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Performance of olive mill solid waste as a constituent of the substrate in commercial cultivation of Agaricus bisporus
International Biodeterioration & Biodegradation 63 (2009) 993–997
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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Performance of olive mill solid waste as a constituent of the substrate in commercial cultivation of Agaricus bisporus Roberto Altieri a, *, Alessandro Esposito a, Francesca Parati b, Arianna Lobianco c, Milva Pepi c a
CNR, Istituto per i Sistemi Agricoli e Forestali del Mediterraneo, Via Madonna Alta, 128, 06128 Perugia, Italy Azienda Agricola Valfungo, Frazione Gricignano, 6, 52037 Sansepolcro, Arezzo, Italy c ` degli Studi di Siena, Via P. A. Mattioli, 4, 53100 Siena, Italy Dipartimento di Scienze Ambientali, Universita b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 May 2009 Received in revised form 11 June 2009 Accepted 11 June 2009 Available online 21 July 2009
The feasibility of using olive mill waste (OMW) as an ingredient in the substrate used for cultivation of Agaricus bisporus (Lange) Sing. was studied in a large-scale cultivation trial, concerning 2500 m2 of mushroom growing area, at a specialized mushroom farm. Standard commercial cultivation technique involving compost preparation, spawning, casing and harvesting was used. The performance indicators such as mushroom yield, biological efficiency, market quality as well as horticultural value of the spent compost showed that the compost prepared with OMW was superior to the control compost in all the categories. The OMW-amended substrate supported higher populations of beneficial microorganisms especially, actinomycetes which enabled the breakdown of the compost ingredients. It is suggested that OMW is a suitable ingredient for the preparation of mushroom substrate. We have demonstrated that conversion of OMW (a liability) into value-added mushroom substrate (an asset) is an effective waste management tool in oleaculture. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Olive mill waste Recycling Composting Agaricus bisporus cultivation Biological efficiency Organic matter loss Spent mushroom compost
1. Introduction The increasing growth in olive oil production worldwide poses several ecological challenges. The lack of feasible and cost-effective olive mill waste (OMW) management technology compounds this problem. Olive mills produce a lignocellulosic residual solid (olive husk, OH) waste and a dark liquid effluent (olive mill wastewater, OMWW) rich in polyphenols and organic load (Niaounakis and Halvadakis, 2006). OH and OMWW are recalcitrant wastes with significantly high biological oxygen demand (BOD) and chemical oxygen demand (COD). Spreading OH and OMWW on farmlands may be a cost-effective solution to OMWW disposal provided lack of suitable and accessible land close to the mill and high cost of transportation can be overcome (Altieri and Esposito, 2008). Therefore, methods to transform OMW into value-added products, have recently received increasing attention (Felipo´, 1996; Sequi, 1996). With this in mind, the institute of ISAFoM-CNR recently developed a new technology, called MATReFO (WO/2005/082814), which has the capacity to convert raw olive mill effluents (OH and OMWW) into a non-leaching and non-odorous organic matter
* Corresponding author. Tel.: þ39 075 5014540. E-mail address: [email protected] (R. Altieri). 0964-8305/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2009.06.008
useful for agronomic applications. The MATReFO involves system occurs by mixing destoned OH hygroscopic additives such as straw, wool waste, sawdust, olive leaves, twigs and olive pruning residues. This relatively dry organic product is subjected to a short period of aerobic storage and maturation before use. Such type of substrate contains sugars, tannins, lignin, polyphenols, polyalcohols, pectins, lipids and proteins, which serve as carbon, nitrogen and energy sources for growth of mushroom (Zervakis et al., 1996; Sanjust et al., 1991; Kalmis et al., 2008). The selection and management of starting ingredients and the proper conditions for composting make growing Agaricus species so demanding (Sanchez, 2004). Among ingredients, chicken manure is probably the most common and economical source of nitrogen, although many mushroom growers are recently more and more concerned about hygienic (i.e. exposition to Avian influenza virus and other pathogens) and smell problems due to the use and management of animal derivatives (Pecchia et al., 2000). Hence, increasing attention has been devoting to find out and check different source of nitrogen to make mushroom substrate (Noble et al., 2002). Although several papers, mainly relating to Pleurotus spp., have been published on the use of olive mill effluents in the cultivation of edible mushrooms (Kalmis and Sargin, 2004; Zervakis, 2005), there are no published reports on commercial-scale studies on Agaricus bisporus (Lange) Sing. The aim of this paper is to determine whether
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MATReFO mixture can be used as an ingredient in the preparation of substrate for the cultivation of A. bisporus on a commercial-scale.
relatively high concentration of CO2 is required for optimum growth of the mushroom mycelium (vegetative stage).
2. Material and methods
2.2.4. Supplementation of substrate Soy bean meal that has been specially prepared (treated with formaldehyde 3000 ppm) was added to the spawn-run substrate at the rate of 1% fresh weight. The main aim of supplementation is to boost the protein and free fatty acids in the substrate for increased yield of mushrooms. The supplemented substrate was then transferred to the cropping room.
2.1. Cultivation method Preparation of substrate and cultivation of A. bisporus were conducted using the commercial facilities and procedures at the Valfungo mushroom farm at Sansepolcro, Arezzo, Italy. The cultivation trial was conducted over a period of 66 days starting from preparation of substrate to the completion of mushroom harvest comprising of two flushes. The initial three phases of the cultivation process namely, phase 1 (aerobic substrate preparation), phase 2 (pasteurization and stabilization of the substrate) and phase 3 (inoculation of the substrate with mushroom starter culture, the spawn, and incubation of the inoculated substrate) were carried out in concrete ‘tunnels’ (concrete bunkers). Environmental conditions such as ventilation, ambient and substrate temperature and moisture content were controlled during the substrate preparation. The spawn-run substrate was transferred when ready to a 500 m2 cropping room, having a total growing surface area of 13.200 m2 using the Dutch shelf system. The experiment was carried out using a standard mushroom substrate (control) normally used in the Valfungo farm and compared with an experimental substrate (ES) containing olive mill waste (OMW) with added organic ingredients as in the CNRMATReFO recycling system (Altieri and Esposito, 2008). The MATReFO mixture consisted of 72% destoned olive husk taken from OMW produced by two phase decanters in the olive mill, 14% waste wool and 14% wheat straw. 