Biodegradation 3: 189-205, 1992. © 1992KluwerAcademic Publishers. Printedin the Netherlands. Utilization of lignocellulosic waste by the edible mushroom, Pleurotus Yitzhak Hadar, Zohar Kerem, Barbara Gorodecki & Orly Ardon The Otto Warburg Center for Biotechnology in Agriculture, Faculty of Agriculture, P. O. Box 12, Rehovot, 76100 Israel Key words: lignin degradation, Pleurotus, lignocellulose utilization, solid state fermentation Abstract Lignocellulosic waste represents huge amounts of unutilized renewable resource. The use of the polysaccharides in the lignocellulosic complex is limited due to their high lignin content. White rot fungi are capable of selectively degrading lignin, thereby upgrading it. The focus of this article is on the potential utilization of edible mushrooms of the genus Pleurotus, via solid state fermentation, using cotton plant stalks as a substrate. This material poses agrotechnical problems since the stalks have a fibrous structure similar to that of hardwood. Potential uses for this material are as a fuel in rural areas, a substrate for mushrooms, an animal feed and substrate for paper making. In this study, degradation of cotton stalks by Pleurotus is described using chemical analyses and scanning electron microscopy. During four weeks of solid state fermentation, lignin content significantly decreased and in vitro digestibility was increased. The fermentation product was consumed by ruminants at a level of up to 40% of their diet. Introduction The lignocellulosic complex constitutes a major portion of the total carbon fixed by photosynthesis. However, only a small part of the cellulose, hemicellulose and lignin produced as agricultural or forestry by-products is utilized, while most of it is considered waste material. In Europe, for example, the amount of unexploited lignocellulosic byproducts is immense. Cereal straw, the major byproduct, shows a yearly surplus of 24.106 tons (Zadrazil & Reinger 1988). Most of the straw is either burned in the field creating environmental and ecological problems, or wasted by being ploughed back into the soil. The direct use of lignocellulosic residue as animal feed, or as a component of such feeds, represents one of its oldest and most widespread applications and as such it plays an important role in the ruminant diet. However, lignocellulosic residues are not high value feeds: they are classified instead as low quality roughage, i.e. high in fiber, low in protein, vitamins and minerals. The lignin component creates the barrier to efficient utilization, conversion or degradation of the polysaccharides in lignocellulose to useful products or high value animal feed (Buswell & Odier 1987). Lignin is defined as a polymeric product arising from an initial enzymatic step followed by chemically driven dehydrogenative polymerization of primary precursors possessing a p-hydroxycinnamyl alcohol structure (Monties 1989). Due to its complex and heterogeneous structure, lignin degradation is slow and limited to a relatively small number of microorganisms (Buswell & Odier 1987). The white rot fungi can degrade all the major components of wood and are generally considered to be the main agents of lignin degradation in nature (Buswell & Odier 1987). The best studied organism of this group is Phanerochaete chrysosporium (Kirk & Farrell 1987; Schoemaker & Leisola [65] 190 1990). Most of the knowledge regarding physiological, biochemical and genetic factors associated with lignin biodegradation as well as their practical uses, mainly in the pulp and paper industry, has been obtained from this model organism. Another group of interesting white rot fungi that can utilize lignocellulose is comprised of the edible mushrooms. These saprophytic basidiomycetes have been successfully cultivated at a commercial level worldwide (Wood & Smith 1987). This paper focuses on the edible mushrooms of the genus Pleurotus and their potential in the utilization and upgrading of lignocellulosic wastes during solid state fermentation (SSF). Cultivation of Pleurotus spp. on lignoceilulosic waste Pleurotus spp. are wood-degrading saprophytic fungi which are widely distributed and cultivated in many countries throughout the world. Pleurotus cultivation is gaining popularity in Europe, America and the Far East, and from the standpoint of annual production, it has become the third most important mushroom, after Agaricus bisporus and Lentinus edodes (Wood & Smith 1987). Commercial production techniques for this basidiomycete are well developed (Tautorus 1985; Wood & Smith 1987; Zadrazil 1978) and relatively simple