New substrates for the cultivation of Pleurotus ostreatus using exhausted compost

Several materials have been used in the cultivation of the edible mushroom Pleurotus ostreatus. However, little is known about the reuse of the exhausted compost. This study evaluated the utilization of used substrates. Four formulations of composts were evaluated: C1 with no exhausted compost, and C2, C3 and C4 with 26, 45 and 64% of exhausted compost, respectively. Loss of organic matter, biological efficiency and mass of basidiomata were evaluated by means of the results of the chemical analysis of the initial and final composts and the nutritional assessment of the basidiomata. The data obtained were submitted to statistical analysis. The results of the chemical analysis of the composts show an increase of nitrogen between the initial and the exhausted compost and a decrease of the carbon/nitrogen ratio. The loss of organic matter and biological efficiency of composts C2, C3 and C4 were lower than the traditional compost. The mass of fresh basidiomata of composts 1 and 2 were not significantly different, being superior to other treatments. Treatment C3 showed a higher amount of protein. The conclusion was that the exhausted compost can be reused up to a certain amount without affecting the production and the nutritional value.


INTRODUCTION
With the advent of the second-generation ethanol, crushed sugarcane, one of the main substrates used in mushroom cultivation in the State of São Paulo, has become scarcer in the market, once it has been used by industries as an energy source in boilers.Thus, fungi producers have found difficulties to obtain this product and, when it is available, its price has become continuously inviable.
Another common problem in mushroom cultivation regions is the correct discharge of the exhausted compost in order to avoid environmental damages.After harvesting, it is very common to find producers who pile all the exhausted substrate somewhere else in the property, attracting flies and other agronomic pests which can eventually cause damages for the next mushroom cultivations, according the distance from the production site and the environmental factors (rain, wind, humidity, etc.).
Several materials have been used in the preparation of the compost for the cultivation of Pleurotus ostreatus, *Corresponding author.E-mail: mcnandrade@hotmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License such as sawdust, banana tree straw, coffee husks and several cellulosic residues (Bonatti et al., 2004;Fan et al., 2006;Tisdale et al., 2006;Das and Mukherjee, 2007;Sales-Campos et al., 2010a;Carvalho et al., 2010).However, little is known about the reuse of these materials for new cultivation cycles.Royse (1993) evaluated the performance of exhausted compost used to produce Shiitake by adding wheat bran and corn in the production of Pleurotus sajor-caju.Kilpatrick et al. (2000) used formulations in the cultivation of Lentinula edodes added with exhausted compost of Agaricus, with several grains, wheat flour and calcium carbonate ratios.Mamiro and Royse (2008) evaluated portions of exhausted substrate added to the traditional compost of A. bisporus.
Thus, the objective of the present work was to find a noble destination for the exhausted compost (mushrooms production) and reduce the accumulation of this material in the environment by using it in new cultivation cycles of P. ostreatus.

