is an interesting question for speculation (Amer & Drew, 1980; Zeikus, 1981; Kirk, 1982). The fact that lignin degradation is secondary metabolic means that primary growth on lignin does not occur for regulatory reasons. Although this could perhaps be overcome, evidence suggests that lignin degradation provides insufficient carbon and energy even for the secondary stage (Jeffries, Choi & Kirk, 1981). This limits the options for bioconversion of waste lignins and lignocellulosics (e.g. conversion to protein; section 11.7 below). Neverthe-less, understanding of lignin degradation as related to energy needs and to secondary metabolism is rudimentary; new potential applications of bio-ligninolytic systems will undoubtedly become apparent as knowledge becones more complete.

The enzymes of lignin degradation have not been identified. Many past studies focused on phenol-oxidizing enzymes such as laccase and peroxidase, but it is unlikely that this activity is important in structural degradation (Kirk, 1971, 1983).

Because the lignin polymer is attacked by an extracellular non-specific oxidizing agent(s), it is possible that enzymes may not be directly involved. Hall (1 980) suggested that ‘diffusible species’ derived from molecular oxygen, such as superoxide (O2 · ), may participate, and Zeikus (1981) simi-larly suggested ‘chemical agents.’ Koenigs (1972) speculated that H2O2 might in some way be involved. Recent investigations have shown that either catalase or superoxide dismutase, which destroy H2O2 and O2 · , inhibit lignin degradation by P. chrysosporium (B. D. Faison and T. K. Kirk, unpublished). Both H2O2 and O2 · can give rise to highly reactive species through interaction with transition metals (Fee, 1980).

Recent studies of the degradation of lignin substructure model compounds by P. chrysosporium have supported the concept that active oxygen species are involved. A side chain cleavage reaction in a ‘β-1’ compound has been shown to be non-stereoselective and to result in the incorporation of oxygen from O2 (Nakatsubo, Reid & Kirk, 1982). The fungal system responsible is labile and has not yet been isolated. Interestingly, it has been possible to mimic the fungal reaction with a Fenton system chelated (Fe+2 + H2O2) (T. K. Kirk, unpublished results). Further comparisons of this biomimetic system with cultures will determine whether Fenton-type chemistry is involved in lignin degradation. It should also be possible to stabilize and characterize the system responsible for ligninolytic reactions in cultures. Despite indications that active oxygen species are involved in ligninolysis, it is not yet known whether these are non-enzyme-associated, or enzymes such as cytochrome P450 oxygenases.

Further metabolism of low molecular weight products of the initial degradation of lignin is probably via more classical modes. Thus, vanillic acid, a prominent product of the fungal degradation of lignin (Chen, Chang & Kirk, 1982), is degraded enzymatically by P. chrysosporium (Buswell & Eriksson, 1979; Yajima et al., 1979).

Although brown-rot fungi are poor degraders of lignin (Fig. 11.8), they do


apparently possess the basic degradative machinery. The main effect they have on lignin is demethylation of aryl methoxyl groups (Kirk & Adler, 1970), although oxidative changes, including some cleavage of aromatic rings, occur (Kirk, 1975). Indeed, limited oxidation of aromatic and propyl side chain carbon, as well as methoxyl carbon to CO2 has been demonstrated (Kirk et al., 1975; Haider & Trojanowski, 1980). Extensive depolymerization apparently does not occur (Brown, Cowling & Falkehag, 1968; Kirk, Brown & Cowling, 1969), and it seems unlikely that the limited degradation is sufficient to open up the wood structure to polysaccharidases. As discussed above, these fungi apparently oxidatively depolymerize cellulose non-enzymatically as the initial degradative step, and this is what opens up the wood structure.