as compared to those used with the most commonly cultivated mushroom, A. bisporus. Pleurotus ostreatus, popularly known as the "oyster mushroom" is the most common artificially cultivated species. The substrate is usually partially shredded, mixed with water (up to 70%) and placed in containers such as bags, trays or frames. However, unlike A. bisporus, no composting or casing layer is required. Because P. ostreatus is a wood-degrading fungus, it was first cultivated on logs (Falck 1917). Today it has become common practice to prepare Pleurotus substrate from shredded wheat straw, which can be supplemented with protein-rich materials such as alfalfa meal or soybean flour. Since Pleurotus spp. can decompose lignocellulose efficiently without chemical or biological pre-treatment, a large variety of lignocellulosic wastes can be utilized and recy[66] cled. Some examples of the agricultural wastes studied as substrates for Pleurotus spp. are presented here. In coffee-growing regions, coffee pulp is considered to be one of the most abundant, as well as one of the hardest to handle agricultural wastes. To date, the majority of the by-product remains unused, causing contamination problems (Guzman & Martinez 1986). A most economical way of dealing with the excessive amount of pulp and of solving the environmental problem would be to use it for animal feed. However, chemical components such as lignin, phenolics and caffeine hamper its utilization by animals. Caffeine and phenolics are known to exert detrimental effects to both the rumen's microfiora and the host animal. Biological pre-treatment could, however, improve the value of this waste product. The use of coffee by-products as a substrate for Pleurotus has been studied by Leon et al. (1983). Guzman & Martinez (1986) successfully cultivated P. ostreatus on coffee pulp on a semi-industrial scale, at a coffee farm in Mexico. Sharma (1987) studied the utilization of flax shive, a woody by-product of little value. For every ton of flax fiber produced, 2.5 tons of shive are left over. Shive has a high lignin content (24.9%), preventing its direct utilization. Pleurotus grown on this substrate was able to reduce the lignin content of the shive to 12.4%. Cassia spp. are tropical shrubs with medicinal, e.g. laxative, properties. The residue of extracted cassia plants was tested for cultivation of Pleurotus by Muller (1987). The utilization of Pleurotus in the recycling of medicinal plants could be of additional importance in cases where toxic components are present in the material. The reduction of such components by the fungus would simplify the problem of waste disposal (Muller 1987). Oriaran et al. (1989) compared lignin degradation of hard and soft wood chips by P. ostreatus, L. edodes and Phanerochaete chrysosporium. All three white rot fungi selectively degraded the lignin on glucose-supplemented chips, an observation which could be useful in biopulping by the paper industry. A large variety of other wastes, such as corn cobs 191 and leaves, sugar cane bagasse and leaves, citronella bagasse, rice hulls, water hyacinth leaves, cocoa shells and cotton gin waste, could easily be used as substrates for Pleurotus spp. The substrates used in each region would depend on the available agricultural wastes. In our study, the use of cotton stalks as a substrate for SSF by P. ostreatus was analyzed. Platt et al. (1981) and Balasubramanya (1981) have successfully grown P. ostreatus and P. sajor-caju on this substrate. Utilization of cotton stalks as a substrate for Pleurotus fermentation, as animal feed and in paper production Cotton is Israel's main field crop and generates the largest proportion of local agricultural waste. Its worldwide importance is illustrated by the 10 million tons of cotton stalks reported for India yearly (Balasubramanya et al. 1989). Those remaining in the field, 5 tons/ha, are ploughed under the soil surface. In addition to wasting a potential agricultural resource, this treatment could lead to an increase in cotton diseases and pests, as well as to difficulties in cultivation due to slow decomposition in the soil (mainly in dry regions). The major obstacle to using cotton stalks as a mushroom substrate lies in its difficult preservation. This is due mainly to the high water content and to the high level of soluble carbohydrates (2-4%, as compared to 0.4-1.4% in wheat straw) (Silanikove & Levanon 1986). The substrate is rapidly overgrown by molds, resulting in spoilage and aerobic degradation. The anaerobic preservation of cotton stalks and the production of silage has been