2.2. Composting process 2.2.1. Phase 1 The individual ingredients of the substrate were mixed thoroughly by mechanical means to achieve a homogenous mixture. Two phase 1 tunnels were each filled separately with the two substrates uniformly to a depth of 4 m. The tunnels were sealed and the substrates were ventilated by forcing air through the slatted floor of the tunnel to promote aerobic decomposition of the substrate. The oxygen concentration of the compost piles was controlled in the range 8–15%. The substrate and ambient temperature of the tunnel were monitored by probes located at different points in the tunnel and substrate. The total duration of phase 1 was eleven days. However, six days after filling the tunnel the substrate was removed from the tunnel, mixed for homogeneity and adequate moisture and, placed back in the tunnel for a further period of five days. 2.2.2. Phase 2 This phase was characterized by pasteurization of the substrate at 60 C for 8–9 h. The temperature was lowered to 45–50 C after pasteurization and held at that temperature for five days to ensure total conversion of ammoniacal nitrogen and dissipation of ammonia. The substrate was then allowed to cool down to 25 C. 2.2.3. Phase 3 The pasteurized substrate was removed from the phase 2 tunnel by means of slat bed conveyors into a clean work area. This area was kept under positive pressure bypassing the incoming air through absolute filters (1 mm). The substrate was then inoculated (spawned) with a commercial strain (A15, Sylvan Spawn Ltd.) of A. bisporus at the rate of 0.8% wet weight. The spawned substrate was put into phase 3 tunnel and incubated at 24–26 C, relative humidity 90% and without ventilation (CO2 approx. 3000 ppm in the tunnel) for thirteen days. A
2.2.5. Casing ‘Casing’ is the term used for covering the surface of substrate that has been fully colonized by the mushroom mycelium (spawnrun substrate). The function of casing in mushroom production is not known; however, if the substrate is not cased the switch from vegetative to reproductive stage (production of sporophore initials and sporophores) will be significantly affected with the loss of crop. A mixture of peat moss (Grunig, Vipiteno, Italy) and aged sugar beet lime at 7.5 pH was spread over the substrate to a depth of 5 cm. 2.2.6. Production of mushrooms The cased control and ES substrates were placed in a cropping room fitted with 25 cm deep Dutch shelves arranged in multilayered rows. An additional room was used where the control and ES substrates were placed in shelves of 333 m2 and 167 m2 respectively. Soon after the mycelial growth was detected at the top of the casing surface, a cold shock was applied to the crop by reducing the room temperature to 17–18 C and decreasing the ambient CO2 concentration to 300 ppm by increasing ventilation (number of air changes per hour) in the room. Environmental control of all facilities employed in the trial were performed by automatic control systems. Temperature by means of PT100 probes controlled by PLC; RH through measuring temperature on dry and wet sensors; CO2 by an infra red gas analyzer (IRGA). 2.3. Mushroom production parameters Timing of first flush, total yield and biological efficiency (BE) were evaluated. Total yield, evaluated per square meter or per tons of substrates, and BE were analyzed statistically by the Student– Newman–Keuls test at a significance level of P < 0.05. Market qualities such as appearance (shape, size and blemishes) and shelf life, and nutritional qualities such as dry matter, ash, total nitrogen, protein, fat, total and soluble carbohydrates and polyphenols were analyzed. Shelf life was estimated at 2–4 C in the dark for 10 days on mushrooms collected within the first flush and packaged in commercial boxes. The main mushroom pests and diseases (moulds and insects) were monitored from the stage of sporophore initiation to the end of harvest (second flush). 2.4. Substrate analysis 2.4.1. Microorganisms Total counts of heterotrophic microorganisms and actinomycetes were made using Triptic Soy Agar (TSA) (Difco) and Actinomycete Isolation Agar (AIA) (Difco) of substrate samples collected after pasteurization using standard procedures. One gram of each sample was added to 9 ml of buffer (0.9% NaCl) and serial (1 in 10) dilutions were prepared. One hundred microliters of each dilution was spread by using a sterile spatula, in duplicate, on the surface of TSA and AIA media in Petri dishes. The plates were then incubated at 28 C, and colony forming units (CFU) per gram of fresh weight counted after 2 and 7 days of incubation.
R. Altieri et al. / International Biodeterioration & Biodegradation 63 (2009) 993–997 Table 1 Composition and fresh weight of the substrates used in the A. bisporus cultivation trial. Substrate ingredients
Straw CNR-MATReFO mixture Chicken manure Gypsum Waste wool Ammonium sulfate Urea Starting total carbon Starting total nitrogen
Control substrate
Experimental substrate
tons
%
tons
%
43.4 – 18.5 4.0 – 0.6 0.2 – –
65.1 – 27.8 6.0 – 0.8 0.3 42.5 1.7
46.4 20.0 5.0 4.0 2.4 1.1 0.4 – –
58.5 25.2 6.3 5.0 3.0 1.4 0.5 44.3 1.6
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Table 3 Total yield of A. bisporus from first and second flushes and assessment of market quality. Control substrate
2.4.2. Physical/chemical factors The main chemical–physical parameters (pH, electrical conductivity (EC), ash, total organic carbon (TOC), total nitrogen (TN), ammonium nitrogen (AN), total polyphenols (TP) and phytotoxicity by means of germination index (GI) using Lepidium sativum) were analyzed on the substrates during its preparation, and on spent mushroom substrate (SMS) at the end of the cultivation trial. Substrate organic matter (OM) loss was measured before pasteurization and after incubation by means of initial weight loss. The data were confirmed by calculating the proportion of OM-loss by mineralization, using initial (X1) and final (X2) ash concentration, according to the equation: OM-loss % ¼ 100100[X1(100-X2)]/[X2(100-X1)] (Viel et al., 1987). Samples of substrates were collected in triplicate and the fresh samples were tested for EC and pH in water extract 1:10 (w/v), TOC, AN and TN. TOC was determined by the Springer–Klee method; AN and TN by means of macro-Kjeldahl distillation method (DI.VA.P.R.A., 1992). Moisture content of the fresh samples was determined as weight loss upon drying at 105 C in an oven for 24 h. Ash were determined as sample weight loss (previously ovendried at 105 C) upon ashing at 650 C for 24 h in a muffle furnace. Water-soluble polyphenols were determined in water extracts (1:10 w/v) using Folin-Ciocalteau method as reported by Singleton et al. (1971). A simplified phytotoxicity test based on germination of L. sativum L. was used: deionized water was added to fresh samples to achieve 65% of water content (wet weight); samples were shaken for 1 h and extracts were then obtained by centrifugation and filtration through a 0.45 mm membrane filter. Extracts were then diluted (30%) and used as germination media. A Whatman filter paper (n. 42), placed inside a 9 cm Petri dish, was wetted by 1 ml of the germination media and 9 seeds of L. sativum were placed on the paper. Deionized water was used as a control germination media and five replicates were carried out for each treatment. The dishes
Experimental substrate
tons
%
tons
%
Yield – first flush Yield – second flush
20.2 8.1
71.4 28.6
17.4 10.6
62.1 37.9
Market quality standard 1 Carpophores diameter > 3 cm 2 1.5a < carpophores diameter < 3 cm 3 Open veil and/or visible spot
6.1 9.5 12.7
21.5 33.5 45.0
8.2 8.1 11.7
29.2 29.0 41.8
a
Tight veil and no visible spot.