Analyses of soft-rotted wood have revealed limited depletion of lignin (Savory & Pinion, 1958, Levi & Preston, 1965; Eslyn, Kirk & Effland, 1975). That these fungi can oxidize lignin to CO2 was shown by Haider & Tro-janowski (1975, 1980) using 14C-lignins. Rates did not approach those seen with white-rot fungi, but optimization studies have not yet been conducted. The chemical and biochemical activities of the soft-rot fungi have received relatively little research attention.

13.6 Deterioration of Lignocelluloses Because of their abundance, lignocelluloses play a dominant role in the terrestrial carbon cycle. It follows from this and from the foregoing that the lignin-degrading filamentous fungi play a prominent, and probably pre-dominant, role in the biodegradative part of this all-important cycle. In terms of ultimate import on human affairs, this role of the fungi, of course, far outweighs both the damage they cause, and the few direct economic applications of them. As human activities become more complex and control over agriculture and forestry increases, so must attention to how these practices influence biodegradative processes. A case in point is the recent concern that increasingly close utilization of forest harvesting residues will upset the contribution that the degradative products of these residues makes to future forest health and growth (Harvey, Larsen & Jurgensen, 1979).

The importance of the filamentous fungi in deterioration of diverse materials has been treated by Eggins & Allsopp (1975) in Volume I of this series. Probably the most costly losses are due to the decay of wood in service. Because the use of wood as the major housing construction material is not likely to change, the fact that it decays must continue to be recognized by builders, and increased attention must be paid to decay prevention. The brown-rot fungi are by far the most important destroyers of wood in service, although both white-rot and soft-rot fungi also play significant roles (Scheffer & Verrall, 1973). Prevention of wood decay is best accomplished by designing structures to prevent moisture build-up. Where this is impossible, wood must be treated with fungitoxic chemicals such as penta-chlorophenol, creosote, chromated-copper-arsenate, etc. Further under-standing of the fungal degradative processes, particularly the nonenzymatic oxidative reactions, should lead to more sophisticated treatments than these. Rowell (1980) has recently renewed investigations into chemical deriva-tizations of wood to impart both decay resistance and dimensional stability.


Decay resistance results from increased hydrophobicity and the inability of hydrolytic enzymes to approach their derivatized substrates.

Many plant diseases involve deterioration of lignified tissues. Examples include root rots of trees caused by the white-rot fungi Armillaria mellea (Vahl) Quel., Rigidoporus lignosus (Klotzch.) Imaz. (= Fomes lignosus (Klotzch.) Bref.), Heterobasidium annosum (Fr.) Bref., and Poria Weirii Murr. Polyporus schweinitzii Fr. is one of the few brown-rot fungi that cause diseases, in this case a bole rot of conifers. Despite these and other examples, however, the proportion of lignocellulose-degrading fungi that attack living tissues (i.e. cause tree diseases) is quite low. The relevant biochemical peculiarities of those that do are not yet known.

Without attacking living tissues, some white-rot and brown-rot fungi are able to decay the non-living interiors of trees. Such ‘heartrots’ are by far the most serious cause of volume loss in mature forests, even more serious than fire. The unique biochemical features that enable a relatively small percen-tage of wood decay fungi to cause heartrots are not known (Highley & Kirk, 1979); the success of these few species does not appear to be due to an unusual ability to degrade wood at low O2 and/or high CO2 concentrations (T. L. Highley, S. S. Bar-Lev, T. K. Kirk & M. J. Larsen, unpublished).

Lignification of plant tissues imparts resistance to deterioration by most micro-organisms, and appears to be an important mechanism of active as well as passive resistance to plant pathogens. This, however, is another area requiring considerably more research attention (Vance, Kirk & Sherwood, 1980).

