ABSTRACT
The cocoa and palm oil agro-industries active in the state of Bahia, Brazil, generate high quantities of lignocellulosic wastes that could be recycled through their use in the formulation of substrates to cultivate edible mushrooms. Pleurotus ostreatus, also known as oyster mushroom, is the second most cultivated mushroom in the world due to its highly appreciated gastronomic, nutritional, and medicinal characteristics. This work evaluated the vertical mycelium growth, biological efficiency, mushroom yield, and nutritional composition of P. ostreatus produced in substrates formulated with a combination of palm oil fruit mesocarp (POFM) and cocoa almond peels (CAP) processing wastes. The substrates were formulated with the following POFM/CAP proportions (%/%): S1 – 86.4/9.6; S2 – 76.8/19.2; S3 – 67.2/28.8; S4 – 57.6/38.4, and S5 – 48.0/48.0. Substrates also received 3% powdered charcoal and 1% calcium carbonate. Substrates S1, S2, S3, and S4 were superior for vertical mycelium growth. S2 promoted the best biological efficiency (148.8%) and yield (560.5g·kg-1).The mushrooms produced in all substrates presented good nutritional values, although mushrooms produced using the S2 presented the highest crude protein content. Overall, S1 is the recommended substrate as it results in higher yields of nutrient rich mushrooms. Production of P. ostreatus in substrates composed of POFM and CAP represents a good alternative for recycling these wastes with potential economic and ecological benefits to regions where palm oil and cocoa are grown.
KEYWORDS
oyster mushroom; fungal protein; bioconversion
RESUMO
As indústrias de cacau e óleo de dendê no estado da Bahia, Brasil, geram grandes quantidades de resíduos lignocelulósicos que podem ser reciclados na formulação de substratos para o cultivo de cogumelos comestíveis. Pleurotus ostreatus ou cogumelo ostra é o segundo cogumelo mais cultivado no mundo por apresentar características gastronômicas, nutricionais e medicinais muito apreciadas. Este estudo avaliou o crescimento micelial vertical, a eficiência biológica, a produção e a composição nutricional de P. ostreatus produzido em substratos formulados com a combinação de resíduos do processamento de frutos de dendê (mesocarpo do fruto de dendê – MFD) e de amêndoas de cacau (tegumento de amêndoas de cacau – TAC). Os substratos foram formulados com as seguintes proporções de MFD e TAC (%/%): S1: 86,4/9,6; S2: 76,8/19,2; S3: 67,2/28,8; S4: 57,6/38,4 e S5: 48,0/48,0. Os substratos também receberam 3% de carvão e 1% de carbonato de cálcio. Os substratos S1, S2, S3 e S4 foram superiores quanto ao crescimento micelial vertical. S2 promoveu os melhores resultados para eficiência biológica (148,8%) e produção (560.5 g·kg-1). Os cogumelos produzidos em todos os substratos apresentaram valores nutricionais promissores. Entretanto, os cogumelos produzidos com o substrato S2 apresentaram o maior conteúdo de proteína bruta. De modo geral, S1 é o substrato recomendado por resultar na maior produção de cogumelos ricos em nutrientes. A produção de P. ostreatus em substratos compostos por MFD e TAC representa uma boa alternativa para a reciclagem desses resíduos com potenciais benefícios econômicos e ecológicos para as regiões produtoras de dendê e cacau.
PALAVRAS-CHAVE
cogumelo ostra; proteína fúngica; bioconversão
INTRODUCTION
Agricultural activities generate high quantities of lignocellulosic wastes represented by leaves, stems, roots, straws, and husks of cultivated plants. The increase in wastes from agriculture directly relates to the world’s population growth and the consequent rising demand for food production. A good alternative to recycle these wastes is through their use in the formulation of substrates for the cultivation of edible mushrooms (KUMLA et al., 2020KUMLA, J.; SUWANNARACH, N.; SUJARIT, K.; PENKHRUE, W.; KAKUMYAN, P.; JATUWONG, K.; VADTHANARAT, S.; LUMYONG, S. Cultivation of Mushrooms and Their Lignocellulosic Enzyme Production Through the Utilization of Agro-Industrial Waste. Molecules, Emeryville, v.25, n.12, p.2811, 2020. https://doi.org/10.3390/molecules25122811
https://doi.org/10.3390/molecules2512281...