were wrapped in ParafilmÒ M, to minimize water loss and allow air penetration, and kept in the dark for 42 h at 24 C. At the end of incubation, the number of germinated seeds and primary root lengths were measured and expressed as percentage of the control (germination index). 2.5. Analysis of mushrooms Water content, total nitrogen, ash and water-soluble polyphenols were determined as described above. Fat was determined according to standard procedure (A.O.A.C, 1990); soluble carbohydrates and proteins by using anthrone reaction (Mokrasch, 1954) and Lowry method (Lowry et al., 1951); total proteins were calculated as TNx4.38 and total carbohydrates by difference, as reported by Manzi et al. (2004). 3. Results Composition of the initial mixtures used in the A. bisporus cultivation trial are given in Table 1. The control and ES substrates had a starting total nitrogen 1.7 and 1.6, total carbon 42.5 and 44.3, C:N 25.0 and 27.2, respectively, and were 71–73% saturated. The total yield and BE for each separate fruiting room under trial are presented in Table 2. Although the total weight of ES spawned compost used in the cultivation trial was lower than control on a dry and fresh weight basis, the total overall yields were comparable (28.27 and 27.94 tons for C and ES, respectively), thus signifying a significant higher mean BE of ES spawned compost (100.5%) than control (93.8%). This trend is also true when mean yields, expressed per square meter or per weight of fresh spawned compost, is considered. The yield and fresh market quality assessments (based on prescribed Italian standards) of mushrooms in the first two flushes are given in Table 3. ES produced a higher prime quality fresh
Table 2 Total yield of A. bisporus from two flushes in each cropping room. Cropping room
Cropping area, m2
Fresh SS, tons
Dry SS, tons
Total yield, tons
Yield, kg m2
Yield, kg tons1 fresh SS
20.4 21.3 22.3
251.7 217.6 256.7
21.3 A
242.0 A
24.1 23.9 23.4
282.5 287.6 310.7
100.5 104.0 97.1
23.8 B
293.6 B
100.5 B
1 2 5 mix room
C C C Total Mean
500 500 333 1333
40.50 49.00 28.90 118.40
10.58 11.50 8.04 30.12
10.20 10.66 7.42 28.27
3 4 5 mix room
ES ES ES Total Mean
500 500 167 1167
42.70 41.60 12.60 96.90
12.00 11.50 4.03 27.53
12.06 11.96 3.92 27.94
a
BE,a % 96.4 92.7 92.3 93.8 A
BE ¼ Biological efficiency (sporophores fresh weight/SS dry weight) 100. (C ¼ control substrate; ES ¼ experimental substrate, SS ¼ spawned substrate). Mean data flanked by the same letter are not significantly different according to SNK test for P < 0.05.
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Table 4 Composition (g 100 g1) of A. bisporus sporophores grown in control and experimental substrates.
Dry mattera Ash Total proteinsb Soluble proteins Total fat Total carbohydrates Soluble carbohydrates Polyphenols
Control substrate
Experimental substrate
1st flush
2nd flush
1st flush
2nd flush
7.9 11.1 19.7 13.6 1.4 67.8 7.2 0.4
7.8 11.1 27.6 18.6 1.5 59.9 6.1 0.6
8.1 10.1 17.5 15.9 1.6 70.6 3.5 0.5
7.9 12.7 27.6 15.2 1.4 58.3 3.5 0.6
Data are means of triplicate dry weight, with coefficient of variation (CV) < 5 %. a g 100 g1 fresh weight. b Total nitrogen 4.38.
Table 5 Total microbial counts CFU (108) g1 in control and experimental substrates after pasteurization during phase 1. Time of incubation
2 days 7 days
Control substrate
Experimental substrate
TSAa
AIAb
TSAa
AIAb
110 580
1.3 340
200 1800
1.7 950
active oxygen-driven composting process, pasteurization and incubation phases (data not showed). Total microbial counts CFU (108) g1 in ES and control substrates after pasteurization during phase 1 are reported in Table 5. After an incubation period of two days the ES compost showed approximately twice the total number of microorganisms than the control substrate. Similarly, after two days incubation, ES accounted for 30% more actinomycetes than the control and this increased to about 279% after 7 days. Evolution of main mushroom growth media chemical and biological parameters, determined all along the cultivation trial, and degradation of organic matter during the preparation of compost, are reported in Table 6 and Table 7, respectively. Although at the beginning of composting the nitrogen content was similar in both compost under trial, nitrogen apparently increased more in ES than control from start to the end of spawnrun phase (2.79% and 1.94% in ES and control, respectively). In addition, both composts showed at spawning, ammonium nitrogen content below lethal limit for A. bisporus. Similar trends for ash and TOC content were recorded in both the substrates, while pH was lower in the ES at all stages of the process. ES also showed a slightly higher electrical conductivity than control while total phenols rapidly degraded, with an overall degradation up to 90% of the initial content, in both substrates. SMC from ES showed phytotoxicity, comparable to that from control, while it showed 2.4 times total nitrogen content compared to that from control.
Data are means of triplicate, with coefficient of variation (CV) < 5%. a Triptic soy agar. b Actinomycete isolation agar.
4. Discussion
mushrooms (29.2%) than the control substrate (21.5%) and a lower amount of third quality mushrooms (41.8%) than control (45.0%). Differences were not observed on the occurrence of fungal diseases such as green mould (Trichoderma spp.) and dry bubble (Verticillium fungicola) between the crops grown in ES and control substrates. As regard shelf life, harvested sporophores grown on ES showed a higher degree of color change from white to brown than those grown on standard compost (pictures not showed). This phenomenon was significantly reduced by packaging sporophores under a polyethylene film. At the present time we are unable to explain this result. In general, low differences were observed in the composition of fruit bodies grown on ES and control substrates except for soluble carbohydrates which were about twice in mushrooms grown in the control substrate in both flushes (Table 4). Differences were not observed between the ES and control substrates in temperature and oxygen profiles recorded during the
The practicality of using olive mill solid waste as an ingredient in the substrate used for commercial-scale cultivation of A. bisporus (Lange) Sing. was studied. Data on mushroom yield, production efficiency, market quality and horticultural value of spent substrate showed that the substrate prepared with olive mill waste performed significantly better than the control in these respects. Therefore, the performance of the ES compost was higher than that normally obtained in the Valfungo crops; the higher production of prime quality mushrooms reduced time and consequent cost of manual harvesting. ES showed a better distribution of yield among flushes with reasonable commercial advantages for a farm such as Valfungo concentrating on fresh market sales. The large variability in the overall sporophore composition reported in literature for A. bisporus (Kurtzman, 1997) suggests that the difference found in soluble carbohydrates (Table 5) fall within the normal range and should not affect the organoleptic value of the mushrooms.
Table 6 Characterization of the control and experimental substrates during preparation of substrates and mushroom growth. Days
DM, %
pH
EC, dS m1
Ash, %
TOC, %
TN, %
AN, %
GI, %
TP, g kg1
Control substrate Start of substrate preparation Oxygen-driven phase (phase 1) Pasteurization (phase 1) Spawning (phase 2) Spawn-run compost (phase 3) End of cultivation - SMS
0 6 13 20 33 66
29.0 22.0 23.4 27.8 25.7 28.8
8.33 8.18 8.37 6.98 6.05 7.37
4.71 5.22 6.45 6.71 7.71 6.87
15.0 18.0 24.2 30.2 27.5 36.6
42.5 41.0 37.9 34.9 36.3 31.7
1.70 1.41 1.42 1.83 1.94 1.07
0.58 0.37 0.41 0.00 0.00 0.00
1.90 27.9 28.2 37.7 80.5 78.1
2.63 1.37 1.22 0.37 0.19 0.17
Experimental substrate Start of substrate preparation Oxygen-driven phase (phase 1) Pasteurization (phase 1) Spawning (phase 2) Spawn-run compost (phase 3) End of cultivation – SMS
0 6 13 20 33 66
26.6 24.0 23.5 28.5 29.3 30.8
8.24 7.30 8.27 6.68 5.86 6.69
4.75 6.88 7.10 7.12 8.14 7.95
11.5 14.6 17.9 20.8 24.9 33.8
44.3 42.7 41.1 39.6 37.6 33.1
1.63 1.85 2.10 2.63 2.79 2.54
0.69 0.83 0.69 0.03 0.00 0.00
15.9 29.7 21.9 54.2 68.9 70.4
2.96 1.29 1.32 0.65 0.21 0.23
Data are means of triplicate dry weights, with coefficient of variation (CV) < 5%. (DM ¼ dry matter; EC ¼ electrical conductivity; TOC ¼ total organic carbon; TN ¼ total nitrogen, AN ¼ NH3-nitrogen; GI ¼ germination index; TP ¼ total polyphenols; SMS ¼ spent mushroom substrate).