MATERIALS AND METHODS
The experiment was carried out in two stages: 1. Composting and pasteurization carried out at the Faculdade de Ciências Agronômicas (FCA/UNESP), Botucatu, São Paulo, Brazil.2. Incubation and harvesting carried out at the Universidade do Sagrado Coração (USC), Bauru, São Paulo, Brazil.Three formulations of composts were tested, named based on the exhausted substrate and compared to traditional compost (without the addition of exhausted compost -control) for the cultivation of P. ostreatus.
The P. ostreatus strain used in the experiment was POS-09/101, obtained from the mycology collection of the Módulo de Cogumelos, FCA/UNESP, Botucatu.The inoculum was prepared by using the methodology proposed by Minhoni et al. (2005).
Phase I of the composting was performed in an open shed, with galvanized sheet roof and concrete floor.Before forming the plots, the sugarcane straw was moistened at 75% of average humidity and revolved every two days for a total period of six days (prewetting).
After Phase I of composting, the plots were formed by a moistened straw layer (20 cm high), followed by an exhausted compost layer (20 cm high) (with exception of the control) until reaching 1.8 m of height × 1.8 m of width, respectively.
Limestone, plaster and wheat bran were added in all plots according to each treatment (Tables 1 to 4).All the materials used were previously analyzed in order to obtain a calculated C/N ratio of 67/1, which is the most recommended for the cultivation of P. ostreatus.Limestone was used to correct the pH of the compost.Gypsum was added to improve the physical characteristics of the compost.
The composts were turned over and water was added manually with a hose to keep humidity between 70 and 75%.In Phase I, three overturns were performed in a total of six days.In Phase II, the composts were transferred to lattice boxes and then arranged randomly inside a climatic chamber (Dalsem Mushrooms) for pasteurization (8 hours at 62 ± 2°C) and conditioning (4 days at 48 ± 2°C).
The inoculation of the compost with Spawn from strain POS-09/101 of P. ostreatus was performed manually, inside a Dalsem climatic chamber.The tools used (tray, scissors and dosing glass of Spawn) were cleaned with alcohol 70%.The inoculum ratio used was 40 g Kg -1 of fresh mass of the compost.
The incubation was performed in an experimental greenhouse at USC for 30 days at an average temperature of 25°C and relative humidity of 55 to 70%.The four treatments named C1, C2, C3 and C4 were control, 26% of exhausted compost, 45% of exhausted compost and 64% of exhausted compost (Tables 1 to 4), respectively; they were randomly arranged on shelves and represented by four treatments with twenty repetitions each.After the incubation period, harvesting and weighing of mushrooms were carried out daily for 60 days.
The nutritional analyses of the basidiomata were performed at the Food Laboratory of the USC, in Bauru, São Paulo.Three whole samples of basidiomata from each treatment were dehydrated and ground for analysis.A total of 12 samples of mushrooms were analyzed for raw protein, ash and lipids, according to the methodology of Silva and Queiroz (2002), with some adjustments mentioned ahead.
To evaluate humidity, dry weighting bottles were used in a greenhouse at 105°C for half an hour and then cooled with their respective lids in a desiccator.After cooling, the bottles were weighed empty in analytical scales and then approximately 3.5 g of sample of each treatment were added and the bottles were weighed again.Next, the samples were dried in a greenhouse at 105°C for 6 h, removed, weighed again and placed back inside the greenhouse until reaching constant weight.Ash was determined by incinerating a 5 g sample of each treatment in a muffle at 550°C.The samples were manipulated by using a clamp and a crucible and then cooled in a desiccator and weighed again.