).
Pleurotus ostreatus (Jacq.) P. Kumm. 1871, commonly known as oyster mushroom or white-rot fungi, is the world’s second most cultivated mushroom. This edible mushroom is appreciated worldwide for its flavor, as well as its nutritional and medicinal properties (PÉREZ-MARTÍNEZ et al., 2015PÉREZ-MARTÍNEZ, A.S.; ACEVEDO-PADILLA, S.A.; BIBBINS-MARTÍNEZ, M.; GALVÁN-ALONSO, J.; ROSALES-MENDOZA, S. A perspective on the use of Pleurotus for the development of convenient fungi-made oral subunit vaccines. Vaccine, Rochester, v.33, n.1, p.25-33, 2015. https://doi.org/10.1016/j.vaccine.2014.10.059
https://doi.org/10.1016/j.vaccine.2014.1...
). The commercial production of P. ostreatus using lignocellulosic wastes as substrates has an important role in preserving natural resources and ecosystems by recycling agricultural wastes and removing water micropollutants (HULTBERG et al., 2020HULTBERG, M.; AHRENS, L.; GOLOVKO, O. Use of lignocellulosic substrate colonized by oyster mushroom (Pleurotus ostreatus) for removal of organic micropollutants from water. Journal of Environmental Management, Heverlee, v.272, p.111087, 2020. https://doi.org/10.1016/j.jenvman.2020.111087
https://doi.org/10.1016/j.jenvman.2020.1...
). Pleurotus ostreatus participates in the decomposition of lignocellulose, which is composed of lignin, cellulose, and hemicellulose, through the synthesis of enzymes that act on the cell wall components of woody materials (ALFARO et al., 2020ALFARO, M.; MAJCHERCZYK, A.; KÜES, U.; RAMÍREZ, L.; PISABARRO, A.G. Glucose counteracts wood-dependent induction of lignocellulolytic enzyme secretion in monokaryon and dikaryon submerged cultures of the white-rot basidiomycete Pleurotus ostreatus. Scientific Reports, London, v.10, p.12421, 2020. https://doi.org/10.1038/s41598-020-68969-1
https://doi.org/10.1038/s41598-020-68969...
).
Palm oil (Elaeis guineensis Jacq.) and cocoa (Theobroma cacao L.) are two socioeconomically important crops cultivated in the southern region of the state of Bahia, Brazil. In 2018, Brazil produced 1.565.197 tons of palm oil fruit bunches out of the 272.055.131 tons produced worldwide in the same year (FAO, 2020FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS (FAO). Food and Agriculture Data (FAOSTAT), 2020. Available from: http://www.fao.org/faostat/en/#data/QC. Access on: 6 Sep. 2020.
http://www.fao.org/faostat/en/#data/QC...
). The processing of palm oil fruit bunches generates several wastes such as palm kernel, stems, fruit fibers, and peels. The fibers resulting after palm oil fruit pressing (palm oil fruit mesocarp – POFM) represent 12% of the wastes generated from the palm oil processing industry (ROSA et al., 2010ROSA, M.F. et al. Cellulose nanowhiskers from coconut husk fibers: effect of preparation conditions on their thermal and morphological behavior. Carbohydrate Polymers, Tenbury Wells, v.81, n.1, p.83-92, 2010. https://doi.org/10.1016/j.carbpol.2010.01.059
https://doi.org/10.1016/j.carbpol.2010.0...
).