R. Altieri et al. / International Biodeterioration & Biodegradation 63 (2009) 993–997
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Table 7 Evolution of organic matter, total and organic nitrogen in the control and experimental substrates during phases 1 of substrate preparation and phase 3 spawn-run substrate. Dry matter, tons Organic matter, tons Total nitrogen, tons Organic nitrogen, tons Organic matter, % Total nitrogen, % Organic nitrogen, % Control substrate Start of substrate preparation 56.3 Pasteurization (phase 1) 39.4 Spawn-run substrate (phase 3) 31.4
44.4 30.6 22.5
0.96 0.56 0.61
0.63 0.40 0.61
0.0 35.6 52.5
0.0 41.5 36.4
0.0 36.8 3.4
Experimental substrate Start of substrate preparation 59.6 Pasteurization (phase 1) 41.6 Spawn-run substrate (phase 3) 28.3
52.5 34.4 21.2
0.97 0.87 0.79
0.56 0.59 0.79
0.0 34.4 59.6
0.0 10.2 18.6
0.0 4.6 41.1
The selectivity of the ES and control substrates for A. bisporus cultivation (Fermor and Macauley, 1991) was assessed by evaluating microbial growth, with particular reference to actinomycetes in samples collected after pasteurization. Actinomycetes are known to be stimulated by mesophilic conditions at 45–50 C after pasteurization of the substrate, and play an important role in the conversion of N-ammonia into protein and subsequent reduction of free ammonia content to levels that are not toxic to Agaricus mycelium (Fermor et al., 1985). Moreover, Agaricus mycelium is known to be a scavenger of some actinomycete species. In fact, during the composting, actinomycetes promote decomposition of straws leading to brown substances rich in amino acids, particularly alanine and isoleucine (Huntjens, 1972), which are showed to significantly increase Agaricus yield (Royse and Sanchez, 2008). Our work demonstrated that the OMW-amended substrate supported higher populations of beneficial microbes (actinomycetes), which helped in breakdown of the compost, thus improving compost selectivity and subsequent protection against competitors (Huntjens, 1972). This produced a higher OM degradation in the ES than in the control (59.6% and 52.5%, respectively) and the better ammonium nitrogen fixation by actinomycetes with an overall lower nitrogen loss in ES than in the control substrate (18.6% and 36.4%, respectively, Table 7). Difference in the proportion of OMloss was confirmed by ash analyses (Table 6), with 60.8% and 53.5% OM-loss in ES and control respectively as also showed by Viel et al. (1987). The better nitrogen fixation by mesophilic microorganisms was also confirmed by the increase in the organic nitrogen (41.1%) in ES at spawn run instead of organic nitrogen loss (3.4%) recorded in the control (Table 7). This feature increases the agronomic value of ES and make it a slow-releasing nitrogen source for plants in the preparation of nursery growth media or in the re-cultivation of mushroom (Xiao, 1998; Poppe, 2000). Both SMC under trial showed low phytotoxicity (GI>60%), within the range of safety for agronomic use (Zucconi et al., 1981). In conclusion, the re-use of olive mill waste in the preparation of growth media for A. bisporus (Lange) Sing. has been demonstrated in a commercial-scale cultivation trial, being an efficient technology in olive mill waste management; it was also assessed that the SMC derived from ES showed good physical–chemical and biological properties for different agronomic applications. Acknowledgments This work was carried out with the financial support from the ARSIA Toscana Agency. References Altieri, R., Esposito, A., 2008. Olive mill waste amendments in an intensive olive orchard: effects on soil organic carbon, plant growth and yield. Bioresour. Technol. 99/17, 8390–8393. A.O.A.C., 1990. Official Methods of the Association of Official Analytical Chemists, fifteenth ed. Arlington, VA, USA, p. 79. DI.VA.P.R.A. (DIpartimento di VAlorizzazione e Protezione delle Risorse Agro-forestali, Sez. Chimica Agraria, Universita` di Torino) e I.P.L.A. (Istituto per le Piante
da Legno e l’Ambiente), 1992. Metodi di analisi dei compost - Determinazioni chimiche, fisiche, biologiche. Analisi merceologica dei rifiuti, Assessorato all’Ambiente Regione Piemonte. Collana Ambiente, 29–33. 43–47. Felipo´, M.T., 1996. Compost as a source of organic matter in Mediterranean soils. In: de Bertoldi, M., Sequi, P., Lemmes, B., Papi, T. (Eds.), The Science of Composting. Blackie Academic and Professional, Glasgow, UK, pp. 403–412. Fermor, T.R., Randle, P.E., Smith, J.F., 1985. Compost as a substrate and its preparation. In: Flegg, P.B., Spencer, D.M., Wood, D.A. (Eds.), The Biology and Technology of the Cultivated Mushroom. John Wiley and Sons Ltd., Chichester, UK, pp. 81–110. Fermor, T.R., Macauley, B.J., 1991. Microbiological factors contributing to selectivity and nutritional quality of mushroom compost. In: Nair, N.G. (Ed.), Proceedings of AMGA/ISMS International. Workshop-Seminar on Agaricus Compost, Sydney, Australia, pp. 48–65. Huntjens, J.L.M., 1972. Amino acid composition of humic acyd like polymers produced by streptomycetes and humic acids from pasture and arable land. Soil. Biol. Biochem. 4, 339–345. Kalmis, E., Azbar, N., Yildiz, H., Kalyoncu, F., 2008. Feasibility of using olive mill effluent (OME) as a wetting agent during the cultivation of oyster mushroom, Pleurotus ostreatus, on wheat straw. Bioresour. Technol. 99, 164–169. Kalmis, E., Sargin, S., 2004. Cultivation of two Pleurotus species on wheat straw substrates containing olive mill waste water. Int. Biodeter. Biodegrad. 53, 43–47. Kurtzman, R.H., 1997. Nutrition from mushroom, understanding and reconciling available data. Mycoscience 38, 247–253. Lowry, O.H., Rosebrough, N.J., Lewis Farr, A., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275. Manzi, P., Marconi, S., Aguzzi, A., Pizzoferrato, L., 2004. Commercial mushrooms: nutritional quality and effect of cooking. Food Chem. 84, 201–206. Mokrasch, L.C., 1954. Analysis of hexose phosphates and sugar mixtures with the anthrone. reagent. J. Biol. Chem. 208, 55–59. Niaounakis, M., Halvadakis, C.P., 2006. Olive Processing Waste Management Literature Review and Patent Survey, second ed.. In: Waste Management, Series 5 Elsevier, Amsterdam, NL, pp. 23–64. Noble, R., Hobbs, P.J., Mead, A., Dobrovin-Penninoton, A., 2002. Influence of straw types and nitrogen sources on mushroom composting emissions and compost productivity. J. Ind. Microbiol. Biotechnol. 29 (3), 99–110. Pecchia, J.A., Beyer, D.M., Wuest, P.J., 2000. The effects of poultry manure based formulations on odor generation in phase I mushroom composting. In: Proceedings of the 15th International Congress on the Science and Cultivation of Edible Fungi, Netherlands, 15–19 May, vol. 1, pp. 335–339. Poppe, J., 2000. Use of the agricultural waste materials in the cultivation of mushrooms. Mushroom Sci. 15, 3–23. Royse, D.J., Sanchez, J.E., 2008. Supplementation of 2nd break mushroom compost with isoleucine, leucine, valine, phenylalanine, Fermenten and SoyPlus. World J. Microbiol. Biotechnol. 