The raw protein content was evaluated by the Kjeldahl method by extracting the total amount of nitrogen of the samples and multiplying them by the correction factor (PB% = N × 4.38).
Usually, the correction factor used in this type of analysis is 6.25, considering that proteins have 16% of nitrogen.However, this value is 4.38 for fungi because they have non-digestible nitrogen composts, such as the chitin, in the cell wall (Furlani and Godoy, 2005).
Approximately 0.2 g of each sample was weighted to extract the nitrogen.Ten glass beads in the Kjeldahl tube, 5 ml of H2SO4 and 2.5 g of a catalyzed mixture of CuSO4 and K2SO4 were placed inside the digester; the temperature was gradually increased until reaching 400°C.
7 ml of distilled water and 3 drops of methyl red indicator were dropped in the Kjeldahl tube. 10 ml of 4% boric acid and 3 drops of mixed indicator were poured in an Erlenmeyer flask.20 mL of 40% NaOH were added to the receptacle of the equipment and the tap was opened in order to allow a slow dripping over the Kjeldahl tube of the equipment already adapted.
The button on the left of the equipment was turned on in the beginning of the reflow; the Erlenmeyer flask was placed and left to distill until reaching the volume of 50 ml.The button on the left was turned off when the desired volume was reached; the Erlenmeyer was removed and titrated with standardized HCl 0.1 M until the color changed.
The N percentage was calculated by using the formula "nitrogen % = 0.014 × N × f × V (ml) spent × 100/weight of the sample".
The determination of lipids was performed by using a Soxhlet extractor; approximately 3.5 g of each sample were weighed and placed in Soxhlet cartridges and weighed in a flat bottom round glass bottle.The equipment was assembled with enough petroleum ether for the occurrence of siphonage and left for 8 h with 4 to 5 drops per second; the round glass botlle was dried in a stove at 105°C for half an hour and weighed again.
At the end of Phases I and II of composting, three samples of compost from each plot were removed and dehydrated at 65°C during 48 h for carbon, nitrogen, organic material and pH analysis.The same procedure was repeated at the end of the cultivation cycle.These analyses were performed at the Fertilizers and Correctives Chemical Analyses Laboratory of the Department of Natural Resources of the School of Agronomic Sciences -UNESP, According to Rajarathnam and Bano (1989), the loss of organic matter (LOM) is the index that evaluates the decomposition of the substrate by the fungus, which occurs during the cultivation.This index is based on the loss of organic matter decomposed by the fungus and it is determined by the difference between the dry mass of the initial substrate and the dry mass of the residual substrate (post-harvest).
Productivity was expressed by means of the biological efficiency (BE), which represents the conversion percentage of the substrate into fungal biomass (basidiomata).
Data were submitted to the analysis of variance and the averages were compared by the Tukey test (5%) (Snedecor and Cochran, 1972) using the SISVAR 4.2 software developed by the Department of Exact Sciences from the Federal University of Lavras, Minas Gerais, Brazil (UFLA).

