Fine structure and mechanical properties of straw filaments invaded by Pleurotus ostreatus

Biological Wastes 27 (1989) 87-100

Fine Structure and Mechanical Properties of Straw Filaments Invaded by Pleurotus ostreatus A. Schiesser Dipartimento di Agrobiologia e Agrochimica, Sez. di Microbiologia, Universit~ d'ella Tuscia, Loc. Riello, 01100 Viterbo, Italia

C. Filippi* lstituto di Microbioiogia Agraria e Tecnica, Universit~ degli Studi di Pisa,

Via del Borghetto 80, 56100 Pisa, Italia

G. Totani & A. A. Lepidi Dipartimento Ingegneria delle Strutture, Acque e Terreno, Dipartimento di Scienze, Tecnologie Biologiche, Mediche e Biometria, Universit~i dell'Aquila, 67100 L'Aquila, Italia (Received 4 February 1988; revised version received 17 April 1988; accepted 22 April 1988)

ABSTRACT

Wheat straw filaments attacked by Pleurotus ostreatus under different nutritional conditions were examined by transmission and scanning electron microscopy; their resistance to traction was determined by a ring dynamometer. Micromorphology and mechanical properties o f the filaments were consistent with a mechanism o f fungal attack subdivided into two steps, the first dominated by hyphal growth on the soluble components present in the straw or supplemented with the medium, and the second during which lignocelluloses are degraded at a rate related to the amount o f biomass produced during the first stage and to the nutrients left in the medium. * To whom correspondence should be addressed. 87 Biological Wastes 0269-7483/89/$03-50 © 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain

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INTRODUCTION Degradation of dead plant materials containing ligno-celluloses is accomplished by several micro-organisms among which higher fungi are considered to play a relevant role. Experimental cellulose and lignin hydrolyses are expected to evolve into very promising biotechnologies, such as biological delignification of wood and straw for paper making (Eriksson & Vallander, 1982; Eriksson et al., 1983), cellulose saccharification and ruminant feeding (Chesson et al., 1980; Akin et al., 1984; Reid, 1985), hydrolysis of lignin components of plant tissues into chemicals usable as a feedstock (Kirk & Moore, 1972; Scalbert et al., 1985) or hydrolysis of cellulose to simple sugars (Koshijma et al., 1983). Nutritional and physiological requirements of the organisms growing on ligno-celluloses have been studied in some detail in a few cases, with major attention on the factors affecting the production of cellulolytic and ligninolytic enzymes. Simple sugars are well known as inhibitors of synthesis and activity of the lignin- and cellulose-hydrolysing proteins. In this connection, the soluble materials associated with the native celluloses have been either disregarded or suspected of inhibiting the attack on these polymeric structures (Jeffries et al., 1981). In the present paper, data are reported on the growth of Pleurotus ostreatus strain Floridae on wheat straw under various conditions; on the micro- and fine-structure of straw and fungus cells during microbial attack, and the residual mechanical characteristics of the straw filaments. These data are consistent with a model of attack by Pleurotus ostreatus on native wheat straw starting with a first step during which the soluble materials are the preferential supporters of the fungal growth and maintenance. The fungal biomass resulting from the utilization of the soluble substances is finally responsible for a massive attack on the cellulose and lignin polymers. Pleurotus ostreatus is largely cultivated on straw-containing litters as a potential producer of safe and digestible proteins for humans and animals. The strain Floridae, which was used in this work, is an active producer of lignin- and cellulose-degrading enzymes (Hiroi & Eriksson, 1976; Giovannozzi-Sermanni et al., 1982; Reid & Seifert, 1982; Platt et al., 1983; Giovannozzi-Sermanni, 1986). METHODS

Micro-organism Pleurotus ostreatus strain Floridae was kindly provided by Professor G. Giovannozzi, Director of the Department of Agrobiology and Agrochemistry, Tuscia University. Cultures were transferred monthly to fresh potato

Invasion of straw by P. ostreatus

89

dextrose agar (Difco), supplemented with 0.5% yeast extract (Oxoid). The basic medium (BM) for growth on wheat straw filaments contained (g/litre): K2HPO 4 1.7; KH2PO4 0.68; MgSO47H20 0.2; NaCI 0.14; CaC12H20 0.13 together with (mg/litre): H3BO 3 2-9; FeSO47H20 2.5; NaMoO42H20 2"5; ZnSO47H20 1.2; CoC12 1.0; CuSO45H20 0.01; MnCI24H20 0"09; Biotin 0.02. Where specified, BM was supplemented with either glucose (5 g/litre, BM-G), or ammonium sulphate (200mg/litre, BM-N) or yeast extract (5 g/litre, BM-Y) or combinations of these nutrients.