24, 2011–2017. Sanchez, C., 2004. Modern aspects of mushroom culture technology. Appl. Microbiol. Biotechnol. 64, 756–762. Sanjust, E., Pompei, R., Rescing, A., Rinaldi, A., Ballero, M., 1991. Olive milling wastewater as a medium for growth of four Pleurotus species. Appl. Biochem. Biotechnol. 31, 223–235. Sequi, P., 1996. The role of composting in sustainable agriculture. In: de Bertoldi, M., Sequi, P., Lemmes, B., Papi, T. (Eds.), The Science of Composting. Blackie Academic and Professional, Glasgow, UK, pp. 23–29. Singleton, V.L., Sullivan, A.R., Kramer, C., 1971. An analysis of wine to indicate aging in wood or treatment with wood chips or tannic acid. Am. J. Enol. Vitic. 22, 161–197. Viel, M., Sayag, D., Peyre, A., Andre´, L., 1987. Optimization of in-vessel co-composting through heat recovery. Biol. Wastes 20, 167–185. WO/2005/082814. Method and apparatus for treatment of refuses of oil mills, (Metodo ed Apparato per il Trattamento dei Reflui dei Frantoi Oleari MATReFO). http://www.wipo.int/pctdb/en/wo.jsp?WO¼2005%2F082814. Xiao, C., 1998. Studies on mushroom re-cultivation on use compost waste. In: Proc. Int. Symp. Sci. Cultiv. Mushrooms, 1998, 56. Zervakis, G., 2005. Cultivation of the king-oyster mushroom Pleurotus eryngii (DC.:Fr.) Que´l. on substrates deriving from the olive-oil industry. Int. J. Med. Mushrooms 7, 486–487. Zervakis, G., Yiatras, P., Balis, C., 1996. Edible mushrooms from olive oil mill wastes. Int. Biodeter. Biodegrad. 38, 237–243. Zucconi, F., Forte, M., Monaco, A., de Bertoldi, M., 1981. Biological evaluation of compost maturity. BioCycle 22 (4), 27–29.
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Performance of olive mill solid waste as a constituent of the substrate in commercial cultivation of Agaricus bisporus Roberto Altieri a, *, Alessandro Esposito a, Francesca Parati b, Arianna Lobianco c, Milva Pepi c a
CNR, Istituto per i Sistemi Agricoli e Forestali del Mediterraneo, Via Madonna Alta, 128, 06128 Perugia, Italy Azienda Agricola Valfungo, Frazione Gricignano, 6, 52037 Sansepolcro, Arezzo, Italy c ` degli Studi di Siena, Via P. A. Mattioli, 4, 53100 Siena, Italy Dipartimento di Scienze Ambientali, Universita b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 May 2009 Received in revised form 11 June 2009 Accepted 11 June 2009 Available online 21 July 2009
The feasibility of using olive mill waste (OMW) as an ingredient in the substrate used for cultivation of Agaricus bisporus (Lange) Sing. was studied in a large-scale cultivation trial, concerning 2500 m2 of mushroom growing area, at a specialized mushroom farm. Standard commercial cultivation technique involving compost preparation, spawning, casing and harvesting was used. The performance indicators such as mushroom yield, biological efficiency, market quality as well as horticultural value of the spent compost showed that the compost prepared with OMW was superior to the control compost in all the categories. The OMW-amended substrate supported higher populations of beneficial microorganisms especially, actinomycetes which enabled the breakdown of the compost ingredients. It is suggested that OMW is a suitable ingredient for the preparation of mushroom substrate. We have demonstrated that conversion of OMW (a liability) into value-added mushroom substrate (an asset) is an effective waste management tool in oleaculture. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Olive mill waste Recycling Composting Agaricus bisporus cultivation Biological efficiency Organic matter loss Spent mushroom compost
1. Introduction The increasing growth in olive oil production worldwide poses several ecological challenges. The lack of feasible and cost-effective olive mill waste (OMW) management technology compounds this problem. Olive mills produce a lignocellulosic residual solid (olive husk, OH) waste and a dark liquid effluent (olive mill wastewater, OMWW) rich in polyphenols and organic load (Niaounakis and Halvadakis, 2006). OH and OMWW are recalcitrant wastes with significantly high biological oxygen demand (BOD) and chemical oxygen demand (COD). Spreading OH and OMWW on farmlands may be a cost-effective solution to OMWW disposal provided lack of suitable and accessible land close to the mill and high cost of transportation can be overcome (Altieri and Esposito, 2008). Therefore, methods to transform OMW into value-added products, have recently received increasing attention (Felipo´, 1996; Sequi, 1996). With this in mind, the institute of ISAFoM-CNR recently developed a new technology, called MATReFO (WO/2005/082814), which has the capacity to convert raw olive mill effluents (OH and OMWW) into a non-leaching and non-odorous organic matter
* Corresponding author. Tel.: þ39 075 5014540. E-mail address: [email protected] (R. Altieri). 0964-8305/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2009.06.008
useful for agronomic applications. The MATReFO involves system occurs by mixing destoned OH hygroscopic additives such as straw, wool waste, sawdust, olive leaves, twigs and olive pruning residues. This relatively dry organic product is subjected to a short period of aerobic storage and maturation before use. Such type of substrate contains sugars, tannins, lignin, polyphenols, polyalcohols, pectins, lipids and proteins, which serve as carbon, nitrogen and energy sources for growth of mushroom (Zervakis et al., 1996; Sanjust et al., 1991; Kalmis et al., 2008). The selection and management of starting ingredients and the proper conditions for composting make growing Agaricus species so demanding (Sanchez, 2004). Among ingredients, chicken manure is probably the most common and economical source of nitrogen, although many mushroom growers are recently more and more concerned about hygienic (i.e. exposition to Avian influenza virus and other pathogens) and smell problems due to the use and management of animal derivatives (Pecchia et al., 2000). Hence, increasing attention has been devoting to find out and check different source of nitrogen to make mushroom substrate (Noble et al., 2002). Although several papers, mainly relating to Pleurotus spp., have been published on the use of olive mill effluents in the cultivation of edible mushrooms (Kalmis and Sargin, 2004; Zervakis, 2005), there are no published reports on commercial-scale studies on Agaricus bisporus (Lange) Sing. The aim of this paper is to determine whether
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MATReFO mixture can be used as an ingredient in the preparation of substrate for the cultivation of A. bisporus on a commercial-scale.
relatively high concentration of CO2 is required for optimum growth of the mushroom mycelium (vegetative stage).
