STUDIA FORESTALIA SUECICA The degradation of cellulose and the production of cellulase, xylanase, mannanase and amylase by wood - attacking microfungi Cellulosanedbrytning och produktion av cellulas, xylanas, mannanas och arnylas hos vedangripande mikrosvampar. THOMAS NILSSON Department of Forest Products SKOGSHOGSKOLAN ROYAL COLLEGE OF FORESTRY STOCKHOLM Abstract Thirty-six species of wood-inhabiting microfungi have been assayed for cellulase, xylanase, mannanase and amylase activity by the use of different test methods. The wood-degrading capabilities of the test organisms were previously known. Three of the species were unable to degrade birch wood. The other species represented three different degradation patterns, viz. I) formation of soft rot cavities (Type 1 attack); 2) erosion of the wood cell walls (Type 2 attack);and 3) simultaneous Type 1 and Type 2 attack. 'With one exception, all species were able to degrade starch. The production of the enzymes cellulase, xylanase and mannanase was demonstrated for twenty of the wood-degrading species. Five of the wood-degrading species, which all produced soft rot cavities in birch wood, failed to exhibit any of the enzyme activities. The remaining wood-degrading species could be shown to produce one or two of the enzymes. One of the species which was unable to degrade birch wood produced xylanase. Cellulase and mannanase were not produced by any of these species. Twelve of the wood-degrading species appeared unable to degrade pure cellulose substrates when grown on agar media or in liquid cultures. Their only exhibition of cellulolytic activity was found in solid birch wood, where they formed soft rot cavities. These species have been referred to as "non-cellulolytic" soft rot fungi. Various explanations of the anomalous behaviour of these species are discussed. Ms received 27th February, 1974 Allmanna Forlaget ISBN 91-38-01913-2 Tryck: Tryckindustri AB, Solna, 1974 Contents 1 Introduction . 5 2 Materials and methods . 6 2.1 Organisms . 6 2.2 Substrates . 6 2.3 Assay of cellulase, xylanase, mannanase and amylase with the RautelaCowling technique 6 2.4 Cultivation on cellulose agar plates and assay of the cellulase produced . 7 2.5 Assay of cellulase and xylanase present in wood blocks attacked by the test organisms 9 2.6 Cultivation in liquid media. Weight losses of cellulosic substrates and assays of cellu- lase and xylanase . 9 3 Results . 3.1 Type of attack produced in birch wood . 3.2 Clearing produced in agar columns with cellulose, xylan, glucomannan and starch (Rautela-Cowling technique) . 3.3 Growth and cellulase production on cellu- lose agar plates . 3.4 Presence of cellulase and xylanase in birch wood attacked by the test organisms . 3.5 Growth onmedium EP and B-VII-L in liquid cultures with glucose as source of carbon 3.6 Degradation of cellulose N, Avicel and cot- ton and the production of cellulase and xy- lanase on these substrates in liquid cultures 3.7 Degradation of birch wood meal and the production of cellulase and xylanase on this substrate in liquid cultures . 3.8 Degradation of cotton and jute fibres and the production of cellulase and xylanase on these substrates in liquid cultures . 3.9 Effect of various amounts of glucose added to medium EP on the production of cellulase and xylanase . 3.10 Comparison of the results obtained with different test methods . 4 Discussion . 22 4.1 Wood degradation and enzyme production 22 4.2 Test methods and results . 24 4.3 The anomalous behaviour of the "non- cellulolytic" soft rot fungi . 31 Summary . 35 Acknowledgements . 37 Sammanfattning . 3 8 References . 40 Tables . 43 1 Introduction In a previous paper (Nilsson 1973) the method for cellulolytic activity mentioned celluloly tic activity of 160 species of micro- above had been used. fungi was compared with their ability to degrade birch wood (Betula verrucosa In order to investigate whether this anoma- Ehrh.). The Rautela and Cowling (1966) lous behaviour is a consistent feature of this method was used for assays of celluloly- group of fungi or whether it is an effect due tic activity. With this method cellulolytic to the test method used for assaying cellulo- activity is measured as the depth of clearing lytic activity, further studies have been of cellulose beneath fungal cultures growing carried out with other assay methods. The on the top of agar columns in test tubes. The studies have also been extended to assays of ability to degrade birch wood was deter- xylanase, mannanase and amylase. It is likely mined by microscopic observations on sec- that xylanase and mannanase are involved in tions from wood which had been attacked. the degradation of wood and it was intended Loss in weight of the birch wood was also to examine whether fungi which had failed determined for several of the species. to show cellulolytic activity also would fail to exhibit xylanase and mannanase activities. When using the decay method employed Amylase activity was assayed only for the (Nilsson 1973), it was found that the micro- purposes of comparison. fungi could be classified into