RESULTS AND DISCUSSION
The chemical analyses of the composts used for the cultivation of P. ostreatus at the end of the composting Phases I and II and at the end of the cultivation cycle (exhausted compost) are shown in Tables 5 to 7.
The results of the four treatments at the end of Phases I and II of composting were not significantly different among each other (Tables 5 and 6).There was a little decrease in the nitrogen values in this period.On the other hand, the values presented in the substrate after the cultivation showed an increase in the nitrogen percentage.Sales-Campos et al. (2010b) noticed an increase in the amount of nitrogen in the exhausted substrate of P. ostreatus.It is possible to observe the reduction of organic material (OM%) and carbon (C%) among the three stages (Table 5 to 7).
Organic matter and carbon values were not significantly different between treatments at the end of phases I and II, but these values varied for the exhausted substrate, showing a non-uniform consumption of carbon and organic matter among the four treatments (Table 7).
The C/N ratio of the treatments varied between 49.0 and 37.0 at the end of Phase I.These values declined in the end of Phase II and in the end of the cultivation cycle due to the carbon consumption from the fungal capability of degrading lignin and cellulose, with a posterior release of CO 2 and H 2 O.The highest values of the C/N ratio were provided by the treatments C1 (40.66) and C2 (46.66), indicating that these values are influenced by the amount of exhausted compost mixed with the traditional one.
According to Sales-Campos et al. (2010a), the ideal value of the C/N ratio for P. ostreatus is 80/1 in axenic cultivation (without composting) and around 25 to 50/1 for agroindustrial waste taken to composting and pasteurization process, according to Duprat (2012).
A decrease in the substrate humidity was verified in the results of the three composting phases (Tables 6 to 8); however, it was not significantly different in the analyses in the end of Phase I and in the end of the cultivation cycle.It was observed that this decrease occurs as the amount of exhausted substrate increases.
PH decreased during the colonization of the substrate by P. ostreatus, except for Treatment C3, which showed an increase.According to Chang and Miles (1989), the decrease of pH during the colonization process of the substrate by the fungus occurs due to the production of substances, such as fatty acids.The increase of pH in treatment C3 may be explained by the production of metabolites by P. ostreatus, such as the fatty acids affecting the concentration of the compost.
The biological efficiency (BE%) and the loss of organic matter (LOM%) were significantly different among each other (Table 8); the highest averages were obtained by treatment C1 (54.20%).There was a gradual decrease in terms of BE% and LOM% as the levels of exhausted substrate increased.However, the mass of fresh basidiomata (MFB) were not significantly different between treatments C1 and C2.
The decreasing rates of BE% and LOM% might be related to the decreasing rates of the C/N ratio in the end of Phase II, once this was the moment in which the inoculation of P. ostreatus occurred.
In an experiment carried out with sugarcane straw and bocaiuva straw, Cardoso et al. (2013) noticed that the decrease of BE% was proportional to the decrease of the C/N ratio.Bernardi (2010), testing the efficiency of various substrates for P. ostreatus and P. sajor-caju, noticed that P. ostreatus showed a better mycelial growth in elephant grass with a high C/N ratio: 162:1.However, the results for biological efficiency and productivity showed better results in waste of castor bean cultivation (with a C/N ratio of 37:1) and elephant grass mixed with waste of castor bean (with a C/N ratio of 73:1); the biological efficiency was not significantly different.
These results might be compared the treatment C2, which did not obtain a significant BE% when compared to treatment C1, but presented good results in terms of mass of fresh basiodiomata (MFB).It was observed that both treatments showed higher values of C/N ratio in the end of Phase I.
Pardo-Giménez and Pardo-Gonzáles ( 2009) evaluated the efficiency of the cultivation of P. ostreatus in exhausted substrate of P. ostreatus mixed with the exhausted substrate of Agaricus sp, in different proportions when compared to the commercial substrate.It was observed that, in this study, the C/N ratio decreased as the amount of exhausted substrate of P. ostreatus decreased and proportionally to the decrease of BE%.However, the productivity of basidiomata was not significantly different to a certain extent, in terms of the amount relation of the P. ostreatus exhausted substrate and the C/N ratio, as in this study.
The result of the nutritional analysis of the basidiomata can be seen in Table 9. Treatment C3 had the highest values of proteins and ash.On the other hand, treatments C1 and C2 were not significantly different from each other in the parameters analyzed.However, this result showed itself different for ash in the dry base, being the best An increase in the protein content was observed in treatment C3.This increase might have occurred due to the increase in the amount of exhausted substrate; however, this value decreased in treatment C4, showing that this assumption is not completely trustful.According to Furlan and Godoy (2005), the type of substrate used is one of the main factors that influence the proteins content of the mushrooms.The nitrogen content in the substrate in the end of phase II did not vary in the present study; however, the C/N ratio was different among the treatments (Table 6).It is theoretically known that the amount of total nitrogen and organic matter are closely related, probably influencing the amount of proteins and ash in this study.

Conclusions
1.The use of exhausted substrate for the cultivation of P. ostreatus is viable until the amount of 26% mixed to the traditional substrate; 2. The biggest average of raw protein in the basidiomata was provided in the compost with 45% of exhausted substrate mixed to the traditional; 21.06%; 3. The C/N ratio influenced the biological efficiency and the loss of organic material proportionally; the initial composts with higher C/N ratios provided a higher biological efficiency and loss of organic matter.

Table 5 .
Chemical analysis of the substrate at the end of phase I.

Table 6 .
Chemical analysis of the substrate at the end of phase II.

Table 7 .
Chemical analysis of the substrate at the end of the cultivation cycle.(exhausted).

Table 9 .
Nutritional analysis of the basidiomata.with64% of exhausted compost; CV, coefficient of variation; MSD, Minimum significant difference; N%, Nitrogen percentage; OM%, Organic matter percentage; C%, Carbon percentage; pH, hydrogen potential.Averages followed by equal letters in each column are not different among each other (Turkey, 5%).result obtained for treatment C3.Treatments C1 and C2 are right below this value and were not significantly different from each other.The treatment with the lowest value was treatment C2.The results regarding lipids and humidity were not statistically different from each other.