Growth on straw filaments Sections of straw filaments, cut to a length of 5 cm and an average breadth of 2.5mm, in the internodes, were placed in 10ml tubes containing 0.2ml distilled water and sterilized at 120°C for 20 min; 2 ml of sterile medium were then added. Hyphal suspensions for inoculation were obtained from mycelia actively growing on the complete medium (BM-G,N,Y); such mycelia were homogenized for a few seconds at 4°C in an Ultra-Turrax/TP-18N (Janke & Kunkel GmbH & Co IKA-Werk 7813 Staufenim Breisgau) and washed twice by centrifuging from sterile distilled water. The hyphal pellets were resuspended in sterile distilled water and the tubes containing straw and medium were inoculated with 0-2 ml of hyphal suspension.

Chemical analyses Composition of the straw was determined according to Stephen et al. (1981).

Transmission electron microscopy Fragments of c. 0-15 x 0.25 x 0.15cm were cut from the straw filaments submitted to the fungal attack. The fragments were washed twice in phosphate buffer (0-05M, pH 6-8) and fixed for 12h at 4°C with 3% glutaraldehyde in the same buffer. The samples, rinsed several times with the buffer, were post-fixed with OsO4 (1% in the same buffer) for 6 h at room temperature. After dehydration through an ethanol series and two passages in propylene oxide, samples were embedded in Epon-Araldite by conventional methods with a sequential polymerization for 4 h at 37°C and then at 65°C. Sections were cut by a diamond knife using a Reichert U K 12 ultramicrotome, stained with uranyl acetate and then with lead citrate (Reynolds, 1963) and finally observed with a Siemens Elmiskop IA transmission electron microscope.

Scanning electron microscopy Fragments of straw, incubated in various conditions, were fixed overnight with glutaraldehyde (6% in phosphate buffer 0.2si, pH 7"2) and then

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A. Schiesser, C. Filippi, G. Totani, A. A. Lepidi

dehydrated at the critical point (Boyde & Wood, 1969) by an Edwards Sputter Coater S 150 B, gold coated by a Balzers Union Mod. CPD 020 and observed with a Philips SEM 505.

Measurement of mechanical properties Mechanical properties of the wheat straw filaments were measured by a flexible ring dynamometer at constant rate (WF 25.000 by WykehanFarranch, Slough, UK). The straw filament was fixed with two clamps (purposely modified so as not to damage the straw), the first being displaced by an electric motor and the other being connected with an elastic metallic ring. The initial distance between the clamps was 2.5 cm in order to measure the mechanical character of the middle part of the filament. Such a middle part was chosen after the elimination of the bottom part of the filament which was plunged in medium during the incubation and the upper part where soluble components were possibly accumulated by capillary migration. Just before the mechanical measurement, to ensure a uniform humidification filaments were kept for 2 h in a container fully saturated with water in the presence of some liquid water. A longitudinal traction, which is opposed by the ring deformation, is exerted on the filament at a constant rate by the electric motor. The data, elaborated by a computer program developed according to the characteristics of the apparatus, were used to show the filament elongation vs the tractive force exerted on the filament.

RESULTS A N D DISCUSSION The chemical composition of the straw is reported in Table 1. The content of soluble substances is similar to the figures reported in the literature (Stephen et al., 1981) and its organic portion is higher than the nutrient concentration usually adopted to prepare culture media. The reason why the growth of micro-organisms on native straw occurs to a limited extent using such soluble materials is that they are not balanced, and limiting factors of the growth are likely to occur; furthermore, physical accessibility of the individual nutrients is not necessarily uniform. It seems likely that cellulolytic and/or ligninolytic micro-organisms growing as a pure culture on straw could gain some advantage by a preliminary growth on the soluble organic substances in order to attain the starter biomass for the following steps of ligno-cellulose utilization, even if, for the above-cited factors, the microbial growth on the solutes is slow and not uniform. Soluble nutrients are known to influence microbial attack on lignin and cellulose. Soluble sugars having no fl-glucoside linkages inhibit cellulase synthesis and activity