11.7 Lignocellulose Conversion As pointed out above, fully lignified tissues are degraded only by microbes that are able to degrade lignin, with the exception of brown-rot fungi. Brown-rot fungi have not been studied as agents for direct conversion of lignified tissues, research into their activities having been limited to wood decay mechanisms and prevention. Their ability to circumvent the lignin barrier makes them potential candidates for direct bioconversion of the carbohydrates in lignocellulosics, and deserving of investigation. Use of other non-ligninolytic microbes to process lignocelluloses requires chemical or physical disruption of the lignin barrier, or chemical or physical degradation of the tissues to fermentable products. Bioconversion of delignified cellulosics is discussed in Chapter 12. Fermentation of lignocellulose hydrolysates has been treated in recent reviews by Flickinger & Tsao (1978), Gong et al., (1982), and Hajny (1981). The following discussion deals with applications and potential applications of ligninolytic fungi.

Extant and potential uses of fungi for lignocellulose conversion can be divided into five categories: (i) conversion into food or feed; (ii) manufacture of mechanical pulp; (iii) production of microbial chemical products; (iv) production of chemicals from lignin; and (v) treatment of lignocellulose-derived wastes.

Conversion into food or feed The idea of bioconverting indigestible lignocelluloses into ruminant-digestible materials has been studied to a limited extent in recent years,


although the idea is not new. Knoche, Cruz-Coke & Pacotet (1929) described ‘palo podrido, ’ which was said to be decayed wood of several angiosperm trees used as animal feed in southern Chile. Kühlwein (1963) later studied palo podrido, and reported that the responsible organisms were yeasts (Candida, Rhodotomla). It seems far more probable, however, that white-rot fungi were primarily responsible, and, in fact, basidiomycete hyphae were reported to be present. Zadražil (personal communication, 1981) has recently studied samples of palo podrido and concluded that white-rot fungi indeed are the causal agents. Palo podrido is no longer used.

Recent studies have aimed at feed production by controlled cultivation of ligninolytic fungi on lignocellulosic substrates. Substantial increases in crude protein have been reported for woods, barks and lignocellulosic wastes following cultivation of various white-rot fungi (Daugulis & Bone, 1978; Ek & Eriksson, 1980, Matteau & Bone, 1980) and a Chaetomium (Pamment et al., 1978). Similarly, increases in in vitro polysaccharidase digestibility of lignocelluloses has accompanied solid substrate incubation with white-rot fungi (Kirk & Moore, 1972; Detroy et al., 1980; Zadražil, 1980; Zadražil & Brunnert, 1980, 1981). Digestibility increases in several substrates on incubation with white-rot fungi are shown in Table 11.3.

Technical problems in using lignin-degrading fungi to produce feeds by solid substrate ‘fermentation’ appear to be three: (i) in scaling-up with the required careful control of humidity, aeration and temperature necessary for uniform treatment; (ii) in preventing contamination by unwanted microbes; and (iii) in the slowness of degradation of coniferous woods. The relatively low value of the resulting product exacerbates these drawbacks, but might be offset by development of low-technology processes, perhaps analogous to ensiling, for use on a small scale from easy-to-treat lignocellulosics such as Populus spp. and certain agricultural residues. If edible mushrooms are produced in conjunction with feed production, the attractiveness can change dramatically; indeed, the mushrooms become the objective.

Retail sales of edible mushrooms produced from wood and straw probably amount to well over a billion dollars (U.S.) per year (Delcaire, 1978). Throughout Asia, ligninolytic mushroom-forming fungi are used to convert low value lignocelluloses to valuable food crops. The dominant mushroom in Asia is ‘shiitake,’ or the forest mushroom (Lentinus edodes Sing.). Most shiitake is produced by inoculating oak bolts with wood plugs grown through with mycelium; such spawn is available commercially. The cultivation of shiitake has been described briefly by Hajjes & Nair (1975) in Volume I of this series, and is covered in more detail by Ito (1978). Several small commercial ventures are now underway in the United States for producing shiitake. The mushroom has been cultivated on wood chips, on corn cobs, and on cereal grass straws. Although L. edodes is quite effective in increasing the digestibility of its substrate (Table 11.3), utilization of the partially delignified residue as feed has apparently not been explored.

