FEMS Microbiology Reviews46 (1987) 145-163 Published by Elsevier FER 00061

145

Lignocellulose-degrading actinomycetes
A.J. M c C a r t h y
Microbiologlv Department, Life Sciences Building, University of Liverpool, Liverpool L69 3BX, U.K.

Received 12 November 1986 Accepted 9 February 1987 Key words: Ecology; Lignin degradation; Cellulases; Xylanases; Bioconversion; Saccharification

1. S U M M A R Y Increasing interest in the exploitation of plant biomass as a renewable resource has provided an impetus for research on microbial degradation of lignocellulose. This has traditionally been concentrated on the fungi, but lignocellulose-degrading prokaryotes are beginning to receive more attention. Strain improvement by genetic manipulation, and large-scale cultivation are more easily achieved in prokaryotes, and the actinomycetes are no exception. In addition, their growth as branching hyphae is well adapted to the penetration and degradation of insoluble substrates such as lignocellulose. Actinomycetes form an important part of the microbial community responsible for nutrient recycling in natural substrates. Their specific contribution to lignocellulose degradation in the environment has received only limited attention but their importance can be inferred from the ubiquity and diversity of species in which activity against lignocellulose has been demonstrated. Characterisation of the cellulolytic activity of actinomycetes has understandably received most attention, since cellulose is the major component of plant biomass
Correspondence to: Dr. A.J. McCarthy, Microbiology Depart-

ment, Life Sciences Building, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, U.K.

and a potentially utilisable source of glucose. Actinomycete cellulases are inducible extracellular enzymes which appear to attack cellulose in much the same way as fungal hydrolytic cellulases. Actinomycete xylanases similarly conform to the basic patterns of production and activity established in other bacteria and fungi, but they have been relatively little-studied. Evidence for activity against lignin has only recently been obtained for actinomycetes, with the development of sensitive radiometric assays which permit detection of limited ligninolytic activity. There is little doubt that actinomycetes compare poorly with the whiterot fungi in relation to the extent of delignification achieved, particularly with wood lignocelluloses. Nevertheless, their ability to solubilise grass lignins may have a role in humification and biotechnological applications of lignocellulose conversion. The biochemistry of lignin degradation by actinomycetes remains poorly understood and the enzymes involved have yet to be identified. There is more information on the enzymology of cellulose and hemicellulose degradation, but how these different groups of enzymes and their component proteins interact to solubilise lignocellulose is largely unknown. Elucidation of the biochemistry of lignocellulose degradation and development of actinomycete-based systems for lignocellulose conversion should be greatly assisted by the application of the recombinant D N A tech-

0168-6445/87/$03.50 1987 Federation of European Microbiological Societies

146

niques recently developed for this group organisms.

of

2. I N T R O D U C T I O N Actinomycetes are a heterogeneous group of Gram-positive bacteria of which terrigenous saprophytes are the most common forms. The growth of most actinomycetes as branching hyphae is a trait shared by the filamentous fungi, but actinomycetes may be just as important in the primary degradation of organic matter. Isolates representing a diverse range of actinomycete genera and exhibiting the full range of degradative enzyme-mediated activities have been recovered in large numbers from soils and composts [1]. Actinomycetes have been extensively exploited for commercial antibiotic production, and more recently as a source of industrial enzymes such as glucose isomerase. This has encouraged research on the biology of actinomycetes, with emphasis on molecular genetical aspects of the streptomycetes. These organisms account for the vast majority of commercial antibiotic producing strains and while our knowledge of streptomycete biology is advanced relative to other actinomycetes, much of their basic physiology and biochemistry remains poorly understood. This should be rectified by the application of recombinant DNA techniques developed for streptomycetes [2] and we are now in a position to extend this approach to the biodegradative actinomycetes. Plant biomass is by far the most important renewable resource and its bioconversion is of immense ecological, and potentially biotechnological, importance. It is essentially lignocellulose, with low molecular weight compounds and readily degradable polymers such as starches and pectins contributing a relatively small proportion of the total carbon content. Lignocellulose itself is a complex of three primary polymers - - lignin, cellulose and hemicellulose. The plant cell wall is composed of cellulose microfibrils containing both highly crystalline and amorphous regions, embedded in a matrix of lignocarbohydrate comprising polyphenolic lignin covalently bound to hemicellulose [3,4]. In situ degradation of native

lignocellulose is limited by the interactive nature of this structure as well as the physical and chemical properties of the polymers themselves. Cellulose is the most abundant (30 45% w / w ) and although a homopolymer of glucose units linked by a hydrolysable repeating ,8 1-4 linkage, its

Table 1 Summarised classification of genera to which lignocellulose-degrading actinomycetes have been assigned Genus " Circumscription b Chains of arthrospores borne on aerial hyphae; wall chemotype 1. Aerial mycelium absent: single spores on substrate hyphae; wall chemotype II. Longitudinal pairs of spores on aerial hyphae; wall chemotype Ill (madurose present). Single heat-sensitive aleuriospores on aerial hyphae; wall chemotype III (madurose absent). Aerial hyphae bear chains of arthrospores; wall chemotype III (madurose usually present). Aerial hyphae bear chains of arthrospores; spores also produced on substrate hyphae; wall chemotype IV; mycolic acids absent. Single spores on aerial hyphae; wall chemotype IV; mycolic acids absent. Substrate hyphae fragment into bacillary and coccoid elements; aerial hyphae may bear chains of spores; wall c h e m o t y p e IV; mycolic acids present. Cocci or short rods which may develop into f i l a m e n t s or branched hyphae; aerial hyphae a b s e n t or rudimentary; wall chemotype IV; mycolic acids present.

Streptomvces (T) micrornonospora

Microbispora (T)

Thermornonospora (T)

A ctinornadura

Pseudonocardia (T)

Saccharomonospora (T)

Nocardia

Rhodococcus

(T) denotes genera which contain thermophilic strains with activity against lignocellulose. b Wall chemotype as defined by Lechevalier and Lechevalier [33].