studied by Silanikove & Levanon (1986), Levanon et al. (1988) and Danai et al. (1989). Cotton stalks were harvested and chopped into 2-3 cm particles with a forage harvester originally designed to cut corn. The material was taken to a concrete silo with a storage capacity of 450 tons, pressed with a heavy tractor, and then covered with black plastic sheets. After one month of storage, the pH of the preserved cotton stalks stabilized at 5.5 and the material was successfully utilized for commercial Pleu- rotus cultivation, up to nine months after harvest (Danai et al. 1989; Levanon et al. 1988). Chemical treatment for the upgrading of cotton stalks and their subsequent utilization as animal feed has also been investigated (Ben-Ghedalia et al. 1983; Ben-Ghedalia et al. 1980). Apparent digestibility of organic matter in ozone-treated cotton stalks used in a sheep diet was 75%, as compared to 30 and 39% in control and NaOH-treated substrates. Cotton stalks could also potentially be used as raw material for the manufacture of some grades of paper (Balasubramanya et al. 1989; Pandy & Shaikh 1987). The use of Pleurotus to improve digestibility of cotton stalks in ruminants The lignocellulose complex in straw and other plant residues is degraded very slowly by ruminants because of the physical barrier imposed by lignin polymers, which prevent free access of hydrolytic enzymes such as cellulases and hemicellulases to their substrates. Normally, the rates of decay of plant debris are proportional to their lignin content. Biological delignification of straw seems to be the most promising way of improving its digestibility (Kamra & Zadrazi11986; Streeter et al. 1982; Zadrazil & Reinger 1988). Several authors have examined this possibility, using mainly wheat straw and Pleurotus spp. under different conditions and substrate pre-treatments (Kamra & Zadrazil 1986; Lindenfelser et al. 1979; Streeter et al. 1982; Zadrazil 1980). A significant increase in in vitro dry matter digestibility of wheat straw by Pleurotus was reported by Lindenfelser et al. (1979), who used commercial cellulase to compare the release of glucose from untreated straw to that of straw following Pleurotus fermentation. They found that lignin content decreased by 51% during the incubation period (90 days). Similar results were reported by Zadrazil (1985) showing an increase of 23-32% in the in vitro digestibility following fermentation by P. eryngii, P. sa]or-caju, P. ostreatus and P. serotiFLUS. These results encouraged us to examine the effect of Pleurotus on cotton stalks during SSF. SSF [67] 192 1 ~1000 & 4 /~ 5 "~ 800 .~600 400 ~200 0 0 I I I 6 12 I 18 24 I I I I I I I I I I 30 36 42 6 12 18 24 30 36 42 Time (d) Time (d) Fig. 1. Pleurotus respiration during SSF on ground (A) and chopped (B) cotton stalks. 1-5 are the treatments as described in the text. Bars represent standard errors. seems to be the most appropriate method, both for conducting basic research and for applied, largescale processes involving selective lignin biodegradation. SSF was carried out in the laboratory to define optimal conditions for increasing the in vitro digestibility of cotton stalks and improving the SSF process. Five pre-treatments were compared: 1. ground cotton stalks (particle size 2-5 mm), 70% water content; 2. ground cotton stalks, 70% water content plus / A "1" ~. 1 -- 3 the following nutrients: (g/Kg dry cotton stalk) 6.0 KzHPO4; 2.0 MgSO4.7H20; 4.0 C a C O 3 ; 0.008 thiamine.HC1; 4.0 Bacto-peptone, pH 5.6; 3. ground cotton stalks, 75% water content; 4. chopped cotton stalks (particle size 2-7 cm), 70% water content; 5. chopped cotton stalks, 70% water content plus the nutrients as in treatment 2. The chopped cotton stalks were harvested in the ] B A 4 -~0 "~0 6 12 18 24 Time (d) 30 36 42 6 12 18 24 30 36 42 Time (d) Fig. 2. Fluoroscein diacetate hydrolysis during SSF of Pleurotus on ground (A) and chopped (B) cotton stalks. 1-5 are the treatments as described in the text. Bars represent standard errors. [68] 193 40 0 6 12 18 24 30 36 42 10 Time (d) 20 30 40 Time (d) Fig. 3. Ethylene generation from KTBA during SSF of Pleurotus on ground (A) and chopped (B) cotton stalks. 1-5 are the treatments as described in the text. Bars represent standard errors. field using agricultural equipment, with no further treatment. Polystyrene bags containing 95 g (d.w.) of cotton stalks were either wetted with water or supplemented with nutrient solution and sterilized once for one hour, then again 24 h later. At various stages after inoculation with P. ostreatus, bags from each treatment were sampled for enzymatic and chemical analyses. Methods used in this study have been described previously (Kerem et al. 1992). Fungal activities and chemical composition of the fermentation product were followed. 