World Journal of Microbiology & Biotechnology 17: 191±200, 2001. Ó 2001 Kluwer Academic Publishers. Printed in the Netherlands.
191
Bioconversion of agricultural lignocellulosic wastes through the cultivation of the edible mushrooms Agrocybe aegerita, Volvariella volvacea and Pleurotus spp. A. Philippoussis1,*, G. Zervakis2 and P. Diamantopoulou1 1 National Agricultural Research Foundation, Institute of Agricultural Engineering, Edible Fungi Research Laboratory, 61 Democratias St., 135 61 Ag. Anargyri, Athens, Greece 2 National Agricultural Research Foundation, Institute of Kalamata, 85 Lakonikis St., 241 00 Kalamata, Greece Fax: 0030-721-27133, E-mail:
[email protected] *Author for correspondence: Fax: 0030-1-2619202, E-mail:
[email protected] Received 24 October 2000; accepted 7 February 2001
Keywords: Agricultural wastes, Agrocybe aegerita, basidiomycetes, bioconversion, mycelium growth, Pleurotus eryngii, Pleurotus ostreatus, Pleurotus pulmonarius, Volvariella volvacea
Summary Ten selected wild and commercial strains of Pleurotus ostreatus, Pleurotus eryngii, Pleurotus pulmonarius, Agrocybe aegerita and Volvariella volvacea were cultivated on three agricultural wastes, i.e. wheat straw (WS), cotton waste (CW) and peanut shells (PS). All species demonstrated signi®cantly higher colonization rates on WS and CW than on PS. WS supported faster growth of A. aegerita and Pleurotus spp., whereas V. volvacea performed better on CW. Comparison of growth rates on composted and non-composted WS and CW substrates revealed that in the latter case faster colonization was achieved, particularly for Pleurotus spp. However, one commercial strain of V. volvacea presented higher growth rates when the composted CW medium was used. Furthermore, earliness in the fructi®cation of P. ostreatus, P. pulmonarius and V. volvacea strains was promoted in CW substrates, while WS favoured earliness of P. eryngii and A. aegerita. Similarly, high sporophore yields were obtained by P. ostreatus and P. pulmonarius on both wastes, whereas WS enhanced yield and basidioma size of P. eryngii and A. aegerita strains and CW production of V. volvacea. The substrates cellulose:lignin ratios were found to be positively correlated to mycelial growth rates and to mushroom yield of P. ostreatus and P. pulmonarius; in addition, positive correlation was also detected for carbon:nitrogen ratio and mushroom yield in P. eryngii and A. aegerita and between cellulose content and mushroom yield for V. volvacea strains. Introduction Most agricultural residues are rich in lignocellulosic compounds whose handling and disposal are often problematic, due to their chemical structure and decomposition properties. Cotton wastes (CW) including stem-leaf residues and gin trash, wheat straw (WS) and peanut shells (PS) are of particular interest for the agricultural economy of temperate and subtropical countries, since they are produced in large quantities and their post-harvest treatment is mainly accomplished through burning or incorporation into the soil. The potential of bioconversion of lignocellulosic wastes into value-added products is emphasized in recent studies (Philippoussis & Zervakis 2000a; Poppe 2000). In particular, edible mushroom fungi, which possess the appropriate enzymatic mechanisms for the transformation of complex organic macromolecules into simple compounds, have been exploited as the means for the biodegradation of a wide range of plant litter due to their particular ability for selective deligni®cation (May-
son & Verachtert 1991; Martinez et al. 1994). This type of biotechnological approach results in the conversion of agricultural wastes that are useless and potentially hazardous for the environment into food of high organoleptic properties and nutritive value (Rambelli 1985), or into good quality fodder by enriching the waste protein content and by improving their digestibility (Martinez et al. 1991; Tripothi & Yadar 1992), or in the production of plant fertilizers and soil conditioners bene®cial to overall soil fertility and stability, as well as vegetable yield. In addition, the production of mushroom species other than Agaricus could bene®t diversi®cation of local markets, along with the promotion of employment and social development in rural areas (Philippoussis & Zervakis 2000b; Zervakis & Venturella 2000). Although for the commercial production of Pleurotus mushrooms the principal substrate used is WS (Olivier 1994), there are numerous past studies indicating the need for the examination of other agro-industrial wastes (e.g. wood chips, sawdust, paddy straw, sugarcane
192 bagasse, CW, olive-oil mill wastes, orange peel, grape stalks etc.) as positive alternative substrates for their cultivation (Cho et al. 1981; Nicolini et al. 1987; Bahukhandi & Munjal 1989; Zervakis & Balis 1992; Ragunathan et al. 1996; Zervakis et al. 1996; Thomas et al. 1998; Philippoussis et al. 2000). Similarly, the use of several lignocellulosic by-products, such as barley, maize and WS, orange peel, grape stalks, reed, rice husks and sun¯ower, has been reported for the cultivation of Agrocybe aegerita (Nicolini et al. 1987; Zadrazil 1994). In the case of the paddy straw mushroom Volvariella volvacea, the substitution of the traditional composted cotton or rice straw substrates has been examined through the use of dierent residues e.g. oil palm pericarp, CWs, sugarcane baggase etc. (Chang 1974; Hu et al. 1974; Khan et al. 1991). Along this line of approach, three selected lignocellulosic substrates (Ioannidou et al. 2000; Philippoussis et al. 2000), have been