147 crystalline structure requires concerted action by several different classes of enzyme to effect hydrolysis [5]. Hemicellulose is somewhat less abundant (20-40% w / w ) and is the collective name for a group of branched heteropolysaccharides e.g. arabinoxylan, the degradation of which again involves several classes of enzyme [6]. Lignin is the most recalcitrant and variable of these polymers and comprises three types of aromatic monomer linked by a range of non-hydrolysable bonds of which fl-aryl-ether linkages are the most common [7]. It is generally accepted that lignin degradation is the important rate-limiting step in lignocellulose biodegradation. Activity against lignin has been demonstrated in a relatively limited range of microorganisms, and even in the best-studied white-rot fungus Phanerochaete chrvsosporium, the enzymology is only beginning to be elucidated [8]. This is largely a reflection of the inherent difficulties in studying the degradation of such a complex and ill-defined polymer. In contrast, the action of fungal cellulases, particularly those of Trichoderma spp., has been relatively well-characterised [9]. Xylan is the most abundant hemicellulose, and while many xylanolytic microorganisms have been described [10], further characterization of xylanases has attracted much less research effort than the corresponding cellulases. Lignocellulose degradation by mixed populations, the cooperative action of which is undoubtedly responsible for the natural degradative process, has received little attention. Improving our understanding of native lignocellulose degradation requires that a wide range of microorganisms are studied, possibly leading to the development of efficient systems for utilisation of waste lignocellulose as a source of chemicals and energy. Two particular groups of prokaryotes, the actinomycetes and anaerobic clostridia, have potential but only in the actinomycetes is there any evidence of activity against lignin. It is the object of this review to present an account of lignocellulose degradation by a group of actinomycetes which include both mesophiles and thermophiles from a range of genera. 3. ECOLOGY Soil is the primary reservoir of degradative actinomycetes and lignocellulose one of the most abundant carbon sources available to support growth in this environment. Nutrients are not uniformly distributed in soil and so it is presumed that routine recovery of actinomycetes from soil samples in numbers exceeding 10 s per gram is attributed to the survival of spores and possibly mycelial fragments. These will have resulted from previous colonisation of organic material and dissemination by air, water and arthropods [11]. Actinomycetes are also commonly encountered in aquatic environments but here their contribution to nutrient recycling, even in sediments, is debatable. In any case, their capacity for sporulation and dispersal is such that there are few environmental niches which do not contain a diverse population of actinomycetes in active or dormant form. Aerobic conditions and neutral to alkaline pH are general prerequisites for the growth of saprophytic actinomycetes. The former is related to water content in soil and while acidophilic streptomycetes can be recovered from acid soils [12] there is no evidence that they are particularly active against lignocellulose. One important ecosystem where anaerobic degradation of lignocellulose by prokaryotes predominates is the rumen. Although there have been reports of anaerobic cellulolytic micromonosporas [13,14], their identity as actinomycetes requires confirmation. 3.1. Isolation Adaptation of standard procedures for the recovery of actinomycetes by incorporation of lignocellulose or related substrates in enrichment and isolation media is the usual approach. Suppression of bacterial and fungal growth is desirable and in the case of the latter, is routinely achieved by including cycloheximide in the medium. Bacterial growth can rarely be excluded but its interference with actinomycete recovery can be minimised in a number of ways. These include heat treatment of samples at temperatures which actinomycete spores can survive [15] and the use of an Andersen sampler and sedimentation

148 chamber in which suspension of particles in air favours recovery of actinomycete spores [16]. The latter approach is particularly useful for isolating species with relatively complex nutritional requirements from substrates where heat-resistant Bacillus spp. predominate. Dilution plating on standard actinomycete isolation media with or without lignocellulose tends to yield mainly streptomycetes. This does not necessarily indicate that they are predominant but could be a reflection of the emphasis placed on this group when isolation media are formulated. In some cases, highly selective isolation media have been developed for specific taxa and later used to isolate Micromonospora, Thermomonospora and Actinomadura strains with activity against grass lignocellulose [17]. A number of strategies have been used to obtain isolates active against lignocelluloses. In most cases, the primary objective is to select strains for further study but some studies have been directed towards enumeration of cellulolytic and xylanolytic actinomycetes in natural substrates [18,19]. The simplest approach is to directly observe clear zones around colonies on isolation media containing insoluble xylan or cellulose preparations. In this way, xylanolytic strains of Streptomyces [20] and cellulolytic strains of Streptomyces [21], Thermomonospora [22] and Micrornonospora [23] have been selected. Other workers have preferred to screen pools of purified isolates without any prior indication of activity on isolation plates [18,24]. Enrichment techniques in which samples are incubated in a liquid medium containing lignocellulose or a related carbon source have also been used and are particularly appropriate to the isolation of ligninolytic actinomycetes. Phelan and coworkers [25] isolated both cellulolytic and ligninolytic streptomycetes from various soils and organic materials by incubating samples in broth containing extracted heartwood, prior to isolation by dilution plating. Repeated passage through enrichment broth containing vanillic acid as carbon source resulted in isolation of a Nocardia strain which could also release 14C02 (5-15%) from methoxyl, side-chain and ring-labelled [14C]lignin maize lignocellulose [26]. The difficulties in detecting ligninolytic activity directly on isolation plates and the complexity of screening procedures for lignin degradation promote the use of enrichment techniques but in some instances the nature of the sample suggests such an approach. The isolation of cellulolytic Streptomyces and Micromonospora spp. from the hind gut of termites by enrichment in Avicel cellulose broth [27] is one example.

3.2. Identification There can be few groups of microorganisms whose taxonomy has been so intensely studied in the last 10 years with such significant results. The traditional view that actinomycetes are a group of Gram-positive bacteria whose common feature is the formation of a branching mycelium throughout or at some stage in their life cycle has been seriously challenged. This stems largely from the construction of phylogenetic trees based on comparative sequencing of 16s rRNA [28] with the result that actinomycetes now encompass all Gram-positive bacteria whose DNA has a G + C ratio of > 55 Mol% [29]. In practical terms, this has led to the inclusion of the coryneform bacteria in this group and the exclusion of thermoactinomycetes, which are more closely related to the Bacillaceae. There is no evidence for activity against lignocellulose in the genus Thermoactinomyces [30], despite some misidentification of cellulolytic thermomonosporas [31]. One genus of coryneform bacteria, Cellulomonas (see [32]), has been extensively studied in relation to cellulose and xylan degradation but will not be considered here. In practice, identification of lignocellulose-degrading isolates as actinomycetes can usually be achieved by observation of a mycelial colony morphology, and in some cases this has been the extent of identification applied. This can be of little or no value in the identification of nocardioform and related actinomycetes, where the formation of hyphae may be only transitory. Where appropriate, colony morphology and spore arrangement observed directly under high magnification can indicate generic assignment e.g. streptomycetes and micromonosporas. Whole-cell analysis for characteristic wall components [33] is now mandatory for identification at the genus level and in some cases additional criteria such as lipid profiles are required. Species identification,