95 E om i 85 75 6~ 65 0 6 12 18 24 30 36 42 Time (d) Fig. 4. Mineralization of organic matter during SSF of Pleurotus on cotton stalks. 1-5 are the treatments as described in the text. Bars represent standard errors. [69] 194 95 75 55 35 0 6 12 18 24 30 36 42 Time (d) Fig. 5. Lignin and cellulose degradation by Pleurotus during SSF of ground cotton stalks with 70% (1) and 75% (3) water content. Bars represent standard errors. Fungal activities related to colonization: Fungal respiration was recorded as CO2 evolution per hour, as determined by gas chromatograph (Fig. 1). Respiration rates rose for the first 15 days, then stabilized. On day 18, the contents of the bags were mixed to disperse the colonized bulks and on day 22, respiration rates had decreased. This could have been due to dispersion of the already colonized bulk. However, mixing showed no effects on the biodegradation process. Treatment 5 showed no resumption of fungal activity after mixing. Fungal activity defined by extracellular hydrolysis of fluorescein diacetate (Schnurer & Rosswall 1982) showed a similar trend for all treatments (Fig. 2). There was a slow rise in the extracellular activity towards day 12, a definite increase towards day 15 and stabilization towards the end of the experiment (day 42). Treatment 3 showed a second peak towards the end of the experiment. Ethylene generation from KTBA (a-keto- 7thio-ethyl butyric acid): KTBA is a water-soluble molecule which, in the presence of free (. OH centered) radicals, is cleaved to release gaseous ethylene (Kelley 1988). Cleavage activity was observed [70] from day 12, reaching a maximum on day 15 and continuing at a low level from day 29 to the end of the experiment (day 42) (Fig. 3). Treatment 3 showed the highest activity level. Treatments 1 and 2 produced slightly lower activities than treatment 3 and treatments 4 and 5 were found to have the lowest levels of activity. Organic matter degradation: Mineralization of organic matter was calculated from changes in the ash content of the substrate throughout the experiment. In treatments 2, 3 and 5, a steady mineralization rate was observed, with treatment 3 producing the highest rate (Fig. 4). In treatments i and 4 an acceleration was observed between days 12 and 15. By the end of the experiment (day 42), 35% of the organic matter had been mineralized in treatments 3 and 4, as compared to 30% in treatment 1 and 20% in treatments 2 and 5. A correlation was evident between mineralization patterns and respiration rates. Differentiation of fiber composition and in vitro digestion: Cellulose and lignin, the major fiber components of the substrate, were analyzed. Since chemical analysis of these components 195 ~, -- 3O ~ 1 3 25 2o 15 0 I I I I I I I 6 12 18 24 30 36 42 Time (d) Fig. 6. In vitro digestibilityof ground cotton stalks with 70% (1) and 75% (3) water content during SSF of Pleurotus. Bars represent standard errors. is time-consuming, only treatments 1 and 3 were studied in detail. Results are shown as residual lignin and cellulose (Fig. 5). Lignin degradation patterns were similar in both treatments, as were those of cellulose. During the first 15 days, lignin degradation rates were highest, whereas cellulose degradation accelerated from day 22. Thus, after 21 days of fermentation, the amount of degraded lignin was higher than that of cellulose. In vitro digestibility of the substrate was measured for treatments 1 and 3 (Fig. 6). Results are presented as dry weight of digested matter per bag. Digested matter in treatment 1 (70% water) decreased for the first 15 days, increased thereafter until day 22 and then stabilized until the end of the experiment. The level of digested matter in both treatments peaked on days 22 and 36. On those days, the amounts of digested matter per bag were found to be similar in both treatments. It was concluded that fermentation for 22 to 36 days produces the highest levels of digestibility, rendering the fermentation product suitable for use as animal feed. Day 22 represents the lowest percentage of digested matter, while day 36 represents the longer fermenta- tion period and a higher ash content due to overall organic matter degradation by the fungus. Chemical analyses of the substrates from the five treatments were therefore conducted on days 22 and 36. Acid insoluble fibers, cellulose, lignin and ash contents, are presented in Table 1, along