Several other commercial mushrooms are grown on untreated wood or other lignocelluloses (Table 11.4). Increased cultivation of these fungi in the West could enrich the variety of available foods and provide an economically attractive outlet for currently unused lignocellulosic residues and low-value trees (Kurtzman, 1979). The favourable impact on agriculture and forestry could be significant.


Manufacture of pulp The knowledge that white-rot fungi do not cause a rapid depolymerization of cellulose as they decay their substrates and that many, such as L. edodes, lower the lignin content of wood, has led to the suggestion that they might be used as pulping agents. Limited research in recent years indicates that the idea has merit and deserves serious research consideration. Complete or nearly complete delignification has not been the objective because of the time involved and the fact that the polysaccharides are also degraded in time. Instead, research has demonstrated than even slight delignification with fungi makes wood much easier to pulp mechanically. (Mechanical pulps account for about 9% of all U.S. pulp production.) Energy savings might be substantial, and some studies have indicated that mechanical pulp properties are improved.

Partial delignification without loss of cellulose has been demonstrated. Eriksson and co-workers (Ander & Eriksson, 1978) have developed ‘cellulase-less’ mutants of Polyporus adustus Willd. ex Fr. ( = Bjerkandera adusta (Willd. ex Fr.) Karst.), S. pulverulentum, and other white-rot fungi that no longer degrade cellulose; lignin and hemicelluloses are degraded. A United States patent has been obtained for the use of such mutants as a ‘method for producing cellulose pulp’ (Eriksson et al., 1976). Other research has shown that addition of glucose, as expected, forestalls polysaccharide degradation; over 30% of the lignin in red alder mechanical pulp was removed without polysaccharide depletion (Yank, Effland & Kirk, 1980).

The potential technical difficulties in biomechanical pulping would appear to be the same as those for feed production: (i) in scaling up with the required careful control of environmental conditions; (ii) in preventing contamina-tion; and (iii) in the slowness of fungal treatment of conifer woods (due to their high lignin content). However, the product and the energy savings are more valuable in pulping than in animal feed, and the extent of delignifica-tion required is much less. Ander & Eriksson (1978) have suggested that large silos might be used as fungal treatment chambers, with wood chips and inoculum being introduced at the top and treated chips removed at the bottom following suitable residence times. Treatment times of one week or less would be sufficient with many hardwoods.

Results of research on practical ‘biomechanical’ pulping can only be considered preliminary. Whereas the limited investigations have produced


ample support for reduction in pulping energy requirements by fungal treatment, the evidence for strength property maintenance or improvement is contradictory (Samuelsson et al., 1980; Ear-Lev, Chang & Kirk, 1982).

Production of microbial chemical products Lignocellulose-degrading fungi, like other groups, produce a wide variety of chemical products, but to the author’s knowledge none at present of commercial significance. Any potential of these fungi for chemicals production from lignocelluloses is diminished not only by the comparatively low yields inherent in any aerobic process, but also by further decreases in yield caused by the consumption of polysaccharides for lignin degradation.

Production of chemicals from lignin By-product lignins from the chemical pulping of wood are currently used in polymeric form to make a variety of commercial products, including dispersants, binders, oil well drilling muds, etc., and via chemical degradation to make vanillin, dimethyl sulphoxide, and a few other chemicals (Goheen, 1971; Hoyt & Guheen, 1971; Glasser, 1981). Only minor portions of the by-product lignins, however, are converted to commercial products, most of the lignin being burned for recovery of inorganic pulping reagents, with the return of only a small net yield of the potential energy because of the high water contents. Large quantities of ‘hydrolysis’ or ‘solvolysis’ by-product lignins are anticipated in the future as lignocelluloses are converted to fuels and chemicals via fermentation of the saccharide hydrolysates.