149 while often claimed, usually requires the application of diagnostic tests beyond the interest or possibly expertise of the investigator. Numerical taxonomic studies have provided some good species classifications for genera containing lignocellulose-degrading actinomycetes, e.g. Streptomvces [34] and Thermomonospora [30], yielding practical identification systems which have yet to be fully exploited. Consequently, species names must often be considered as having been loosely applied to lignocellulose-degrading actinomycetes. Nevertheless, it is clear that activity against lignocellulose is exhibited by a diverse range of both mesophilic and thermophilic actinomycetes. A summary classification of lignocellulose-degrading actinomycetes is presented in Table 1.
3.3. Ecological role The involvement of actinomycetes in lignocellulose decomposition in natural substrates is usually implied by their recovery in significant numbers with subsequent demonstration of appropriate enzyme activities in the laboratory. However, actinomycetes may be present only as dormant spores, and even when active their contribution relative to that of fungi and other bacteria could be minimal. These limitations on our interpretation of laboratory-acquired data are to a large extent unavoidable and continue to plague reviews on actinomycete ecology in general [1,11]. Nevertheless, there have been a number of observations which support the view that actinomycetes have a role as primary degraders of lignocellulose. Development of in vivo 14C-labelling techniques [35,36] has provided evidence that actinomycetes can attack lignocellulose in its native form. Degradation of lignin and cellulose components of Douglas fir and the lignin component of red maple by Streptomyces strains [21,25], maize stalk lignin degradation by Nocardia strains [26,37] and wheat lignin degradation by several actinomycete strains [17] are good examples. Colonisation of wood by streptomycetes has been the subject of a number of electron microscopy studies, some of which have produced direct evidence for plant cell wall degradation [38,39]. Penetration of primary and secondary lignified walls of wheat

straw by actinomycete hyphae has similarly been observed in compost [40]. Indeed composts are the most likely environments where lignocellulose degradation by actinomycetes could supersede the contribution of fungi. Both the mesophilic and thermophilic growth phases of the composting process involve the development of a diverse actinomycete population [17,41] and lignocellulose degraders have been isolated from a range of composted materials [17-19,22,42-44]. Thermophilic actinomycetes are invariably numerous and in compost production for mushroom cultivation, the numbers of cellulolytic thermomonosporas increase during composting [41]. Evidence that cellulose decomposition in mushroom compost involves a positive interaction between cellulolytic actinomycetes and certain bacteria [45] reinforces the importance of considering natural lignocellulose degradation as the product of an interactive heterogeneous microflora. An alternative approach to the study of ecological roles in compost is to correlate the evolution of enzyme activities and microbial populations. This has proved difficult [46] but in one case the major cellulase detected in municipal refuse compost extracts had identical pH and temperature optima to that produced by a Thermomonospora curvata isolate from the same substrate [47]. A more tenuous correlation exists between suggestions that grass lignin solubilisation by actinomyceres involves oxidation at the Ca position of lignin phenyl propane side chains [48] and observation of the corresponding signal on [a3C]NMR spectra of both composted wheat straw and actinomycete solubilised lignin [49]. Cellulolytic micromonosporas may have an important role in lignocellulose degradation in soil and freshwater sediments. These organisms are slow to develop colonies on isolation plates, often requiring up to 21 days incubation at 25-30C, and consequently can be overlooked in surveys of lignocellulose-degrading actinomycetes. Sandrak [23] recovered up to 3 103 cellulolytic micromonosporas per gram of wheat rhizosphere samples while Johnston and Cross [50] found Micromonospora to be the dominant actinomycete genus in freshwater lakes, having also demonstrated the presence of both mycelium and spores in lake

150 muds [51]. Cellulolytic micromonosporas have also been isolated from decaying wood [52] and implicated in the biodeterioration of timber foundation piles [53]. A role for lignocellulose-degrading actinomycetes in soil humification can also be envisaged. Humus and its fractions, humic and fulvic acids, are chemically ill-defined but can be regarded simply as the recalcitrant residue of the decomposition processes occurring in soil. While it has been claimed that actinomycetes can both synthesise and degrade humic substances (see [11]), their possible role in humification via lignocellulose degradation is more important. Haider and coworkers [54] showed that a significant proportion of the carbon in 14C-labelled lignins and related aromatic compounds could be recovered in the humic acid fraction of soil after 28 days incubation. The ability of nocardiae and rhodococci to attack lignin and related aromatic monomers in soil is well documented [37,55-58] and could provide phenolic structural units for humus formation. The view that actinomycetes are intimately involved in the natural humification process has also been suggested by analysis of the lignocarbohydrate complex solubilised from cereal straw by Thermomonospora mesophila. The acidinsoluble nature of this material and its elemental composition demonstrated a striking similarity to humic acids [49]. A specific ecological niche where actinomycetes may have a role in lignocellulose degradation is as symbionts in the gut of higher termites. Direct observation of filamentous microorganisms in situ suggested that actinomycetes were not simply transitory but had formed a stable association [59]. More recently, cellulolytic actinomycetes have been isolated from the hindgut of four different termites [27]. ity controls the degree and rate of enzymic hydrolysis and depends on the treatment used to prepare the substrate. For example, cotton is the purest natural form comprising approximately 90% highly crystalline cellulose, while solvent or alkali treatment to generate wood pulps or rayon yields cellulose which is much more readily degraded [5]. Soluble derivatives such as carboxymethylcellulose are even further removed from native cellulose and can often be degraded by microorganisms which show little or no activity against cellulose per se. Hemicelluloses are more variable and chemically complex than cellulose. There are three principal groups: xylans, mannans and galactans, their names deriving from the sugar which makes up the fi 1-4 backbone. Xylans are the most common and extensively studied and are the principal hemicellulose c o m p o n e n t of grasses and hardwoods. They usually occur as arabinoxylans where, in addition to arabinose units, residues of glucuronic acid and arabinoglucuronic acid are present together with varying degrees of acetylation of xylose units [6]. For a detailed description of hemicellulose structure see Whistler and Richards [60]. In principle, three types of enzyme are involved in the degradation of cellulose and hemicellulose: endo-enzymes which cleave intermonomer bonds at random; exo-enzymes which sequentially remove monomer or dimer units from one end of the chain; enzymes which hydrolyse dimers. Enzymes which attack branch points on hemicelluloses can also be involved. In practice, few enzyme systems have been sufficiently characterised, particularly hemicellulases, to present such a unifying model. In Trichoderma reesei, the best studied cellulolytic organism, endoglucanases, exoglucanases (cellobiohydrolases) and /~-glucosidase (cellobiase) have been described and some progress made towards understanding how they interact to effect complete degradation of crystalline cellulose [9]. Endoxylanases and /~-xylosidases have been described in a range of microorganisms [10] but the existence of exoxylanases as distinct entities has yet to be demonstrated. The enzymology of side-chain liberation and deacetylation of xylan is also poorly understood [6].