comparatively evaluated as regards their suitability in enhancing mushroom production of the following edible species: Pleurotus ostreatus, P. pulmonarius, P. eryngii, A. aegerita and V. volvacea. Ten wild and commercial strains were subjected to cultivation experiments and all essential parameters for growth and fructi®cation were recorded and analysed. The results are interpreted and discussed in the view of establishing viable mushroom production methodologies capable of supporting relevant agro-economic activities. Materials and Methods Organisms The identity of the ten dikaryotic strains which were used in the present study is as follows: (i) P. ostreatus (Jacq.: Fr.) Kumm. (wild strain LGAM P69 originating from Greece; commercial strain HK35 provided by Somycel), (ii) P. pulmonarius (Fr.) QueÂl (wild strain LGAM P26 originating from Greece; commercial strain S3014, provided by Somycel), (iii) P. eryngii (D.C.: Fr.) QueÂl. (wild strains LGAM P101 and AMRL HER3, both originating from Greece), (iv) A. aegerita (Bring.) Sing. (wild strain SIEF 0834 originating from China; commercial strain S4021 provided by Somycel), (v) V. volvacea (Bull.: Fr.) Sing (wild strain SIEF 1318 originating from China; commercial strain SLC originating from Indonesia). These cultures (maintained at the authors' Institutes of the National Agricultural Research Foundation) are registered in the Fungal Culture Collection of Athens Mushroom Research Laboratory with the accession numbers AMRL 134, 138, 176, 177, 161, 164, 101, 104, 186 and 193 respectively. Culture media and spawn preparation The culture media used for routine culture and storage purposes were complete yeast medium (CYM) and
A. Philippoussis et al. potato dextrose agar (PDA, Difco). Grain spawn of individual species was prepared in 500 ml Erlenmeyer ¯asks ®lled with 180 g of boiled wheat (Triticum durum) grains supplemented with 3% calcium carbonate and 1.5% gypsum (w/w, in terms of dry weight). The contents of the ¯asks were thoroughly mixed and autoclaved at 15 psi for 1 h. Each ¯ask was inoculated with mycelium scraped o the surface of a growing colony in a Petri dish (9 cm diam). Flasks were incubated at 26 °C for 2±3 weeks, in the dark, with periodical shaking. Substrate preparation and constituent analysis Composted substrates of WS and CW were prepared in a 2 m3 capacity rotating drum composting system (KompoRot, Umwelttechnik A. Kreutner GmbH) at NAGRF's Athens Soil Science Institute. About 250 kg of raw waste materials were ®lled in the drum, watered thoroughly, allowed to drain to obtain 65% moisture and subjected to a 12-day composting process. Mixing and aeration was achieved by periodic drum revolutions. Throughout composting, temperature and moisture level were monitored daily just before turning and water was added as necessary to maintain 65±70% moisture content. Temperature raised at 65±70 °C in the ®rst 3 days, kept in the range of 50±65 °C for the following 6 days and gradually dropped to 40 °C by the completion of the process. Non-composted substrates were prepared as follows: residues with particles size of 2±3 cm were soaked in water for 24 h. After the surplus water had been drained o, substrates were mixed with calcium carbonate (2% dw) to obtain a pH value of 6±7 and were supplemented with wheat bran at a 90:10 ratio (w/w, in terms of dry weight) to obtain a C/N value within the range of 30±60/ 1. The moisture content of the sterilized substrates was 70%. For substrate analysis, samples of the raw materials were dried to a constant weight in a 60 °C oven, milled and sieved. Five to ®fteen milligram aliquots were used for total carbon and nitrogen determination in an elemental analyser (Carlo Erba EA1108). The cellulose and lignin content was measured by the method described by Van Soest & Wine (1968). Measurement of mycelial growth rates The measurements of linear growth rates were used to test the suitability of non-composted or composted wastes to support growth of the mushroom strains under study. Glass `race' tubes (200 ´ 30 mm) were uniformly ®lled to 100 ml volume with the substrates as described by Ioannidou et al. (2000) and sterilized twice for 1 h at 121 °C. Five replicates per strain and substrate were inoculated with one agar plug, taken from the actively growing periphery of a fungal colony and incubated at 26 °C. Mycelium linear growth was recorded daily in two perpendicular directions as
Mushroom cultivation on lignocellulosic wastes described by Zervakis & Balis (1996). The extension rate Kr (mm/day) was calculated after the mycelium front had reached more than 30 mm. Cultivation conditions for mushroom production For fructi®cation and productivity evaluation, ®ve replicates per substrate (non-composted) and strain were used. Polypropylene-autoclavable bags were ®lled with 1.2 kg of substrate, and were sterilized twice for 1 h at 15 psi. Inoculation was carried out along the central vertical axis of the bag, at a rate of 3% (w/w). Colonization of the substrates took place in growth chambers, in the darkness, at 35 °C for V. volvacea and at 26 °C for the rest of the species examined. After complete colonization of the substrate, polypropylene bags were removed and environmental conditions (temperature, relative humidity, aeration and light intensity) were adjusted for basidiomata induction and maintained at the appropriate levels during the entire length of the production cycle. During fructi®cation, the light intensity was set at 700 lux (12 h/day, ¯uorescent lamps), air exchange rates were controlled to maintain low CO2 level (