2. Material and methods
2.2.4. Supplementation of substrate Soy bean meal that has been specially prepared (treated with formaldehyde 3000 ppm) was added to the spawn-run substrate at the rate of 1% fresh weight. The main aim of supplementation is to boost the protein and free fatty acids in the substrate for increased yield of mushrooms. The supplemented substrate was then transferred to the cropping room.
2.1. Cultivation method Preparation of substrate and cultivation of A. bisporus were conducted using the commercial facilities and procedures at the Valfungo mushroom farm at Sansepolcro, Arezzo, Italy. The cultivation trial was conducted over a period of 66 days starting from preparation of substrate to the completion of mushroom harvest comprising of two flushes. The initial three phases of the cultivation process namely, phase 1 (aerobic substrate preparation), phase 2 (pasteurization and stabilization of the substrate) and phase 3 (inoculation of the substrate with mushroom starter culture, the spawn, and incubation of the inoculated substrate) were carried out in concrete ‘tunnels’ (concrete bunkers). Environmental conditions such as ventilation, ambient and substrate temperature and moisture content were controlled during the substrate preparation. The spawn-run substrate was transferred when ready to a 500 m2 cropping room, having a total growing surface area of 13.200 m2 using the Dutch shelf system. The experiment was carried out using a standard mushroom substrate (control) normally used in the Valfungo farm and compared with an experimental substrate (ES) containing olive mill waste (OMW) with added organic ingredients as in the CNRMATReFO recycling system (Altieri and Esposito, 2008). The MATReFO mixture consisted of 72% destoned olive husk taken from OMW produced by two phase decanters in the olive mill, 14% waste wool and 14% wheat straw. 2.2. Composting process 2.2.1. Phase 1 The individual ingredients of the substrate were mixed thoroughly by mechanical means to achieve a homogenous mixture. Two phase 1 tunnels were each filled separately with the two substrates uniformly to a depth of 4 m. The tunnels were sealed and the substrates were ventilated by forcing air through the slatted floor of the tunnel to promote aerobic decomposition of the substrate. The oxygen concentration of the compost piles was controlled in the range 8–15%. The substrate and ambient temperature of the tunnel were monitored by probes located at different points in the tunnel and substrate. The total duration of phase 1 was eleven days. However, six days after filling the tunnel the substrate was removed from the tunnel, mixed for homogeneity and adequate moisture and, placed back in the tunnel for a further period of five days. 2.2.2. Phase 2 This phase was characterized by pasteurization of the substrate at 60 C for 8–9 h. The temperature was lowered to 45–50 C after pasteurization and held at that temperature for five days to ensure total conversion of ammoniacal nitrogen and dissipation of ammonia. The substrate was then allowed to cool down to 25 C. 2.2.3. Phase 3 The pasteurized substrate was removed from the phase 2 tunnel by means of slat bed conveyors into a clean work area. This area was kept under positive pressure bypassing the incoming air through absolute filters (1 mm). The substrate was then inoculated (spawned) with a commercial strain (A15, Sylvan Spawn Ltd.) of A. bisporus at the rate of 0.8% wet weight. The spawned substrate was put into phase 3 tunnel and incubated at 24–26 C, relative humidity 90% and without ventilation (CO2 approx. 3000 ppm in the tunnel) for thirteen days. A
2.2.5. Casing ‘Casing’ is the term used for covering the surface of substrate that has been fully colonized by the mushroom mycelium (spawnrun substrate). The function of casing in mushroom production is not known; however, if the substrate is not cased the switch from vegetative to reproductive stage (production of sporophore initials and sporophores) will be significantly affected with the loss of crop. A mixture of peat moss (Grunig, Vipiteno, Italy) and aged sugar beet lime at 7.5 pH was spread over the substrate to a depth of 5 cm. 2.2.6. Production of mushrooms The cased control and ES substrates were placed in a cropping room fitted with 25 cm deep Dutch shelves arranged in multilayered rows. An additional room was used where the control and ES substrates were placed in shelves of 333 m2 and 167 m2 respectively. Soon after the mycelial growth was detected at the top of the casing surface, a cold shock was applied to the crop by reducing the room temperature to 17–18 C and decreasing the ambient CO2 concentration to 300 ppm by increasing ventilation (number of air changes per hour) in the room. Environmental control of all facilities employed in the trial were performed by automatic control systems. Temperature by means of PT100 probes controlled by PLC; RH through measuring temperature on dry and wet sensors; CO2 by an infra red gas analyzer (IRGA). 2.3. Mushroom production parameters Timing of first flush, total yield and biological efficiency (BE) were evaluated. Total yield, evaluated per square meter or per tons of substrates, and BE were analyzed statistically by the Student– Newman–Keuls test at a significance level of P < 0.05. Market qualities such as appearance (shape, size and blemishes) and shelf life, and nutritional qualities such as dry matter, ash, total nitrogen, protein, fat, total and soluble carbohydrates and polyphenols were analyzed. Shelf life was estimated at 2–4 C in the dark for 10 days on mushrooms collected within the first flush and packaged in commercial boxes. The main mushroom pests and diseases (moulds and insects) were monitored from the stage of sporophore initiation to the end of harvest (second flush). 2.4. Substrate analysis 2.4.1. Microorganisms Total counts of heterotrophic microorganisms and actinomycetes were made using Triptic Soy Agar (TSA) (Difco) and Actinomycete Isolation Agar (AIA) (Difco) of substrate samples collected after pasteurization using standard procedures. One gram of each sample was added to 9 ml of buffer (0.9% NaCl) and serial (1 in 10) dilutions were prepared. One hundred microliters of each dilution was spread by using a sterile spatula, in duplicate, on the surface of TSA and AIA media in Petri dishes. The plates were then incubated at 28 C, and colony forming units (CFU) per gram of fresh weight counted after 2 and 7 days of incubation.