In 2018, the worldwide production of cocoa was 5.252.377 tons, with Brazil occupying the sixth position in the global ranking, with 239.387 tons produced (FAO, 2020FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS (FAO). Food and Agriculture Data (FAOSTAT), 2020. Available from: http://www.fao.org/faostat/en/#data/QC. Access on: 6 Sep. 2020.
http://www.fao.org/faostat/en/#data/QC...
), of which almost 50% were from the state of Bahia (IBGE, 2019INSTITUTO BRASILEIRO DE GEOGRAFIA E ESTATÍSTICA (IBGE). Sistema IBGE de Recuperação Automática. Tabela 5457 – Área plantada ou destinada à colheita, área colhida, quantidade produzida, rendimento médio e valor da produção das lavouras temporárias e permanentes. 2019. Available from: https://sidra.ibge.gov.br/tabela/5457#resultado. Access on: 7 Sep. 2020.
https://sidra.ibge.gov.br/tabela/5457#re...
). In 2014, it was estimated that the state of Bahia, alone, generated approximately 16.2 thousand tons of cocoa almond peels (CAP). Indeed, 80-120 kg of the waste from CAP are produced from every 1,000 kg of cocoa almonds with 7% humidity (SILVA et al., 2015SILVA, R.B.; FONTES, C.M.A.; LIMA, P.R.L.; GOMES, O.F.M.; LIMA, L.G.L.M.; MOURA, R.C.A.; TOLEDO FILHO, R.D. Cinzas de biomassa geradas na agroindústria do cacau: caracterização e uso em substituição ao cimento. Ambiente Construído, Porto Alegre, v.15, n.4, p.321-334, 2015. https://doi.org/10.1590/s1678-86212015000400053
https://doi.org/10.1590/s1678-8621201500...
). Fruit peels are the main waste produced by the industrial processing of cocoa almonds to obtain the liquor (raw material to make chocolate).
Various agricultural wastes have been used in formulations of substrates to grow P. ostreatus, straw from alfalfa, pecan nuts, wheat, rice, corn, coffee, coconut, eucalyptus, sugarcane, cocoa, cotton, sawdust from bamboo, and several woods (CONDÉ et al., 2017CONDÉ, V.F.; OLIVEIRA, J.E.Z.; OLIVEIRA, D.M.F. Farinha de Pleurotus ostreatus (Jacq.) P. Kumm. (cogumelo Hiratake) enriquecido em ferro. Ciência e Natura, Santa Maria, v.39. n.1, p.1-6, 2017. https://doi.org/10.5902/2179460X21437
https://doi.org/10.5902/2179460X21437...
; RODRÍGUEZ, 2018RODRÍGUEZ, S. Material biobasado compuesto por el micelio de hongos descomponedores de madera y residuos agroindustriales. Revista Chilena de Diseño, RChD: Creación y Pensamiento, Chile, v.3, n.5, p.1-15, 2018. https://doi.org/10.5354/0719-837X.2018.50632
https://doi.org/10.5354/0719-837X.2018.5...
; YAMAUCHI et al., 2019). However, the combination of the agro-industrial wastes POFM and CAP as substrates for the production of high-protein foods such as P. ostreatus has not been investigated so far.
Therefore, the present study aimed to evaluate the mycelium growth and production of P. ostreatus in substrates formulated with different proportions of POFM and CAP. Besides the obvious environmental impact, studies involving the recycling of these wastes are warranted because mushroom production has the potential to increment food production, jobs, and income generation in regions that produce cocoa and palm oil in Brazil and other countries, and, consequently, contribute to food security and poverty reduction. Importantly, these are amongst the objectives for sustainable development of the 2030 Agenda of the United Nations (UN, 2020UNITED NATIONS (UN). The Sustainable Development Agenda. Sustainable Development Goals. 2020. Available from: https://www.un.org/sustainabledevelopment/development-agenda. Access on: 29 Sep. 2020.
https://www.un.org/sustainabledevelopmen...
).