4. C E L L U L O S E DEGRADATION

AND

HEMICELLULOSE

Both of these substrates are essentially similar in that they are linear polymers of /7 1-4 linked sugar residues, susceptible to hydrolysis by acid and enzymes. In cellulose, the degree of crystallin-

151 The methods used to study cellulose and hemicellulose degradation depend on the form in which the substrate is used and the degree of quantitative information required. Evidence for the presence of cellulolytic activity can be provided by observing clear zones around colonies on agar containing cellulose powder or ball-milled lignocellulose. Some xylans are sufficiently insoluble to permit a similar approach. Precipitation of undegraded carboxymethylcellulose to yield clear zones indicates endoglucanase activity and can be modified for the identification of enzymes on polyacrylamide gels [61]. Weight loss of filter paper, reduction in the tensile strength of cotton threads and measurement of dye release from celluloseazure can be used to obtain semi-quantitative data on cellulose degradation by whole-cells or enzyme preparations. Evolution of 14CO2 from [14C]glucan-labelled lignocellulose by cellulolytic actinomycetes [25] has not been applied extensively but is a potentially useful technique for studies on the physiology of cellulose utilisation. Most quantitative data on cellulase or xylanase activity in cell-free preparations are generated by measuring the release of reducing sugar from native or prepared substrates. Colorimetric techniques [62,63] are invariably used to express units of enzyme activity as ~mols of glucose or xylose equivalents released/rain. Identification of the reaction products is essential for elucidating the mechanism of attack and can be achieved by thin-layer or paper chromatography (e.g. [64,65]). More precise analysis of reaction products is also possible using high-performance liquid chromatography (e.g. [66,67]). Colorimetric assays are available for fl-glucosidase and fl-xylosidase activity, in which release of p-nitrophenol from pnitrophenol fi-D-glucoside or xyloside is measured (e.g. [68,69]). received comparatively little attention and there has been little progress beyond the early demonstration of five electrophoretically distinct endoglucanases in Stm. antibioticus [70]. While simultaneous p r o d u c t i o n of extracellular endoglucanase, cell-bound fl-glucosidase and, in some cases, extracellular exoglucanase, during growth on cellulose has been established in many strains, the number of forms of each enzyme is largely unknown. There are however a number of reports on partial purification of actinomycete cellulase components. Separation of endoglucanase and exoglucanase activity in culture filtrates of Thin. curvata has been described [64,71] and Hfigerdal and coworkers [72] identified at least three extracellular cellulases in Thm. fusca. Two Thm. fusca fl 1-4 endoglucanases were later purified to homogeneity [73] and found to have molecular weights of 94000 and 46000. The gene for the larger enzyme has been cloned in E. coli [74] as a first step towards construction of a vehicle for the production of high levels of thermostable cellulases. Another thermophilic actinomycete, Microbispora bispora, produces both endo- and exoglucanases and in this case the latter has been positively identified as a cellobiohydrolase [75]. While some progress has been made towards understanding the synergistic action of Trichoderma cellulases [76] and synergism between cellulases from different fungal species has been observed [77], no such studies have been made on cellulolytic actinomycetes. The application of recombinant DNA technology to enable expression of synergistic cellulases by a single strain is attractive and forms at least part of the justification for development of genetic manipulation systems for cellulolytic thermophilic actinomycetes [78]. Actinomycete cellulases are inducible enzymes produced in response to growth on cellulose. Hemicellulose in the form of xylan can also induce cellulase production by Stm. flavogriseus [79] and Stm. lividans [80], and in both cases cellulase yields were higher than on cellulose. Whether this applies to other cellulolytic actinomycetes has yet to be determined but since natural substrates comprise a mixture of both polymers, some form of coordinated regulation could be expected. Cellulase production can also be represented by glu-

4.1. Actinomycete cellulases True cellulolytic activity has been reported in a range of actinomycete species but has been further characterised in relatively few. The mesophilic streptomycete Stm. flavogriseus and the thermophilic species Thm. fusca and Thin. curvata are the best-studied. Characterisation of the protein composition of actinomycete cellulase complexes has

152

cose [81,82]. The effect of cellobiose on cellulase production by actinomycetes appears to be more complex. Cellobiose is comparable to cellulose as a substrate for cellulase production in Thin. fusca [81]. In Thm. curvata, cellobiose has been reported as both a poor [82] and a good [84] cellulase inducer. It can either stimulate or repress production of/~-glucosidase in streptomycetes, depending on the strain [85]. One possible explanation is that the role of cellobiose as an inducer or repressor of cellulase production is concentration-dependent and that thresholds vary in different strains. In Thm. curvata it has been shown that a sufficient concentration of cellobiose to support rapid growth leads to repression of cellulase biosynthesis [86]. The basic hypothesis for regulation of cellulase production is that low constitutive levels of cellulase activity generate soluble oligosaccharides and it is these which can enter the cell and effect induction. Enhanced cellulase production by actinomycetes has been achieved by mutation [87,88] and in Thin. curvata [82] and a Streptomyces sp. [89] the mutants have been identified as catabolite repression-resistant. There is some evidence which implicates cyclic AMP as the mediator of catabolite repression of cellulase bio-

synthesis in Thrn. curvata [86]. Various parameters exert effects on both enzyme production and activity. Cellulose degradation products are involved in the regulation of both enzyme synthesis (discussed above) and enzyme activity through end-product inhibition. Thus, actinomycete cellulase activity is generally inhibited by cellobiose [68,80,90]. Addition of cellobiase (/?-glucosidase) in the form of culture solids can relieve this inhibition and lead to improved cellulase activity and glucose production [91]. In the same way, glucose itself inhibits cellobiase activity in actinomycetes [66,68]. Optimisation of cellulase production is usually achieved by empirical manipulation of growth conditions and is often species- or strain-specific. Actinomycete cellulases are produced and released into the medium during exponential growth, while /?-glucosidase activity remains associated with culture solids throughout [43,44,72]. Cellulose degradation or cellulase production by cultures can be improved by altering the cellulose source and nitrogen source supplied [81,83,92,93]. The cellulolytic fungus Trichoderma reesei is invariably used as the reference standard and a number of authors have claimed comparable levels of cellulase activ-

Table 2 T e m p e r a t u r e a n d p H r e l a t i o n s h i p s of a c t i n o m y c e t e cellulases Species E n z y m e activity ~ O p t i m a for Production Activity 55 o C 40 (7, p H 6.5 50 C, p H 6.0-7.5 40 4 5 C ~ p H 5.3-6.0 40 o C, p H 6.5-7.5 4 5 - 5 5 C, p H 5.0 6.0 Thermostability (half-life) 30 min at 60 o C < 10 min at 40 o C 2 h at 40 C '/ 2 h at 40 C ~ < 10 rain at 40 C t > 24 h at 50 C t ! < 24 h at 50 C ] > 60 min at 70 C [80] [85} Reference

Stm. lividans

Endoglucanase fi-Glucosidase Endoglucanase Cellulase /~-Glucosidase Endoglucanase Cellulase Endoglucanase Cellulase Endoglucanase Cellulase [}-Glucosidase

34 37 o C 31 o C

Stm. flavogriseus

30 C, p H 7

[43,68]

Stm. albogriseus Strn. nitrosporeus Mim. rnelanospora Thm. curvata Thermomonospora sp.