with in vitro digestibility and the amount of residual dry substrate per bag. Since treatments 2 and 5 were supplemented with nutrients, their initial composition differed from that of the other three treatments, leading to higher ash and nitrogen contents in the fermentation products. During the first 22 days, a 5% decrease of the fibers was observed in treatments 1-4 and a 10% decrease in treatment 5. An additional 4% decrease in treatment 1 and 1% in treatment 2 were observed during a further 15 days of fermentation. Cellulose content decreased slightly in the five treatments throughout the fermentation period, at a rate similar to that of overall organic matter degradation. A decrease of 5-6% in substrate lignin content was observed in treatments 1, 3, 4 and 5, 3% in treatment 2, during the first 22 days. On day 36, lignin content reached levels of between 15.7 [71] 196 and 17.5%, with the lowest level being recorded in treatment 4. In vitro digestibility was increased by 12-15% in all treatments during the first period, and by an additional 5% during the following two weeks. Protein content and composition were analyzed on days 22 and 36 in the five treatments (Table 2). Crude protein was determined by the Kjeldahl method. An increase in its nitrogen content shows that no crude protein was lost from the system. Furthermore, the content of the true protein fraction, i.e. the amino acid fraction (Table 2) increased beyond the increase in crude protein content, indicating the fungus' ability to decompose nonprotein nitrogen, probably from lignoprotein compounds, and to incorporate the released nitrogen into its own true protein. A source of nonprotein nitrogen in the final product could be chitin, a major component of the fungal cell wall. Digestibility of the five treated substrates was similar. Treatment 4, requiring the least pre-treatment, was therefore chosen for experiments on a larger scale. One ton of chopped cotton stalks was fermented in an industrial mushroom production plant. The fermentation product was fed to ruminants - goats and heifers, at levels of up to 40% of their diets. No side effects were observed in animals consuming this diet. The in vivo nutritional value of this feed remains to be evaluated. Cotton stalks, harvested using commercial equipment, preserved using silage technology and fermented with P. ostreatus, exhibited increased digestibility and could therefore be utilized by ruminants as a dietary supplement. Table 1. Ash, ADF, cellulose and lignin contents, in vitro digestibility and residual dry weight in treated cotton stalks after 0, 22, and 36 days of fermentation by P. ostreatus. Treatment A - 0 days B - 22 days 1. Ground, 70% water content 2. Ground, 70% water content + nutrients 3. Ground, 75% water content 4. Chopped, 70% water content 5. Chopped, 70% water content + nutrients C - 36 days 1. Ground, 70% water content 2. Ground, 70% water content + nutrients 3. Ground, 75% water content 4. Chopped, 70% water content 5. Chopped, 70% water content + nutrients [721 Ash (%) ADF (%) Cellulose (%) Lignin (%) In-vitro digestibility (%) Residual dry weight (g/bag) 6.4 68.2 46.5 22.1 21.8 100 8.4 63.4 46.4 17.2 34.4 83 9.8 59.3 40.3 19.1 35.1 83 7.7 63.2 45.3 17.6 36.2 80 8.5 59.4 42.4 16.9 35.4 78 10.4 58.8 42.2 16.3 37.0 81 8.9 59.4 41.5 17.5 41.1 73 10.3 57.7 40.5 17.4 38,8 80 9.4 62.4 45.2 16.6 41;4 72 8.3 59.8 44.2 15.7 38.1 73 10.8 57.5 40.6 16.8 40.7 75 197 Ultrastructural changes in cotton stalks during Pleurotus growth Pleurotus spp. can degrade lignin in a variety of lignocellulosic waste materials (Kamra & Zadrazil 1986; Kerem et al. 1992; Platt et al. 1984). In addition, Pleurotus has been found to degrade 14C-labeled DHP to 14CO2 (Platt et al. 1983b; Trojanowski & Huttermann 1987) and to decolorize the polymeric dye Poly-B411 (Platt et al. 1985). Even though lignin degradation by white rot fungi has received a great deal of attention over the last two decades, a direct method for following this degradation is not available. One of the techniques which is increasingly being used in studies of the micromorphological changes taking place during the delignification process is scanning electron microscopy (SEM) (Blanchette 1991). Otjen and Blanchette (1986) reviewed variations in wood decay by observing its microscopic appearance. They demonstrated that white rot fungi cause several recognizable patterns of cell wall decomposition, influenced by the host cell type and the fungal species. With the aid of SEM they distinguished between three decay patterns: a white pocket rot, a mottled decay