MATERIAL AND METHODS
Wastes from the agro-industries of palm oil and cocoa
Palm oil fruit mesocarp waste was collected from the Kidendê palm oil production plant, located in the municipality of Taperoá, Bahia, Brazil (13°19’40.7”S; 39°02’47.0”W). Cocoa almond peel waste was donated by the cocoa almond processing plant Barry Callebaut Company, located in the municipality of Ilhéus, Bahia, Brazil (14°47’35.6”S; 39°03’07.2”W).
Growth of P. ostreatus
Pleurotus ostreatus isolate Plo 02 was donated from the culture collection of the Laboratory of Mycorrhizal Associations, Department of Microbiology, Universidade Federal de Viçosa, Minas Gerais, Brazil. The culture was preserved in test tubes with potato dextrose agar (PDA, Acumedia) at 4 °C before being reactivated by mycelium inoculation onto PDA medium and incubation for 7 days at 25 °C.
Preparation of P. ostreatus spawn
Sorghum grains were soaked in tap water for 2 h, and the excess of water was drained with a common household plastic sieve. Then, 100 g of the soaked grains were transferred to 200 mL-glass flasks, closed with a metallic cover, and sterilized in an autoclave at 121 °C for 55 min. After cooling, each flask, containing the sterilized grains, was inoculated with three agar plugs (0.5 mm diameter) of the P. ostreatus culture (grown as mentioned above) and incubated at 25 °C for 20 days, without photoperiod.
Substrates formulation
For substrate formulation, POFM and CAP were combined as described in Table 1. The substrate mixture received 3% of triturated charcoal and 1% CaCO3, to maintain the humidity and pH, as recommended by some mushroom producers in Brazil. Activated carbon or charcoal is also recommended for grain-based substrate casing in Agaricus bisporus production (BECHARA et al., 2009BECHARA, M.A.; HEINEMANN, P.H.; WALKER, P.N.; DEMIRCI, A.; ROMAINE, C.P. Evaluating the addition of activated carbon to heat-treated mushroom casing for grain-based and compost-based substrates. Bioresource Technology, Lucknow, v.100, n.19, p.4441-4446, 2009. https://doi.org/10.1016/j.biortech.2008.12.064
https://doi.org/10.1016/j.biortech.2008....
).
Vertical mycelium growth of P. ostreatus
To evaluate the growth of P. ostreatus, the formulated substrates (Table 1) were transferred to test tubes 20 cm long and 3 cm wide. Each tube was filled with 28.5 g of fresh substrate, which allowed forming a substrate column of 12 cm length within the tubes. The tubes were than covered with a cotton plug and wax paper and sterilized in an autoclave at 121 °C for 55 min. After sterilization, inoculation was conducted by transferring 1 g of the prepared spawn (inoculum) to the substrate surface within the tubes. The tubes were closed with the cotton plug and wax paper and incubated at 25 ± 1 °C, in the dark, in an incubation room. Mycelium vertical growth was measured along the substrate by using a ruler placed over the tubes, at every two days. The measurements of mycelium growth for all treatments were stopped when growth reached the total substrate length (12 cm), for the first time in one of the tubes. Mycelium vigor was analyzed through the non-microscopic observation of its growth characteristics following the scale described by PEDRA (2006)PEDRA, W.N.; MARINO, R.H. Cultivo axênico de Pleurotus spp. em serragem da casca de coco (Cocos nucifera Linn.) suplementada com farelo de arroz e/ou de trigo. Arquivos do Instituto Biológico, São Paulo, v.73, n.2, p.219-225, 2006. Available from: http://www.biologico.sp.gov.br/uploads/docs/arq/V73_2/pedra.PDF. Access on: 8 Feb. 2020
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: 1 – weekly dense, 2 – medium dense, and 3 – highly dense.
Proportions of palm oil fruit mesocarp (POFM) and cocoa almond peels (CAP) processing wastes used to formulate substrates for the cultivation of P. ostreatus. All substrate formulations received 3% of powdered charcoal and 1% of CaCO3.