2 5 - 3 5 o C, p H 6.7-7.2

[44]

45 55 C, p H 8.0

65 o C, p H 6.0-6.5

[64,71,83]

5 5 C , p H 7.4

70 C, p H 6.0 6 5 C , p H 7.0 55 C, p H 6.5

24 h at 65 o C 4 h at 6 5 C ~ < 1 h at 55 o C J

[31]

E n d o g l u c a n a s e is a measure of activity against c a r b o x y m e t h y l c e l l u l o s e ; cellulase is a measure of activity against Avicel or filter paper; fl-glucosidase is a m e a s u r e of activity against cellobiose or nitrophenol B-D-glucoside.

153

ity in actinomycetes (e.g. [43,91]). However, different sources of cellulose can have a dramatic effect on the extent of hydrolysis achieved [91,94]. Temperature and pH are parameters which affect production, activity and stability of actinomycete cellulases. This information is presented for a range of species in Table 2. In general, pH and temperature optima for cellulase production correspond to the conditions required for optimal growth. In both the mesophiles and thermophiles, optimum cellulase activity is exhibited at temperatures significantly higher than those required for optimum enzyme production. Although cellulases from mesophiles may be active at high temperature, their thermostability relative to those of the thermophilic species is poor. The thermostability and temperature optima for activity are generally higher for endoglucanase than for Avicel or filter paper cellulase in both mesophiles and thermophiles. Intracellular or cell-associated fi-glucosidase activity is generally less active and stable at high temperature than extracellular cellulase activities. Furthermore, slight increases in pH tend to markedly decrease the thermostability of actinomycete fl-glucosidases [31,68,71]. The pH relationships of actinomycete cellulases exhibit a uniform

pattern distinct from fungal cellulases i.e. optimal activity is expressed around neutrality and is significantly reduced at pH values < 5. An interesting interaction between pH and temperature has recently been reported by Stutzenberger and Lupo [71]. The cellulase complex of Thm. curvata was found to be activated by heat treatment at 70 C and pH 8.0, and inactivated by the same treatment at pH 4-6. It was also shown that this thermal activation occurred in the purified major endoglucanase component. Thermostability, particularly in the presence of substrate, is an attractive feature of thermophilic actinomycete cellulases since the ability to recycle enzymes is an important criterion in the development of commercial saccharifications. Furthermore, the pH profile of actinomycete cellulases may be advantageous for the saccharification of alkali-treated lignocellulosic biomass.
4. 2. A ctinomycete xylanases Xylanase activity is more widespread amongst the actinomycetes than cellulase activity and although it is an inherent property of cellulolytic strains, xylanases are clearly a distinct group of enzymes. High proportions of xylanolytic strains

Table 3 Temperature and pH relationship of actinomycete xylanases Species Enzyme activity Optima for Production Activity 55 66 o C, pH 5.0-7.0 4 0 C , pH 6.5 50 o c , pH 6.5 40 C, pH 6.5 / 55 o C, pH 5.5 65 8 0 C , pH 5.5-7.7 7 0 C , pH 5-8 30 min at 60 o C 24 h at 6 5 C 24 h at 6 0 C Thermostability (half-life) 30 min at 60 C Reference

Stm. fieidans

Xylanase ,8-Xylosidase Xylanase fl-Xylosidase Xylanase Xylanase ,8-Xylosidase

40 C 31 o C 30 C, pH 7.0 35 C, pH 6.0 7.0 55 o C, pH 7.6 55 C, pH 7.6

[80}

Stm. flauogriseus

[95] [97] [67] [69]

Streptomyces sp. Thermomonoa;oora sp. Thin. curt~ata Thin. fusca Thin. chromogena Saccharomonospora viridis

Xylanase

50 o C, pH 8

60 75C, pH 5-8

> 60 rain at 70 C

[65]

Xylanase ,8-Xylosidase ~

50 C, pH 8 50 C, pH 8

60-70 o C, pH 5-8 4 0 - 5 5 C , pH 7 9

< 60 min at 70 o C

[65]

" Data for Sam. t,iridis ,8-xylosidase activity from McCarthy and French (unpublished).

154 have been identified in populations of actinomycetes from natural substrates [24,30]. Xylans are more readily degraded than cellulose, and this may partly explain improved production of streptomycete cellulases in cultures containing xylan rather than cellulose as carbon source [79,80]. In Stm. flavogriseus, simultaneous production of glucose (xylose) isomerase in addition to cellulases and xylanases has also been described [95]. Coproduction of cellulases and xylanases has been observed in other actinomycetes [44] but in Thermomonospora strains optimum production of each activity was achieved when grown on its respective substrate [67]. The one conclusion that can be drawn is that actinomycete xylanases, like those of other bacteria, are induced by growth on xylan. Soluble oligosaccharides, generated by low constitutive levels of endoxylanase, are the probable inducers. The effects of cellobiose on cellulase production, discussed above, provides a model but studies are hampered by the commercial unavailability of xylobiose. However, in a Streptomyces sp., induction of xylanase production has been achieved with synthetic xylosides [96]. In Saccharomonospora viridis, a thermophilic actinomycete which degrades xylan but not cellulose~ both extracellular xylanase and cell-bound /3-xylosidase were induced by growth on xylan but not glucose or xylose. Addition of glucose a n d / o r xylose stimulated growth but depressed xylanase production by approximately 50% (French and McCarthy, unpublished). There have been comparatively few studies on actinomycete xylanases. For example, Thermomonospora strains which have been so intensely studied in relation to their cellulolytic activity have only recently been examined for xylanase activity [65,67,69]. Streptomycetes are the only other group of actinomycetes whose xylanase activity has been studied in any detail, and endoxylanase has been purified from several Streptomyces strains [97,98] including a cellulasenegative mutant of Stm. lividans [99]. Streptomyces lividans is an important host for the expression of cloned Streptomyces genes, including those coding for extracellular agarase [100] and amylase [101] activities. In both sets of experiments, the multicopy plasmid pIJ702 was used as the vector and recently Iwasaki and coworkers have cloned an extracellular xylanase from a Streptomyces sp. into both Stm. fividans and Stm. kasugaensis by applying the same strategy [102]. One interesting, and fortuitous, observation was that the host xylanase activity of Stm. lividans [99] was not expressed in the presence of pIJ702. The use of a multicopyplasmid vector resulted in the production of 10-70 times higher levels of extracellular xylanase than the donor strain and, in contrast to the use of E. coli as a host for actinomycete genes for extracellular enzymes [74], the transformants showed a high level of xylanase secretion into the medium. Actinomycete xylanase activity comprises extracellular endoxylanase and cell-bound /?xylosidase. There is no evidence for exoxylanase activity in bacteria in general and even in fungi, the distinction between this and /~-xylosidase activity is not well-defined [6]. Culture filtrates yield a mixture of oligosaccharides ultimately hydrolysed to xylobiose, although xylose is also detectable. Liberation of arabinose from arabinoxylan has not been demonstrated by an actinomycete xylanase preparation. Regulation of actinomycete xylanase activity has been little-studied but xylose inhibition of/~-xylosidase has been demonstrated in a Thermomonospora strain [69]. The pH and temperature relationships of xylanases from different strains are summarised in Table 3, and have much in common with those of cellulases (Table 2). Similarities include optimal activity in the pH range 5-8, increased thermostability of enzymes from thermophilic strains and lower temperature optima for cell-bound dimer hydrolases. Actiuomycete xylanases are sufficiently active to produce some direct saccharification of lignocellulose, as evidenced by the generation of xylose from bagasse by Streptomvces and Micromonospora spp. [44] and from straw by Thm. curvata [65].