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TABLE 1 The Chemical Components of the Internode Zone of Wheat Straw

Component

Percentage of dry weight

Hot-water-soluble Lignin Hemicelluloses Cellulose Ash Total N P20 5 K20 Ca Mg

14-92 15.30 29.83 37.70 11.00 0"30 0"29 0"12 0"40 0"36

at concentrations lower than those of sugars found in straw tissues (Montenecourt, 1983; Eriksson & Hamp, 1978; Sterneberg, 1976). The expected mode of microbial growth on the straw (and similar matter such as woody materials), inoculated with a single ligno-cellulosic microorganism in the presence of added balanced sources of mineral nutrients, would be: a rapid growth on organic soluble nutrients usable by the inoculated micro-organisms and then the utilization of cellulose and lignin after induction of the proper enzymes. In fact, in c o m m o n experience the ligno-cellulose hydrolysis always proceeds very slowly and large biomasses are found on straw only after a long incubation even with added mineral nutrients (Knapp et al., 1983; P l a t t e t al., 1983). These facts suggest that the localization of the soluble compounds in ligno-cellulosic materials could affect their utilization by micro-organisms. Soluble organic nutrients are not homogeneously distributed in the straw tissue, being concentrated mainly inside the cell lumen from where they migrate at a very low rate even when the tissue is saturated with water. Hyphal cells, or hyphal segments, need to penetrate the cell lumen to be able to use the soluble content and consequently they need to hydrolyse at least a minor portion of the cell wall. On this basis, microbial invasion of the straw tissues should show the following characters at the micro- and ultrastructural-levels: hyphae would be mainly concentrated in the lumen of the straw cells and in the intercellular spaces, irrespective of the medium used, but the cell walls of straw filaments supporting the mycelial growth would remain on a macroscopic scale apparently uninvaded; the hyphae would pass directly from cell to cell rather than to diffusely invade the cell wall itself.

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Fig. 1. Scanning EM of wheat straw filaments after 10 days of incubation in the presence of glucose and combined nitrogen (BM-G-N, see Methods). (A) Control (uninoculated); (B), (C) and (D)---on the external (inner and outer) surfaces of the straw filament, a hyphal coat can be observed (B). After mechanical removal of the hyphal coat from the inner surface a moderate invasion of the tissue can be observed (C), the hyphae being located in the intra- and intercellular spaces. Such hyphae are occasionally responsible for minor damages to the plant cell wall (see also Fig. 2(13)). Removal of hyphal coat from the outer surface of the straw leaves an undamaged surface (D).

O

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Fig. 2. Scanning EM of wheat straw after 10 days of incubation with the medium with combined nitrogen (BM-N). Relationships between the fungal hyphae and the inner surface of the straw filament. Notice the micelial appendages passing through the plant tissue cells wall (C), see also Fig. 4(D) and (F) and the hyphae invading intra- and intercellular spaces (A), (B), (D).

?,

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The results of microstructural examination of straw filaments inoculated with Pleurotus ostreatus after addition of different combinations of nutrients (Figs 1, 2) are consistent with such a scheme. A model describing the geometry ofmycelial growth in the straw has been developed by Laukevics et al. (1985). According to figures calculated by these authors, at high nutrient concentrations the geometry of hyphal growth (branching, average distance among hyphal filaments) will be the limiting factors of the biomass growing on a solid porous substrate. According to such a model, the final amount of biomass which can be expected to be produced by a fungus during the first colonization of the straw tissues is related to the utilization of the soluble components only with no contribution from cellulose. The pattern of the first stage of straw tissue colonization by the hyphae is dominated by the search for and utilization of organic solutes, but this will determine, in further stages, the rates of cellulose and lignin utilization (provided that sufficient mineral nutrients are available). In our experiments, the damage to the ligno-cellulosic structures was measured by the loss of mechanical properties of the straw filaments. The results, expressed as filament elongation vs the applied force, are reported in Fig. 3 and indicate greater decreases in the mechanical characteristics after incubation in rich media (Table 2). The native straw (Fig. 3(a)) reacts to elongation effort like a homogenous and elastic material. It elongates 4.88% the original length and tolerates a force higher than 0.7 kg/mm 2. After incubation in the absence of exogenous combined nitrogen, irrespective of the presence of glucose (Fig. 4(B), (D)), the 0.8

a

0.7 0.6 E

0.5 0.4 0.3

~

0.2 0.1

e

0.0 0

0.120.240.360.490,61 0.730.85098

1.1 1.22

elongation (m m)