four groups with respect to their type of attack in birch Thirty-six different species of microfungi wood: 1) fungi producing no attack, or were selected from our culture collections producing no other attack than small bore for these studies. Their type of wood attack holes through the cell walls; 2) fungi produ- was known and the species were selected so cing only soft rot cavities (Type 1 attack); that representatives from all of the four 3) fungi producing only a type of erosion of groups of the fungi mentioned above were the cell walls (Type 2 attack); and 4) fungi included. producing both soft rot cavities and erosion. The objects of the present investigation were A comparison of the results from the assays two; 1) to demonstrate the production of of cellulolytic activity and the results from cellulase, xylanase and mannanase, enzymes the wood decay tests showed that there was which a priori were assumed to be produced a good correlation between the actual clear- by the wood-degrading species, and 2) to ing of cellulose and the ability to degrade obtain information about the conditions birch wood. Of the 109 species which under which these enzymes are produced in produced clearing of cellulose, 105 (96.3 %) order to achieve a better understanding of were able to degrade birch wood. However, the mechanisms of wood degradation. No several of the species tested (9.4 %), which specific studies of the activities of the were able to produce soft rot cavities in the various enzymes were attempted. The pur- wood, failed to produce any clearing of pose was merely to demonstrate the presen- cellulose. They would thus have been consi- ce or absence of the various enzymes by the dered as non-cellulolytic if only the assay use of different test methods. 2 Material and methods 2.1 Organisms chloric acid according to a method described by Bose ( 1963). Thirty-six different species of microfungi were studied. All species are listed in Table 1. Cotton wool. Chemically pure (Kebo AB). Data on isolation, type of wood attack, weight losses of wood and cellulolytic activity Cellulose N (Nattraby cellstoff). This cellulo- can be obtained from a previous paper se is a commercial product prepared from (Nilsson 1973) for all but four of the equal amounts of bleached pine sulphate species, viz. Ceratocystis albida (Mathiesen- pulp and bleached pine sulphite pulp. Accor- Kaarik) Hunt, Ceratocystis stenoceras (Ro- ding to the manufacturer (Molnlycke AB) bak) C. Moreau, an unidentified Cladospo- this cellulose contains only small amounts of rium species, tentatively called Cladospo- hemicellulose. rium sp. A, and an unidentified imperfect fungus called Fungus D. Fungus D is very Jute fibres. From Corchorus olitorius (red common in Sweden in chip piles and preser- jute). Received from Dr. N.J. Poole at the vative treated poles. The strain used here was School of Agriculture, Aberdeen. isolated 1968 from spruce pulpwood chips. Ceratocystis albida (strain B-23) was isola- Birch wood meal. 80 mesh. Prepared from ted 1952 by Dr. A. Kaarik from galleries of Betula verrucosa. Pissodes pini in a pine log. Ceratocystis stenoceras (strain B-104) was obtained Larch xylan. (Koch-Light Laboratories from "Centraalbureau voor Schimmelcultu- Ltd.). res" in Baarn, and Cladosporium sp. A (strain SP78-4) was isolated 1971 from a Glucomannan. Prepared from Pinus silvestris pine foundation pile. at Swedish Forest Products Research Labo- ratory. The preparation contained 78.2 per- cent mannose, 17.8 percent glucose, 1.4 percent galactose, 1.4 percent xylose and 1.2 2.2 Substrates percent arabinose. Avicel. A microcrystalline cellulose prepara- Starch. Soluble starch. (E. Merck, Darm- tion. Average particle size 38 ,U (Kebo AB). stadt). Walseth cellulose. Cellulose swollen in 85 percent o-phosphoric acid. Prepared from cellulose powder (Whatman CF 11) accor- 2.3 Assay of cellulase, xylanase, mannanase ding to the description by Rautela and and amylase with the Rautela-Cowling Cowling (1966). technique Ball-milled cellulose. This cellulose was ob- The technique employed by Rautela and tained from Dr. H.O.W. Eggins and Bernard Cowling (1966) for testing the cellulolytic King at the Biodeterioration Information activity of fungi was adopted. Vertical agar Centre, University of Aston in Birmingham. columns (approx. height 40 mm), containing the various substrates were prepared in test HCI-cellulose. Cellulose powder (Whatman tubes (18 mm diam.). The two media descri- CF 11) treated with concentrated hydro- bed in a previous study (Nilsson 1973) were also used here. The media had the following Walseth cellulose 0.25 R-C compositons: Walseth cellulose 0.125 R-C Walseth cellulose 0.25 B-VII R-C medium (after Rautela and Cowling Walseth cellulose 0.125 B-VII 1966) Ball-milled cellulose 0.2 B-VII NHqH2P04 2.0 g, KH2P04 0.6 g, K2HP04 Ball-milled cellulose 0.2 A 0.4 g, MgS04' 7 H20 0.89 g, thiamine HC1 Ball-milled cellulose 0.2 B 100 pg, yeast extract 0.5 g, adenine 4.0 mg, HC 1-cellulose 0.125 B-VII adenosine 8.0 mg, agar 17 g and distilled Xylan 0.125 B-VII water to make 1 liter.