5. L I G N I N D E G R A D A T I O N BY ACTINOMYCETES Lignin is normally regarded as the component of lignocellulose which must be removed to allow efficient use of cellulosic material for saccharification, paper production or upgraded fodder. Bio-

155 logical delignification has the potential to provide an alternative to largely chemical processes which are energy-intensive, polluting and do not exploit utilisation of lignin and its products. Improvements in our understanding of lignin degradation by microorganisms would also have important implications for the prevention and treatment of wood decay and the biochemistry of soil humification. Within the last 10 years, earlier claims that actinomycetes can degrade lignin [103,104] have been substantiated to the extent that they are the best-studied prokaryote lignin degraders. The problem of obtaining lignin preparations which have not undergone significant chemical modification has necessitated the development of radiometric assays to demonstrate that actinomycetes can attack native lignin in situ. Methods for the preparation of [14C]lignin-labelled lignocelluloses have been described in several reviews [7,105,106]. Introduction of the radiolabelled precursor can be achieved by direct injection or by immersing cut stems in a solution of the precursor. The former method is preferable because it minimises disruption of lignin biosynthesis as a response to injury and permits continued cultivation of the radiolabelled plants under a natural d a y / n i g h t cycle. Incorporation of radiolabel into phenolic acids ester-linked to carbohydrate or lignin fractions [4] can also interfere, particularly in grasses, and it is important that these components are either taken into account or removed by alkali extraction. It has been suggested that etherlinked phenolic acids could also be present [107] and the stability of the ether bond would make their removal difficult. After extraction to remove low molecular weight material and protease treatment (if [UC]phenylalanine has been used as the precursor), detailed analysis is required to determine the specificity of radiolabelling. A combination of acid, alkali and cellulase treatments together with the application of thin-layer, reversephase and gel permeation chromatographic techniques provide a comprehensive profile of the radiolabelled substrate [108]. Nitrobenzene oxidation to demonstrate that 14C is present in the products derived from lignin has also been applied [36]. The best model substrates are dioxane-extracted lignins (milled wood lignins) and synthesised dehydropolymers of coniferyl alcohol (DHP), both of which may be prepared in radiolabelled form [7]. While these may overcome some of the problems of non-specific labelling in native [14C]lignin lignocellulose preparations, biodegradation data should be interpreted with caution. For example, Crawford [21] found that dioxaneextracted [14C]lignins were more susceptible to degradation by streptomycetes than the native substrates from which they were prepared. Lignin-degrading streptomycetes have been identified by measuring 14CO2 evolution from radiolabelled wood lignocelluloses prepared by incubating cut stems with ~4C-labelled precursors [21,25]. Direct injection of maize seedlings with [14C]ferulic acid provided a substrate which enabled Trojanowski and coworkers to obtain evidence for lignin degradation by strains of Nocardia [37] and Rhodococcus [26]. In both cases, 4 15% of the radiolabel in maize lignins and [14C]DHP was evolved as 14CO: in 15 days, with methoxyl, and to a lesser extent, side-chain-labelled preparations being more susceptible to attack than the ring-labelled substrates. By comparison, the whiterot fungus Coriolus versicolor released, during a 30-day period, approximately 45%, 25% and 18% of methoxyl, side-chain and ring-labelled D H P as CO:, respectively [109]. The use of an identical substrate, uniformly labelled [14C]lignin wheat lignocellulose, to determine ligninolytic activity in both actinomycetes and white-rot fungi allows a more meaningful comparison of degradation rates. Thus. Thin mesophila and Stm. cvaneus released 7-8% of the 14C in this substrate as 14CO~ in 14 days [17] compared to 20-50% 14CO2 evolved in 15 days by 3 species of white-rot fungi grown on the same substrate [108]. The kinetics of lignin degradation to CO2 by actinomycetes generally conforms to a uniform pattern distinct from that observed in Phan. chrvsosporium. Lignin degradation always accompanies growth in actinomycetes and is therefore presumed to be a primary metabolic activity. Lignin degradation by Phan. chrvsosporiurn occurs during the secondary growth phase and is a classical secondary metabolic activity induced particularly by nitrogen starvation [110]. High concentrations of both organic and inorganic nitrogen do not