pattern and a uniform white rot. Blanchette (1984) identified selective lignin degradation in wood in his study of 26 white rot fungi. SEM was also used by Agosin et al. (1990) to characterize ultrastructural changes in the Palo Podrido. Ultrastructural changes during lignin degradation by various white rot fungi in different lignocellulosic materials such as sugar cane bagasse and wheat straw, have also been reported (Agosin et al. 1990; Agosin et al. 1987; Johnsrud et al. 1987). We followed the pattern of lignin degradation in cotton stalks during SSF. Sterile cotton stalks were inoculated with P. ostreatus and incubated for six months. Each week a sample was prepared as follows: radial and tangential sections of the cotton stalks were mounted on brass stubs and fixed by vapors of osmium oxide (OsOs) and glutaraldehyde. A radial section of sterile cotton stalk which had not been incubated with P. ostreatus (control) is shown in Fig. 7. There are no signs of lignin degradation in the domains where lignin is abundant. In cotton stems lignin is distributed throughout the cell wall layers. Greater quantities of lignin are found in the tracheal walls, the middle lamella and between the pith and the xylem. The xylem layers (Fig. 7-X) are intact to the pith (Fig. 7-P) and there is no degradation of the middle lamella. A tangential section after one week of incubation with P. ostreatus clearly shows fungal hyphae inhabiting the meristematic cells (Fig. 8). A whole radial section of a cotton stalk, incubated with P. ostreatus for two weeks, can be seen in Fig. 9. The pith is detached from the xylem and many holes appear due to lignin degradation, since this is a tissue Table 2. Amino acid and crude protein content of cotton stalks inoculated with P. ostreatus after 0, 22 and 36 days of fermentation. Treatment Total amino Essential acids amino acids (mg/g (mg/g substrate) substrate) Crude protein (mg/g substrate) A - 0 days 22.7 10.6 64.5 26.9 13.6 79.9 33.0 16.4 87.1 28.4 13.5 77.7 31.7 15.7 66.8 34,6 17.2 83.2 34.2 17.3 85.6 31.9 15.5 88.7 27.4 13.7 95.1 25.4 12.4 70.8 35.0 17.9 85.0 B - 22 days 1. Ground, 70% water content 2. Ground, 70% water content + nutrients 3. Ground, 75% water content 4. Chopped, 70% water content 5. Chopped, 70% water content + nutrients C - 36 days 1. Ground, 70% water content 2. Ground, 70% water content + nutrients 3. Ground, 75% water content 4. Chopped, 70% water content 5. Chopped, 70% water content + nutrient [73] 198 Fig. 7. Radial section of a control cotton stalk demonstrating the different areas, which have not been degraded. P - pith ; X - xylem ; C cambium ; Ph - phloem. Fig. 8. Tangential section of a cotton stalk, after one week of incubation with P. ostreatus. The fungal hyphae populate the cells and grow through them. [74] 199 which is rich in lignin. Figure 10 shows radial sections in which areas of delignification in the middle lamella can be observed. Cells are separated from each other and degradation of the cell walls is apparent. Another domain of lignin deposition is in the tracheal cell walls. A tangential section of the trachea cells, after a three-week incubation with P. ostreatus (Fig. 11), shows areas of complete decay. Fungal hyphae populate the cells and are destroying the tracheal cell wall (Fig. ll-DT). Figure 12 is a radial section of a cotton stalk after six months of incubation with P. ostreatus. Most of the lignin has been degraded and the cells have lost their shape and collapsed. These observations provide us with a record of the sequence of events occurring during P. ostreatus colonization on cotton stalks. A section of a uninoculated cotton stem shows a complete structure with no signs of delignification. After one week of incubation, fungal hyphae were colonizing the cells and lignin degradation was taking place. After the third week delignification had proceeded to the lignin-rich tissues. The pith was detached from the xylem and the trachea cells were degraded. The same phenomenon was also observed in the middle lamella. These observations are in agreement with results of chemical analyses of fermented cotton stalks and with the rate of [14C]lignin mineralization (Kerem et al. 1992). Enzymes involved in lignin degradation by Pleurotus spp. Extracellular enzyme activity related to lignin degradation has been reported by Platt et al. (1984) and Kerem et al. (1992) during SSF of cotton stalks by Pleurotus. The major activity was that of laccase, peaking between days 6 and 8. Similar trends were observed by Sharma (1987), who followed Pleurotus activities on flax shive and by