R. Altieri et al. / International Biodeterioration & Biodegradation 63 (2009) 993–997 Table 1 Composition and fresh weight of the substrates used in the A. bisporus cultivation trial. Substrate ingredients
Straw CNR-MATReFO mixture Chicken manure Gypsum Waste wool Ammonium sulfate Urea Starting total carbon Starting total nitrogen
Control substrate
Experimental substrate
tons
%
tons
%
43.4 – 18.5 4.0 – 0.6 0.2 – –
65.1 – 27.8 6.0 – 0.8 0.3 42.5 1.7
46.4 20.0 5.0 4.0 2.4 1.1 0.4 – –
58.5 25.2 6.3 5.0 3.0 1.4 0.5 44.3 1.6
995
Table 3 Total yield of A. bisporus from first and second flushes and assessment of market quality. Control substrate
2.4.2. Physical/chemical factors The main chemical–physical parameters (pH, electrical conductivity (EC), ash, total organic carbon (TOC), total nitrogen (TN), ammonium nitrogen (AN), total polyphenols (TP) and phytotoxicity by means of germination index (GI) using Lepidium sativum) were analyzed on the substrates during its preparation, and on spent mushroom substrate (SMS) at the end of the cultivation trial. Substrate organic matter (OM) loss was measured before pasteurization and after incubation by means of initial weight loss. The data were confirmed by calculating the proportion of OM-loss by mineralization, using initial (X1) and final (X2) ash concentration, according to the equation: OM-loss % ¼ 100100[X1(100-X2)]/[X2(100-X1)] (Viel et al., 1987). Samples of substrates were collected in triplicate and the fresh samples were tested for EC and pH in water extract 1:10 (w/v), TOC, AN and TN. TOC was determined by the Springer–Klee method; AN and TN by means of macro-Kjeldahl distillation method (DI.VA.P.R.A., 1992). Moisture content of the fresh samples was determined as weight loss upon drying at 105 C in an oven for 24 h. Ash were determined as sample weight loss (previously ovendried at 105 C) upon ashing at 650 C for 24 h in a muffle furnace. Water-soluble polyphenols were determined in water extracts (1:10 w/v) using Folin-Ciocalteau method as reported by Singleton et al. (1971). A simplified phytotoxicity test based on germination of L. sativum L. was used: deionized water was added to fresh samples to achieve 65% of water content (wet weight); samples were shaken for 1 h and extracts were then obtained by centrifugation and filtration through a 0.45 mm membrane filter. Extracts were then diluted (30%) and used as germination media. A Whatman filter paper (n. 42), placed inside a 9 cm Petri dish, was wetted by 1 ml of the germination media and 9 seeds of L. sativum were placed on the paper. Deionized water was used as a control germination media and five replicates were carried out for each treatment. The dishes
Experimental substrate
tons
%
tons
%
Yield – first flush Yield – second flush
20.2 8.1
71.4 28.6
17.4 10.6
62.1 37.9
Market quality standard 1 Carpophores diameter > 3 cm 2 1.5a < carpophores diameter < 3 cm 3 Open veil and/or visible spot
6.1 9.5 12.7
21.5 33.5 45.0
8.2 8.1 11.7
29.2 29.0 41.8
a
Tight veil and no visible spot.
were wrapped in ParafilmÒ M, to minimize water loss and allow air penetration, and kept in the dark for 42 h at 24 C. At the end of incubation, the number of germinated seeds and primary root lengths were measured and expressed as percentage of the control (germination index). 2.5. Analysis of mushrooms Water content, total nitrogen, ash and water-soluble polyphenols were determined as described above. Fat was determined according to standard procedure (A.O.A.C, 1990); soluble carbohydrates and proteins by using anthrone reaction (Mokrasch, 1954) and Lowry method (Lowry et al., 1951); total proteins were calculated as TNx4.38 and total carbohydrates by difference, as reported by Manzi et al. (2004). 3. Results Composition of the initial mixtures used in the A. bisporus cultivation trial are given in Table 1. The control and ES substrates had a starting total nitrogen 1.7 and 1.6, total carbon 42.5 and 44.3, C:N 25.0 and 27.2, respectively, and were 71–73% saturated. The total yield and BE for each separate fruiting room under trial are presented in Table 2. Although the total weight of ES spawned compost used in the cultivation trial was lower than control on a dry and fresh weight basis, the total overall yields were comparable (28.27 and 27.94 tons for C and ES, respectively), thus signifying a significant higher mean BE of ES spawned compost (100.5%) than control (93.8%). This trend is also true when mean yields, expressed per square meter or per weight of fresh spawned compost, is considered. The yield and fresh market quality assessments (based on prescribed Italian standards) of mushrooms in the first two flushes are given in Table 3. ES produced a higher prime quality fresh
Table 2 Total yield of A. bisporus from two flushes in each cropping room. Cropping room
Cropping area, m2
Fresh SS, tons
Dry SS, tons
Total yield, tons
Yield, kg m2
Yield, kg tons1 fresh SS
20.4 21.3 22.3
251.7 217.6 256.7
21.3 A
242.0 A
24.1 23.9 23.4
282.5 287.6 310.7
100.5 104.0 97.1
23.8 B
293.6 B
100.5 B
1 2 5 mix room
C C C Total Mean
500 500 333 1333
40.50 49.00 28.90 118.40
10.58 11.50 8.04 30.12
10.20 10.66 7.42 28.27
3 4 5 mix room
ES ES ES Total Mean
500 500 167 1167
42.70 41.60 12.60 96.90
12.00 11.50 4.03 27.53
12.06 11.96 3.92 27.94
a
BE,a % 96.4 92.7 92.3 93.8 A
BE ¼ Biological efficiency (sporophores fresh weight/SS dry weight) 100. (C ¼ control substrate; ES ¼ experimental substrate, SS ¼ spawned substrate). Mean data flanked by the same letter are not significantly different according to SNK test for P < 0.05.
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Table 4 Composition (g 100 g1) of A. bisporus sporophores grown in control and experimental substrates.
Dry mattera Ash Total proteinsb Soluble proteins Total fat Total carbohydrates Soluble carbohydrates Polyphenols
Control substrate
Experimental substrate
1st flush
2nd flush
1st flush
2nd flush
7.9 11.1 19.7 13.6 1.4 67.8 7.2 0.4
7.8 11.1 27.6 18.6 1.5 59.9 6.1 0.6
8.1 10.1 17.5 15.9 1.6 70.6 3.5 0.5
7.9 12.7 27.6 15.2 1.4 58.3 3.5 0.6
Data are means of triplicate dry weight, with coefficient of variation (CV) < 5 %. a g 100 g1 fresh weight. b Total nitrogen 4.38.
Table 5 Total microbial counts CFU (108) g1 in control and experimental substrates after pasteurization during phase 1. Time of incubation
2 days 7 days
Control substrate
Experimental substrate
TSAa
AIAb
TSAa
AIAb
110 580
1.3 340
200 1800
1.7 950
active oxygen-driven composting process, pasteurization and incubation phases (data not showed). Total microbial counts CFU (108) g1 in ES and control substrates after pasteurization during phase 1 are reported in Table 5. After an incubation period of two days the ES compost showed approximately twice the total number of microorganisms than the control substrate. Similarly, after two days incubation, ES accounted for 30% more actinomycetes than the control and this increased to about 279% after 7 days. Evolution of main mushroom growth media chemical and biological parameters, determined all along the cultivation trial, and degradation of organic matter during the preparation of compost, are reported in Table 6 and Table 7, respectively. Although at the beginning of composting the nitrogen content was similar in both compost under trial, nitrogen apparently increased more in ES than control from start to the end of spawnrun phase (2.79% and 1.94% in ES and control, respectively). In addition, both composts showed at spawning, ammonium nitrogen content below lethal limit for A. bisporus. Similar trends for ash and TOC content were recorded in both the substrates, while pH was lower in the ES at all stages of the process. ES also showed a slightly higher electrical conductivity than control while total phenols rapidly degraded, with an overall degradation up to 90% of the initial content, in both substrates. SMC from ES showed phytotoxicity, comparable to that from control, while it showed 2.4 times total nitrogen content compared to that from control.
Data are means of triplicate, with coefficient of variation (CV) < 5%. a Triptic soy agar. b Actinomycete isolation agar.