156 inhibit lignin degradation in Stm. badius [93] and the elevated concentrations of oxygen required for efficient lignin degradation by Phan. chrvsosporium [111] do not appear to enhance ligninolytic activity by Thrn. mesophila and Stm. cyaneus [17]. Further characterisation of lignin degradation by actinomycetes is mainly confined to Streptomyces strains and to some extent Thm. rnesophila. There is no evidence for significant lignin degradation by any thermophilic actinomycetes described thus far [17,35]. The primary degradative activity of actinomycetes is solubilisation of lignin. Mineralisation to CO 2 is poor in comparison with the levels achieved by Phan. chrvsosporium where a much larger research effort has led to the isolation of extracellular ligninases and detailed characterisation of their biochemically unique mechanism of action [8]. In the actinomycetes, there is some evidence for the involvement of extracellular inducible enzyme activity [49] but its specificity has yet to be proven. While there are some features of lignin solubilisation by actinomycetes which suggest a uniform mechanism of attack, there is also evidence that individual strains have evolved distinct pathways. Working primarily with Stm. viridosporus, Crawford and coworkers have shown that lignin isolated from actinomycete-decayed softwood is extensively demethylated, has undergone side-chain oxidation and is depleted in aromatic rings [112]. Subsequently, attention has focussed on analysis of the products of actinomycete attack in an effort to gain insight into the mechanism of degradation. Some low molecular weight products such as syringic and ferulic acids have been detected [113] but the major product is a high molecular weight complex of lignin, carbohydrate and protein [49,114,115]. Grass lignocelluloses are much more susceptible to actinomycete attack than those of wood [113,114], as indicated by the yields of this water-soluble, acid-precipitable material (APPL) from culture filtrates. Using ~4C-labelled wheat lignocellulose, McCarthy and Broda [17] found that solubilisation of radiolabelled material (30-40%) occurred during primary growth with no evidence of further significant degradation or mineralisation t o C O 2 by fresh cultures of Thin. mesophila or Stm. cvaneus. This suggested an endproduct rather than an intermediate, although Pometto and Crawford [116] have shown that APPL generated by Stm. viridosporus can be further degraded to low molecular weight products. There is also evidence that formation of high molecular weight products does not conform to a universal pattern. Extracellular phenol oxidase produced by Stm. badius is implicated in polymerisation of low molecular weight compounds to produce a chemically distinctive APPL, but there is no evidence for repolymerisation or this enzyme in Thin. mesophila and Stm. uiridosporus [49,115]. Lignocarbohydrate can be solubilised from straw by cell-free preparations of Thm rnesophila grown on straw or xylan [49]. Although pentoses accounted for 80-90% of the carbohydrate fraction of this product, solubilisation was not merely a result of xylanase activity. A similar lack of correlation between lignin solubilisation and levels of endoglucanase and xylanase activity in Stm. ~#ridosporus mutants has been reported [88]. Nevertheless, it is still not clear whether APPL production involves lignin depolymerisation or is simply the result of a number of reactions in which limited oxidation of lignin and attack on crosslinks between lignin and carbohydrate leads to increased solubility. Chemical analyses, including solid-state [L3C]NMR spectroscopy, point at least to an increase in carbonyl content, probably due to oxidation of phenylpropanoid side-chains at the Cc~ position [48,49]. This would tend to increase solubility but it has been suggested that the primary effect is to increase the susceptibility of /~-ether linkages to cleavage by a /~-etherase such as that detected in Stm. ~iridosporus using model compounds [117]. However, this enzyme is cell-associated and therefore would not account for lignocarbohydrate solubilisation by cell-free preparations of Thin. mesophila. Identification of the enzymes involved in lignin degradation by actinomycetes is the most important priority in current and future research. Implication of ~-etherase in lignin degradation by Stm. uiridosporus is supported by the correlation between enhanced APPL production and elevated levels of this enzyme in mutants and recombinants produced by protoplast fusion [88]. The inducible nature of extracellular lignocarbohydrate solubilis-

157

ing activity in Thin. mesophila [49] should also greatly assist the application of recombinant DNA techniques to identification, and ultimately amplification, of the encoding genes.

6. POTENTIAL B I O T E C H N O L O G I C A L APPLICATIONS The feasibility of lignocellulose bioconversion is dependent on many factors and current and potential processes have been comprehensively reviewed by Wood [118]. Apart from political and economic considerations, obtaining maximum p r o d u c t yield and effective scale-up of laboratory-based systems are the most important parameters. In these respects, actinomycetes are well-placed among those microorganisms which can utilise lignocellulose. Although many aspects of actinomycete physiology are poorly understood, there is a strong background of expertise in largescale cultivation of actinomycetes on a variety of substrates for commercial antibiotic production. Lignin degradation by actinomycetes, although limited at present, is subject to less stringent physiological regulation than ligninolytic activity in white-rot fungi, and this would also be advantageous. Furthermore, strain improvement using recombinant DNA techniques can be readily applied to actinomycetes, particularly Streptono'ces spp. A range of genetic manipulation strategies is available, based on the development of protoplast transformation and fusion techniques and the construction of plasmid and phage cloning vectors (see [2]). Improving enzyme production by gene amplification and derepression, and the construction of recombinant strains which can selectively delignify or completely solubilise lignocellulose are amongst the most important objectives. The application of recombinant DNA technology has the potential to overcome problems of insufficient yield, as has been demonstrated for xylanase production by streptomycetes [102], discussed in Section 4.2. In contrast, strain improvement by genetic manipulation is less attainable in fungi. In the streptomycetes, hybrid antibiotic synthesis [119] and the cloning of antibiotic biosynthesis genes [120,121] have already been achieved, indicating

that our understanding of actinomycete molecular genetics has progressed to the point where real biotechnological applications are tenable. Lignocellulose bioconversion processes can be generally subdivided into those which involve the growth of microorganisms directly on the substrate and those in which cell-free enzyme preparations are used. The latter are primarily associated with high-technology applications such as the generation of sugars for commercial ethanol fermentation, while the former could have low-technology applications e.g. upgrading lignocellulose wastes for animal fodder. In fact, research on the biotechnological applications of lignocellulose-degrading actinomycetes has been concentrated on three main areas: the production of useful chemicals from lignin; single-cell protein from cellulosic wastes; saccharification of cellulosic feedstocks. Biological delignification as an alternative to chemical pulping has yet to be achieved on a commercially attractive scale. However, the ligninases of Phan. cho,sosporium could have applications in commercial pulp-bleaching procedures [122]. There is no comparable work with the ligninolytic actinomycetes, which are generally more active against grass lignins. Here the emphasis has been on the use of streptomycetes to generate useful low molecular weight phenolic compounds from waste lignocellulose [113] and the possible use of actinomycete-solubilised lignocarbohydrate (APPL) as an antioxidant or surfactant [88]. The latter workers have also made some progress towards enhancement of APPL production from corn stover lignocellulose by the application of mutagenesis and protoplast fusion programmes. Growth of actinomycetes on lignocellulose is already an important component of the composting process. While little is known of their specific role in the compost microflora, applications where actinomycete growth appears to be vital include the composting of municipal solid waste [22,123], sewage sludge [124] and the creation of an environment suitable for mushroom growth in compost [125]. In these and possible future applications of solid state mixed culture fermentation, process control by environmental manipulation could result from an improved understanding of