Fig. 3. Results expressed as filament elongation vs the applied force after incubation with Pleurotus ostreatus in conditions more, or less, favourable to the biomass growth. (a) Uninoculated control; (b) BM; (c) BM-N; (d) BM-G; (e) BM-Y.

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TABLE 2

Breaking Points (Specific Elongation versus Specific Traction) of Straw Filaments Supplemented with Different Exogenous Nutrients and Incubated for 10 Days with Pieurotus ostreatus Medium

Elongation ( mm/mm )

Traction ( kg/mm 2)

Uninoculated control

0.049

0-76

BMo BM-N BM-G BM-Y BM-N-G BM-N-Y BM-G-Y

0.038 0"020 0.019 0"014 0.019 0"010 0"018

0.23 0-18 0"31 0.07 0"23 0"03 0.28

BM-N-G-Y

0"014

0.13

° F o r m e d i a see M e t h o d s .

straw filament breaks when subjected to a force ofc. 0.25 k g / m m 2 by which it is extended c. 4% of the original length. The incubation with media containing a m m o n i u m or yeast extract promotes a sharp decrease of mechanical performance (Fig. 3(c), (e)). After incubation in BM-Y the filaments are broken by applied forces below 0.1 k g / m m 2 and the maximum tolerated elongation is lower than 2%. The mechanical performance here measured is probably connected with the integrity of the ligno-cellulosic structures. Unfortunately, the break-point reflects merely the weakest site along the filament and the decrease of elasticity and strength cannot be definitely related to a damage uniformly distributed along the filament. It is interesting that more evident damage to ligno-cellulosic structures is found after incubation in richer media. Space occupation by the fungal cells evidenced by T E M (Fig. 4) is in agreement with the model proposed by Laukevics et al. (1985). The attack on the ligno-cellulose structures evidenced by the changes in mechanical characteristics would be aided by the population turnover stimulated by the nitrogen-rich media more than by a m o u n t of primary colonization of the tissue which is likely to occur by the use of soluble substances. As final remarks, the evidence reported here indicates that the colonization of the straw filament by Pleurotus ostreatus can be subdivided into two steps. During the first one, the fungal growth is supported by the soluble materials present in the straw tissue mainly in the intra- and intercellular spaces. The attack on ligno-celluloses occurs as a secondary

0 )0

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stage after the exhaustion o f the available organic matter o f the solutes (of course, at the microsite level), and such an attack is dependent on the a m o u n t o f mycelium already grown and on the availability o f mineral nutrients. The addition o f external sources o f nutrients favours the production o f larger a m o u n t s o f fungal biomass and the residual presence o f minerals promotes the secondary use o f ligno-cellulosic structures.

ACKNOWLEDGEM ENTS Financial support was provided by the National Research Council o f Italy (CNR): special grant I P R A (Sub. Proj. 3, paper no. 1670). The helpful technical collaboration o f Mrs G i o v a n n a Bagnoli and Paola Tamantini is gratefully acknowledged.

REFERENCES Akin, D. E., Rigsby, L. L. & Brown, R. H. (1984). Ultrastructure of cell wall degradation in Panicum species differing in digestibility (and references cited). Crop Sci., 24, 156-63. Boyde, A. & Wood, C. (1969). Preparation of animal tissue for surface scanning electron microscopy. J. Micr., 90, 221-49. Chesson, A., Stewart, C. S. & Wallace, R. J. (1980). Influence of plant phenolic acids on the growth and cellulolytic activity of rumen bacteria. Environ. MicrobioL, 44, 597-603. Eriksson, K. E. & Hamp, S. (1978). Regulation of endo-1-4-fl-glucanase production in S. pulverulentum. Fur. J. Biochem., 90, 183-90. Eriksson, K. E. & Vallander, L. (1982). Properties of pulp from thermomechanical pulping of chips pretreated with fungi. Svensk. Papperstidn., 85, 33-8. Eriksson, K. C., Johnsrud, S. E. & Vallander, L. (1983). Degradation of lignin and lignin model components by various mutants of the white-rot fungi Sporo trichun pulverulenturn. Arch. Microbiol., 135, 161-8.