Kannan et al. (1990), growing P. sajor-caju on paper-mill sludge. Sannia et al. (1986) purified and partially characterized laccase from P. ostreatus. Lignin peroxidase, the most widely studied enzyme in relation to lignin degradation, was not detected in Pleurotus cultures. This observation is in agree- ment with others who were unable to detect lignin peroxidase in Pleurotus at the protein level using antibodies (Waldner et al. 1988) or by using DNA probes (Kimura et al. 1990). The importance of laccase in cleaving phenolic 13-0-4 lignin substructure model compounds has been demonstrated by Higuchi (1990) who suggested that the same chemical principle, i.e. a phenoxy radical as intermediate, is involved in the degradation of phenolic substructure model compounds by both lignin peroxidase and laccase. Platt et al. (1984) suggested that some correlation exists between the level of laccase activity and lignin degradation. P. ostreatus 'florida' activities were compared to those of P. ostreatus 'P3' (Table 3). The results demonstrated a possible relationship between lack of laccase activity and the rate and mode of lignin degradation, DHP mineralization or Poly-B411 decolorization. Nevertheless, laccase is not the only enzyme responsible for lignin degradation in the genus Pleurotus since both strains were capable of degrading lignin. Acidic peroxidase, neutral peroxidase laccase and veratryl alcohol oxidase activities were investigated in cultures of P. sajor-caju (Fukuzumi 1987). The crude extracellular enzymes reduced the brown color of chlorinated oxi-lignin. Bourbonnais and Paice (1988) found two veratryl alcohol oxidizing enzymes in the culture medium of P. sajor-caju. 4-Methoxybenzyl alcohol was oxidized the most rapidly, followed by veratryl alcohol. Not all aromatic alcohols were oxidized. Oxidases may play a role in biodegradation by producing H202 during the oxidation of lignin fragments (Bourbonnais & Paice 1988). These findings are supported by those of Guillen et al. (1990) who found H202 production by aryl alcohol oxidase from P. eryngii. The most rapidly oxidized substrates were anisyl alcohol and veratryl alcohol. It seems that the enzymes described in these two reports have similar substrate preferences. A similar enzyme was discovered in cultures of P. ostreatus (Sannia et al. 1991) and purified. The enzyme is a glycoprotein containing FAD as a prosthetic group. Cinnamyl alcohol was the most rapidly oxidized substrate. Sannia et al. (1991) also suggested that veratryl alcohol oxidase plays a central role in the biodegradation of lignin by Pleuro[751 200 .::.,...:.:.:: Fig. 9. Radial section of a cotton stalk, two weeks after inoculation, shows clear signs of degradation. The pith is detached from the xylem layers. P - pith ; X - xylem ; ~ - areas of degraded lignin. Fig. 10. Radial sections of a cotton stalk, three weeks after inoculation. In all three sections there are clear domains of degraded lignin in the middle lamella. ML - degraded areas of the middle lamella ; C - complete degradation of the cell wall ; ~ - degraded areas. tus. Kim et al. (1986) demonstrated the activity of polyphenol oxidase in P. ostreatus, which is able to oxidize dihydroxyphenylalanine (DOPA). Enzyme activity was evident only after induction with [761 phenolic compounds, such as ferulic and gallic acids. The mechanism of lignin degradation by Pleurotus has been studied significantly less than those of Phanerochaete chrysosporium and some other 201 Fig. 11. Tangential section of trachea cells in a cotton stalk, three weeks after inoculation. H F - fungal hyphae which are seen in a degraded area ; ~ - degraded areas ; T - undegraded trachea cell wall ; D T - degraded trachea cell wall. Fig. 12. Radial section of a cotton stalk after six months of incubation with P. ostreatus, revealing complete destruction of the cells' shape. The cell walls have collapsed, having no lignin to support them. [77] 202 white rot fungi. From studies conducted to date however, a different enzymatic system seems to be responsible for lignin degradation in Pleurotus. The effect of cotton stalk extract on Pleurotus growth and activity Growth and some activities of some Pleurotus strains have been found to be enhanced by the phenolic constituents found in crude water extracts of some plants. Zadrazil (1975) reported the metabolism of flavonoid-type phenolic compounds existing in straw substrates by Pleurotus spp. in the early stages of degradation. Sharma (1987) reported that flax shive extract enhanced degradation of flax shive by four strains of Pleurotus. The extract