158

actinomycete lignocellulose degradation. This could also lead to the development of engineered actinomycete inocula for efficient conversion of lignocellulosic wastes, although the costs associated with any requirement for a sterile system are likely to be prohibitive. Conversion of cellulose to single cell protein in the form of actinomycete biomass is a logical development from observations on the role of actinomycetes in composting but has been studied in conventional liquid monoculture. Thermophilic actinomycetes have attracted most attention. On a laboratory scale, Thm. fusca growth on waste cellulose has been used in feeding trials on chickens [126] and Thin. fusca, Thm. curvata and Pseudonocardia thermophila found to increase digestible protein at the expense of cellulose in fermentations of pig faeces and straw [127]. On a larger scale, Humphrey and coworkers [128] grew a cellulolytic Thermomonospora sp. on Avicel crystalline cellulose in both batch and semi-continuous culture in a 70-1itre fermentor. Their choice of strain for single cell protein production was based on the potential for easy recovery of filamentous growth, optimal growth at 55 C permitting simple control of heat transfer during fermentation, and the high methionine content of Thermomonospora protein. While the results were encouraging, further improvements in cell productivity, yield and cellulose utilisation would be required. At present, the economic feasibility of single cell protein production from lignocellulose is adversely affected by the requirement for pretreatment, e.g. with sodium hydroxide, to improve cellulose utilisation and the competitive price of alternative sources of protein such as soybean and fishmeal (see [118]). Ethanol is the main product associated with the development of systems for the saccharification of lignocellulose. There are essentially two alternatives: single-step conversion to ethanol by a fermentative cellulolytic microorganism such as Clostridium thermocellum or a two-step process in which sugars are first generated by acid or enzyme hydrolysis prior to fermentation, usually by Saccharomyces cerevisiae. Actinomycetes are a potential source of enzymes for the latter process and again thermophilic Thermomonospora spp. have

received most attention. The activity and stability of Thermomonospora cellulases makes them attractive for efficient high-temperature saccharifications in which enzyme recycling is almost mandatory for economic feasibility. Furthermore, Thermomonospora enzymes may have an application in a coupled process in which a manipulated thermophilic Bacillus sp. ferments sugar to ethanol at 70 C, with recovery of ethanol by direct distillation [129]. Improved cellulose saccharification can be achieved by addition of /~-glucosidase to relieve cellobiose inhibition, but this enzyme is more thermolabile than cellulase [66]. Improved cellulase production has also been achieved by mutation of a wild-type Thermomonospora strain [87]. In all cases, efficient saccharification of lignocellulose will require the concomitant production of xylose, with the development of strains which can ferment xylose to ethanol. Characterisation of Thermomonospora xylanases has only recently been instigated [65,67]. Substrates such as bagasse and cereal straw, which are less lignified than wood and contain almost equal amounts of cellulose and xylan, would be the most suitable waste substrates for conversion to sugar feedstocks. In addition to their role in lignocellulose saccharification, xylanases have more specific potential applications in the paper and fibre industries. Purified cellulose is required in viscose rayon production and paper pulps may contain up to 30% xylan. Specific removal of xylan without affecting cellulose yield or, in the case of paper pulps, fibre structure cannot be achieved by chemical treatment and has stimulated studies on the purification of xylanases from fungal cellulase preparations [130,131]. Saccharomonospora viridis is a thermophilic actinomycete which degrades xylan but not cellulose [30] and therefore has the advantage that crude cellulase-free xylanase preparations can readily be prepared. Such a preparation has already been used to specifically remove 20% of the xylan associated with birchwood pulp, prior to the production of paper which exhibited a number of modified properties [132].

159 ACKNOWLEDGEMENTS The author's r e s e a r c h in t h i s a r e a h a s been cellulolytic rumen bacterium. Micromonospora ruminantium sp. nov. J. Gen. Microbiol. 82, 57-65. [14] Hungate, R.E. (1946) Studies on cellulose fermentation. If. An anaerobic cellulose decomposing actinomycete: Micromonospora propionici n. sp. J. Bact. 51. 51-56. [15] Rowbotham. T.J. and Cross, T. (1977) Ecology of Rhodococcus coprophilus and associated actinomycetes in freshwater and agricultural habitats. J. Gen. Microbiol. 100, 231 240. [16] Lacey, J, and Dutkiewicz, J. (1976) Isolation of actinomycetes and fungi from mouldy hay using a sedimentation chamber. J. Appl. Bacteriol. 41, 315-319. [17] McCarthy, A.J. and Broda, P. (1984) Screening for lignin-degrading actinomycetes and characterisation of their activity against [14C]lignin-labelled wheat lignocellulose. J. Gen. Microbiol. 130, 2905-2913. [18] Godden, B. and Penninckx. M.J. (1984) Identification and evolution of the cellulolytic microflora present during composting of cattle manure: on the role of actinomycetes sp. Ann. Microbiol. (Inst. Pasteur) 135B, 69-78. [19] Hankin, L., Poincelot, R.P. and Anagnostakis. S.L. (1976) Microorganisms from composting leaves: ability to produce extracellular degradative enzymes. Microb. Ecol. 2, 296-308. [20] lizuka, H. and Kawaminami, Y. (1969) Studies on xylanase from microorganisms. II. Isolation and selection of xylanase-producing microorgani'sms and the identification of a new species of Streptomvces. Agr. Biol. Chem. 33, 1257-1263. [21] Crawford, D.L. (1978) Lignocellulose decomposition by selected Streptornvces strains. Appl. Env. Microbiol. 35, 1041-1045. [22] Stutzenberger, F.J., Kaufman, A.J. and Lossin, R.D. (1970) Cellulolytic activity in municipal solid waste cornposting. Can. J. Microbiol. 16, 553-560. [23] Sandrak, N.Y. (1977) Cellulose decomposition by micromonosporas. Mikrobiol. 46, 478-481. (In Russian). [24] Sreenath, H.K., Joseph, R. and Murthy, V.S. (1978) Studies on xylan hydrolases from different strains of Streptomvces and their mutual influences in the breakdown of xylan. Folia Microbiol. 23, 299-303. [25] Phelan, M,B., Crawford, D.L. and Pometto, A.L. (1979) Isolation of lignocellulose-decomposing actinomycetes and degradation of specifically 14C-labelled lignocellulose by six selected Streptomvces strains. Can. J. Microbiol. 25, 1270-1276. [26] Trojanowski, J.. Haider, K. and Sundman, V. (1977) Decomposition of 14C-labelled lignin and phenols by a Nocardia sp. Arch. Microbiol. 114, 149-153. [27] Pasti, M.B. and Belli, M.L. (1985) Cellulolytic activity of actinomycetes isolated from termites (Termitidae) gut. FEMS Microbiol. Lett. 26. 107-112. [28] Stackebrandt, E. and Woese, C.R. (1981) The evolution of prokaryotes, in Molecular and Cellular Aspects of Microbial Evolution (Carlile, M.J., Collins, J.F. and M'oseley, B.E.B., Eds.), pp. 1-31. Cambridge University Press, Cambridge.

funded by the British Petroleum Venture Research Unit, the Agriculture and F o o d Research Council a n d the E.E.C. N o n - N u c l e a r Energy research prog r a m m e , a n d t h e i r s u p p o r t is g r a t e f u l l y a c k n o w l edged.

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