Production of P. ostreatus
For mushroom production, the substrates (Table 1) were transferred to 1 kg polypropylene bags fitted with a filter for gas exchange. The bags with the substrates were sterilized in an autoclave at 121 °C for 55 min. After cooling, the surface of the substrates was inoculated with 20 g of P. ostreatus spawn (seed inoculum produced as described above). The bags were sealed and transferred to a mushroom colonization chamber maintained at 25 ± 2 °C in the dark, until complete colonization of the substrate.
After substrate colonization, the bags were transferred to a mushroom growth chamber with automated control of humidity and temperature through a system using nebulization and fans. Initially, the colonized bags received a shock treatment with the reduction of the temperature in the chamber to 20 °C by turning on the nebulization system, three times during the night, for 3 min each. Following this temperature shock, 12 equidistant holes were made in the bags, using a stainless steel scalpel, to allow the emission of mushroom primordia. The air humidity of the growth chamber was maintained at 85–90% and the temperature was kept at 25 ± 2 °C during the entire production period.
Fruiting body (mushroom) harvest was done twice a day, when the fruiting bodies presented the pileus with an almost flat format, which is considered the harvest point for P. ostreatus. The fruiting bodies were weighted and measurements such as the number of bunches, basidiomes, average size of the stipe, and average diameter of the pileus were taken. The fruiting bodies were dried in an oven with ventilation at 45 °C to obtain the dry weight.
Biological efficiency (BE) and yield (Y) were calculated as follows (Eq. 1):
where BE = biological efficiency (%), MFW = mushroom fresh weight (g) and SDW = substrate dry weight (g).
Y was calculated with Eq. 2:
where Y = yield (g·kg-1), MFW = mushroom fresh weight (g) and SFW = substrate fresh weight (kg).
Bromatological analysis of substrates and mushrooms
Substrate samples were analyzed for dry matter (DM), mineral matter (MM), crude protein (CP), ether extract (EE), fiber in neutral detergent (FND), fiber in acid detergent (FAD), and lignin. The analytical methods used are described by SILVA et al. (2017)SILVA, T.E.; DETMANN, E.; CAMACHO, L.F.; SALIBA, E.O.S.; PALMA, M.N.N.; VALADARES FILHO, S.C. Comparação de métodos in vitro para a quantificação da digestibilidade da matéria seca e da fibra em detergente neutro de forragens e concentrados. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, Belo Horizonte, v.69, n.6, p.1635-1644, 2017. https://doi.org/10.1590/1678-4162-9096
https://doi.org/10.1590/1678-4162-9096...
.
Other measurements included the content of carbon (C) to calculate the C/N ratio, as described by CARMO; SILVA (2012)CARMO, D.L.; SILVA, C.A. Métodos de quantificação de carbono e matéria orgânica em resíduos orgânicos. Revista Brasileira de Ciência do Solo, Viçosa, v.36, n.4, p.1211-1220, 2012. https://doi.org/10.1590/S0100-06832012000400015
https://doi.org/10.1590/S0100-0683201200...
. Mushroom crude fiber content was determined by the gravimetric method, as defined by the Association of Official Analytical Chemists (AOAC). Nitrogen (N) content was determined by the micro-Kjeldahl method described by AOAC (1996). Total carbohydrates (C) were calculated with Eq. 3:
Energy was calculated with Eq. 4:
Energy(kcal) = (4 × crude protein) + (9 × fat)
using the Atwater general factor system (FAO, 2003aFOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS (FAO). Calculation of the energy content of foods – energy conversion factors. In: Food energy - methods of analysis and conversion factors. Report of a Technical Workshop, 2003a. Available from: http://www.fao.org/3/Y5022E/y5022e00.htm#Contents. Access on: 7 Sep. 2020.
http://www.fao.org/3/Y5022E/y5022e00.htm...