4. Discussion
mushrooms (29.2%) than the control substrate (21.5%) and a lower amount of third quality mushrooms (41.8%) than control (45.0%). Differences were not observed on the occurrence of fungal diseases such as green mould (Trichoderma spp.) and dry bubble (Verticillium fungicola) between the crops grown in ES and control substrates. As regard shelf life, harvested sporophores grown on ES showed a higher degree of color change from white to brown than those grown on standard compost (pictures not showed). This phenomenon was significantly reduced by packaging sporophores under a polyethylene film. At the present time we are unable to explain this result. In general, low differences were observed in the composition of fruit bodies grown on ES and control substrates except for soluble carbohydrates which were about twice in mushrooms grown in the control substrate in both flushes (Table 4). Differences were not observed between the ES and control substrates in temperature and oxygen profiles recorded during the
The practicality of using olive mill solid waste as an ingredient in the substrate used for commercial-scale cultivation of A. bisporus (Lange) Sing. was studied. Data on mushroom yield, production efficiency, market quality and horticultural value of spent substrate showed that the substrate prepared with olive mill waste performed significantly better than the control in these respects. Therefore, the performance of the ES compost was higher than that normally obtained in the Valfungo crops; the higher production of prime quality mushrooms reduced time and consequent cost of manual harvesting. ES showed a better distribution of yield among flushes with reasonable commercial advantages for a farm such as Valfungo concentrating on fresh market sales. The large variability in the overall sporophore composition reported in literature for A. bisporus (Kurtzman, 1997) suggests that the difference found in soluble carbohydrates (Table 5) fall within the normal range and should not affect the organoleptic value of the mushrooms.
Table 6 Characterization of the control and experimental substrates during preparation of substrates and mushroom growth. Days
DM, %
pH
EC, dS m1
Ash, %
TOC, %
TN, %
AN, %
GI, %
TP, g kg1
Control substrate Start of substrate preparation Oxygen-driven phase (phase 1) Pasteurization (phase 1) Spawning (phase 2) Spawn-run compost (phase 3) End of cultivation - SMS
0 6 13 20 33 66
29.0 22.0 23.4 27.8 25.7 28.8
8.33 8.18 8.37 6.98 6.05 7.37
4.71 5.22 6.45 6.71 7.71 6.87
15.0 18.0 24.2 30.2 27.5 36.6
42.5 41.0 37.9 34.9 36.3 31.7
1.70 1.41 1.42 1.83 1.94 1.07
0.58 0.37 0.41 0.00 0.00 0.00
1.90 27.9 28.2 37.7 80.5 78.1
2.63 1.37 1.22 0.37 0.19 0.17
Experimental substrate Start of substrate preparation Oxygen-driven phase (phase 1) Pasteurization (phase 1) Spawning (phase 2) Spawn-run compost (phase 3) End of cultivation – SMS
0 6 13 20 33 66
26.6 24.0 23.5 28.5 29.3 30.8
8.24 7.30 8.27 6.68 5.86 6.69
4.75 6.88 7.10 7.12 8.14 7.95
11.5 14.6 17.9 20.8 24.9 33.8
44.3 42.7 41.1 39.6 37.6 33.1
1.63 1.85 2.10 2.63 2.79 2.54
0.69 0.83 0.69 0.03 0.00 0.00
15.9 29.7 21.9 54.2 68.9 70.4
2.96 1.29 1.32 0.65 0.21 0.23
Data are means of triplicate dry weights, with coefficient of variation (CV) < 5%. (DM ¼ dry matter; EC ¼ electrical conductivity; TOC ¼ total organic carbon; TN ¼ total nitrogen, AN ¼ NH3-nitrogen; GI ¼ germination index; TP ¼ total polyphenols; SMS ¼ spent mushroom substrate).
R. Altieri et al. / International Biodeterioration & Biodegradation 63 (2009) 993–997
997
Table 7 Evolution of organic matter, total and organic nitrogen in the control and experimental substrates during phases 1 of substrate preparation and phase 3 spawn-run substrate. Dry matter, tons Organic matter, tons Total nitrogen, tons Organic nitrogen, tons Organic matter, % Total nitrogen, % Organic nitrogen, % Control substrate Start of substrate preparation 56.3 Pasteurization (phase 1) 39.4 Spawn-run substrate (phase 3) 31.4
44.4 30.6 22.5
0.96 0.56 0.61
0.63 0.40 0.61
0.0 35.6 52.5
0.0 41.5 36.4
0.0 36.8 3.4
Experimental substrate Start of substrate preparation 59.6 Pasteurization (phase 1) 41.6 Spawn-run substrate (phase 3) 28.3
52.5 34.4 21.2
0.97 0.87 0.79
0.56 0.59 0.79
0.0 34.4 59.6
0.0 10.2 18.6
0.0 4.6 41.1
The selectivity of the ES and control substrates for A. bisporus cultivation (Fermor and Macauley, 1991) was assessed by evaluating microbial growth, with particular reference to actinomycetes in samples collected after pasteurization. Actinomycetes are known to be stimulated by mesophilic conditions at 45–50 C after pasteurization of the substrate, and play an important role in the conversion of N-ammonia into protein and subsequent reduction of free ammonia content to levels that are not toxic to Agaricus mycelium (Fermor et al., 1985). Moreover, Agaricus mycelium is known to be a scavenger of some actinomycete species. In fact, during the composting, actinomycetes promote decomposition of straws leading to brown substances rich in amino acids, particularly alanine and isoleucine (Huntjens, 1972), which are showed to significantly increase Agaricus yield (Royse and Sanchez, 2008). Our work demonstrated that the OMW-amended substrate supported higher populations of beneficial microbes (actinomycetes), which helped in breakdown of the compost, thus improving compost selectivity and subsequent protection against competitors (Huntjens, 1972). This produced a higher OM degradation in the ES than in the control (59.6% and 52.5%, respectively) and the better ammonium nitrogen fixation by actinomycetes with an overall lower nitrogen loss in ES than in the control substrate (18.6% and 36.4%, respectively, Table 7). Difference in the proportion of OMloss was confirmed by ash analyses (Table 6), with 60.8% and 53.5% OM-loss in ES and control respectively as also showed by Viel et al. (1987). The better nitrogen fixation by mesophilic microorganisms was also confirmed by the increase in the organic nitrogen (41.1%) in ES at spawn run instead of organic nitrogen loss (3.4%) recorded in the control (Table 7). This feature increases the agronomic value of ES and make it a slow-releasing nitrogen source for plants in the preparation of nursery growth media or in the re-cultivation of mushroom (Xiao, 1998; Poppe, 2000). Both SMC under trial showed low phytotoxicity (GI>60%), within the range of safety for agronomic use (Zucconi et al., 1981). In conclusion, the re-use of olive mill waste in the preparation of growth media for A. bisporus (Lange) Sing. has been demonstrated in a commercial-scale cultivation trial, being an efficient technology in olive mill waste management; it was also assessed that the SMC derived from ES showed good physical–chemical and biological properties for different agronomic applications. Acknowledgments This work was carried out with the financial support from the ARSIA Toscana Agency. References Altieri, R., Esposito, A., 2008. Olive mill waste amendments in an intensive olive orchard: effects on soil organic carbon, plant growth and yield. Bioresour. Technol. 99/17, 8390–8393. A.O.A.C., 1990. Official Methods of the Association of Official Analytical Chemists, fifteenth ed. Arlington, VA, USA, p. 79. DI.VA.P.R.A. (DIpartimento di VAlorizzazione e Protezione delle Risorse Agro-forestali, Sez. Chimica Agraria, Universita` di Torino) e I.P.L.A. (Istituto per le Piante
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