Fig. 4. Transmission EM of wheat straw filament after 10 days of incubation with medium containing ammonium sulphate (200mg/litre), (A), (B),(D) and (F) or both ammonium sulphate and glucose (200 mg/litre and 5 g/litre), (C) and (E). When only combined nitrogen was added to the medium, a low fungal cell density was found in both plant cell lumen and intercellular spaces, some fungal hyphae were perforating the plant cell wall (D) and (F); the perforation sites were not surrounded by an extensive degradation of the plant cell wall material so that perforating hyphae were possibly exploring for favourable environments more than extensively utilizing the substrate. In the presence of both combined nitrogen and glucose, fungal cells were abundant in the intercellular spaces (C) as well as on the inner face of the straw filament where the plant cell walls are less structurated (E); attacks to and perforations of plant cell wall are apparently missing.

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Giovannozzi-Sermanni, G., Badiani, M. & Luna, M. (1982). Protein production and laccase activity in Agaricus disparus. Biotec. Lett., 4, 507-12. Giovannozzi-Sermanni, G. (1986). Solid state bioreactor for straw metabolization. Int. Conf. Biotecnol. and Agr. in Mediterranea Bas., Atene. Hiroi, T. & Eriksson, K. E. (1976). Microbiological degradation of lignin. Part 1 Influence of cellulose on the degradation of lignins by the white-rot fungus Pleurotus ostreatus. Sven. Papperstidn., 79, 157-61. Jeffries, T. W., Choi, S. & Kirk, T. H. (1981). Nutritional regulation of lignin degradation by Phanerochaete chrysosporium. Appl. Environ. Microbiol., 42, 290-96. I Kirk, T, H. & Moore, W. E. (1972). Removing lignin in wood with white-rot fungi and digestibility of resulting wood. Wood Fiber, 4, 72-9. Knapp, T. H., Elliot, L. F. & Campbell, G. S. (1983). Microbial respiration and growth during the decomposition of wheat straw. Soil Biol. Biochem., 15, 319-23. Koshijima, T., Yaku, F., Muraki, E., Tanaka, R. & Azuma, J. (1983). Wood saccharification by enzyme system without prior delignification. In Proceedings of the Ninth Cellulose Conference H Symposium on Cellulose and Wood as Future Chemical Feedstocks and Sources of Energy and General Papers, ed. A. Sarko. Laukevics, J. J., Apsite, A. F., Viesturs, U. S. & Tengerdy, R. P. (1985). Steric hindrance of growth of filamentous fungi in solid substrate fermentation of wheat straw. Biotechnol. Bioeng., 27, 1687-91. Montenecourt, B. S. (1983). Trichoderma reesei cellulases. Trends in Biotechnology, Vol. I, 156-61. Platt, M. W., Hadar, Y., Henis, Y. & Chet, I. (1983). Increased degradation of straw by Pleurotus ostreatus sp. 'Florida'. Eur. J. Appl. Microbiol. Biotechnol., 17, 140-42. Reid, I. D. (1985). Biological delignification of aspen wood by solid-state fermentation with the white-rot fungus Merulius tremellosus. Appl. and Environ. MicrobioL, 50, 133-9. Reid, I. D. & Seifert, K. A. (1982). Effect of an atmosphere of oxygen on growth, respiration and lignin degradation by white-rot fungi. Can. J. Bot., 60, 252-60. Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Biophys. Biochem. Cytol., 17, 208-12. Scalbert, A., Monties, B., Lallemand, J. Y., Guittet, E. & Rolando, C. (1985). Ether linkage between acids and lignin fractions from wheat straw. Photochemistry, 24, 1359-62. Stephen, H. T., Lynch, H. & Lynch, J. M. (1981). The chemical components and decomposition ofwheat straw leaves, internodes and nodes. J. Sci. Food Agric., 32, 1057-62. Sterneberg, D. (1976). Production ofcellulase by Trichoderma. Biotechnol. Bioeng., 6, 35-53.