Biological approaches to utilizing by-product lignins are only beginning to be researched. Products that retain the polymeric structure and those of low molecular weight can be envisioned. Alteration of the functionality of by-product lignins by microbial action could conceivably provide polymers with increased value. For example, selective demethylation by brown-rot fungi (p. 283) could produce a polymer that might function well in phenol-formaldehyde adhesives. No published research has gone into this or other attempts at useful biomodification. Low molecular weight products are formed during the degradation of lignin by white-rot fungi (Chen, Chang & Kirk, 1981). Practical use of such products, however, would seem to be especially difficult because of their diversity, even if they could be produced in good yields and at attractive rates. Potential bioconversion of lignin to low molecular weight products has been discussed in greater detail by Crawford & Crawford (1980).

Waste treatment The ability of the wood-destroying fungi to degrade cellulose, hemicelluloses and lignin suggests that they might find utility in processes to treat wastes from lignocellulose-using industries. These fungi are not components of the microbial flora of current waste treatment systems.

Forss, Jokinen & Savolainen (1977) in Finland have developed a fungal process for converting the waste liquors from sulphite pulping of wood into protein-rich feed that performs well with poultry. BOD (Biochemical Oxygen Demand) reductions are substantial. The ‘Pekilo’ process utilizes


any of various moulds, but primarily Paecilomyces. Although Paecilomyces spp. can cause soft rot of wood and can degrade lignin (Duncan, 1960; Eslyn, Kirk & Effland, 1975), the lignin sulfonates in sulfite waste liquor are not significantly degraded in the Pekilo process. Simple sugars and oligosaccharides derived from hemicelluloses are the major substrates. The process has been used commercially in Finland.

Ek & Eriksson (1980) have developed a process for converting the waste water from fibreboard manufacture to a protein-rich feed using Sporotrichum pulverulentum. The waste consists of a mixture of sugars and oligosaccharides, low molecular weight acids, and suspended lignocellulosic particles. Reductions in BOD and COD (Chemical Oxygen Demand) during fungal growth are substantial. The product is a satisfactory ruminant feed, and has fairly high nutritional value for monogastric animals as well (Thomke, Rundgren & Eriksson, 1980).

The lignin-degrading ability of white-rot fungi has recently been employed in the laboratory to decolorize the effluent from the chlorine bleach plants of kraft pulp mills. The chromophoric material, derived from lignin via kraft pulping reactions and the oxidations and chlorinations of bleaching, is still polymeric and is resistant to current commercial biological treatment processes. White-rot fungi, however, readily decompose and decolorize this heavily modified waste lignin (Fukuzumi et al., 1977; Lundquist & Kirk, 1977). Decolorization of the effluent with P. chrysosporium has been optimized on a laboratory scale in small flasks, with primary sludge waste (largely cellulose) from pulp and paper mills serving as the required growth substrate (Eaton et al., 1982). A bench-scale thin-film reactor of a rotating disc design has given rates comparable to those achieved on a flask scale, and is currently being evaluated for continuous use. Contamination has not been a problem, even in continuous operation for over 30 days. The notable lack of specificity of the ligninolytic system in white-rot fungi suggests that such a treatment process might have broader applicability in industrial waste management. Waste treatment might well become the first directed use of a bioligninolytic system (Kirk & Chang, 1981).

11.8 References







Kirk, T. Kent. Degradation and conversion of lignocelluloses. In: Smith, J. E.; Berry, D. R.; Kristiansen, B. The filamentous fungi, v. 4, fungal technology. London: Edward Arnold; 1983: 266-295.

U.S. GOVERNMENT PRINTING OFFICE: 1 9 8 3 6 5 4 0 2 5 4 0 0 3

The Filamentous Fungi Volume 4 Fungal Technology

Edited by:

JOHN E. SMITH, D.Sc., F.I. Biol., F.R.S.E. DAVID R. BERRY, B.Sc., M.A., Ph.D. BJORN KRISTIANSEN, B.Sc., Ph.D. Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow

EDWARD ARNOLD

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