also induced a significant increase in the number of primordia produced by these fungi. The infrared spectrum of a chromatogram of flax shive extract showed the presence of a flavonoid-type compound in the aqueous extract of the flax shive. Platt et al. (1981, 1983a) showed that P. ostreatus 'florida' grows faster on cotton straw than on other substrates. After 21 days, 17.1% of the initial dry weight was reduced by the fungi, as compared to a 10.8% reduction in the dry weight of wheat straw. The addition of crude aqueous cotton stalk extract to wheat straw increased its degradation by 33%. When the fungus was grown on water-extracted wheat straw, degradation of both lignin and straw was slower than in the native straw. The crude extract also induced high laccase activity, similar to activity found after induction with known phenolic compounds (Platt et al. 1984). The active fraction in the extract was characterized as a flavonone or a dihydroxyfiavonol-type flavonoid (Platt et al. 1983a). When cotton stalk extract was incorporated into a solid synthetic growth medium, the linear growth rate of the fungal colonies increased, by 14% for P. ostreatus 'florida' F6, 20% for P. ostreatus IMI 341688, 14.3% for P. salmoneo stramineos and 17.6% for P. pulmonarius P3014. Other white rot fungi were also studied: Growth rate of Ganoderma applanatum increased by 17%, and Phlebia tremellosus by 12%. Other fungi, such as Trametes versicolor and Gleophyllum striatum, were not affected, whereas in the case of L. edodes, a 9% inhibition of growth was observed. A similar trend was noted when fungal biomass grown in stationary liquid cultures was monitored. Laccase activity was studied in liquid medium amended with cotton stalk extract. In preliminary studies, all strains tested showed different levels of laccase induction, ranging from a three to flvefold increase in activity Table 3. Fungal activities and lignin degradation by P. ostreatus. Activity Reference P. ostreatus P. ostreatus 'florida' F6 P3 Cotton degradation (%) Lignin degradation (%) Laccase activity (OD/ml/min) Poly-B411 decolorization: absorbance (486nm:553nm) ratio change in 17 56 0.86 9 10 0 Platt et al. (1984) Platt et al, (1984) Plattet al. (1984) 7 days (initial ratio = 3) Release of 14CO2from 14C-ringDHP (% of initial) 0.95 7 7.5 2.9 0.7 4 Release of 14CO2from DHP-O-14CH3 (% of initial) 21 18.1 9 7.5 14COz release from 14C organosolve lignin (% of initial) 14C-water solubles in culture (% of initial) 9.4 5 35.3 11.5 Platt et al, (1985) Platt et al. (1983b) Trojanowski & Hiittermann (1987) Platt et al,(1983b) Trojanowski & Hiittermann (1987) Trojanowski & Hiittermann (1987) Trojanowski & Hiittermann (1987) [78] 203 level within 6 h. T. versicolor and G. applanatum showed a similar increase, while other laccase-producing fungi, such as Rhizoctonia solani, were not affected by the added extract (Ardon & Hadar, unpublished). Mineralization of [14C]lignin by P. ostreatus in the presence of cotton stalk extract was studied. A twofold increase was observed after two weeks of growth, changing only slightly thereafter until the end of the experimental period (75 days). Fortyone and 47% of the total radioactivity was released as 14CO2, from the control and the treated substrate, respectively. These results suggest a change in fungal lignin metabolism, affected by the cotton stalk extract. The role of plant extracts in lignin degradation by Pleurotus remains to be elucidated. ly, as evidenced by electron microscopy and chemical analyses. Our study on its SSF of cotton stalks suggests the usefulness of the fermentation product as a supplement to ruminant diets. Since Pleurotus has been cultivated commercially for human consumption for decades, it can be considered safe for animals as well. This conclusion is in line with findings on SSF of Pleurotus on wheat straw (Zadrazil & Reinger 1988) and other wastes. The mechanism of lignin degradation by Pleurotus should be targeted for future research. The enzymes involved seem to be laccase and aryl alcohol oxidases. However, related activities need to be investigated before any definitive conclusions can be drawn. References Conclusions The utilization of lignocellulosic agricultural wastes as well as forest products via SSF by white rot fungi could be applied in several industries (Buswell & Odier 1987). Its major potential advantages are the upgrading of under utilized resources (agricultural waste) and the introduction of environmentally sound biotechnologies to the pulp and paper industry. 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