). The conversion factor used for N to protein was 6.25 (FAO, 2003bFOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS (FAO). Methods of food analysis. In: Food energy - methods of analysis and conversion factors. Report of a Technical Workshop, 2003b. Available from: http://www.fao.org/3/Y5022E/y5022e00.htm#Contents. Access on: 7 Sep. 2020.
http://www.fao.org/3/Y5022E/y5022e00.htm...
).
Statistical analysis
The experimental design was completely randomized with eight replications for vertical mycelium growth and 15 replications for mushroom production. Data were submitted to analysis of variance (ANOVA) and the grouping of means was done using the Scott–Knott test (p ≤ 0.05). The correlations between the variables were studied using multivariate analysis, the principal component analysis (PCA) and the correlation matrix (correlogram), using the statistical software packages: R v.3.6.3 (R CORE TEAM, 2020R CORE TEAM. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2020. Available from: https://www.R-project.org/. Access on: 3 Oct. 2020.
https://www.R-project.org/...
); RStudio v.1.2.5042 (RSTUDIO TEAM, 2020RSTUDIO TEAM. RStudio: Integrated Development for R. 2020. Available from: http://www.rstudio.com/. Access on: 3 Oct. 2020.
http://www.rstudio.com/...
); the ggplot2 package v3.3.2 (WICKHAM, 2016WICKHAM, H. ggplot2: Elegant graphics for data analysis. 2016. Available from: https://ggplot2.tidyverse.org. Access on: 3 Oct. 2020.
https://ggplot2.tidyverse.org...
); the dplyr package v1.0.2 (WICKHAM et al., 2020WICKHAM, H., FRANÇOIS, R., HENRY, L.; MÜLLER, K. dplyr: A Grammar of Data Manipulation. R package version 1.0.2. 2020. Available from: https://CRAN.R-project.org/package=dplyr. Access on: 3 Oct. 2020.
https://CRAN.R-project.org/package=dplyr...
); the FactoMineR package (LÊ et al., 2008LÊ, S.; JOSSE, J.; HUSSON, F. FactoMineR: An R package for multivariate analysis. Journal of Statistical Software, Innsbruck, v.25, n.1, p.1-18, 2008. https://doi.org/10.18637/jss.v025.i01
https://doi.org/10.18637/jss.v025.i01...
) and the corrgram package v1.13 (WRIGHT, 2018WRIGHT, K. Corrgram: Plot a Correlogram. R package version 1.13. 2018. Available from: https://CRAN.R-project.org/package=corrgram. Access on: 3 Oct. 2020.
https://CRAN.R-project.org/package=corrg...
).
RESULTS AND DISCUSSION
The contents of mineral matter, protein, hemicellulose, cellulose, lignin, and C/N ratio of the wastes used in the formulation of the substrates are shown in Table 2. These values are within the range reported for lignocellulosic wastes commonly used for mushroom production, as described by FIGUEIRÓ; GRACIOLLI (2011)FIGUEIRÓ, G.G.; GRACIOLLI, L.A. Influência da composição química do substrato no cultivo de Pleurotus florida. Ciência e Agrotecnologia, Lavras, v.35, n.5, p.924-930, 2011. https://doi.org/10.1590/S1413-70542011000500009
https://doi.org/10.1590/S1413-7054201100...
, using substrates composed of rice and wheat straw.
Contents of mineral matter (MM), crude protein (CP), hemicellulose, cellulose, lignin, and C/N ratios of palm oil fruit mesocarp (POFM), cocoa almond peels (CAP) and charcoal used to formulate the substrates for the cultivation of P. ostreatus.
Mycelium growth is an important initial phase for mushroom production, since it leads to substrate colonization and allows mushroom growers to determine if growth conditions are adequate (POKHREL et al., 2013POKHREL, C.P.; KALYAN, N.; BUDATHOKI, U.; YADAV, R.K.P. Cultivation of Pleurotus sajor-caju using different agricultural residues. International Journal of Agricultural Policy and Research, Irbid, v.1, n.2, p.19-23, 2013. Available from: http://journalissues.org/wp-content/uploads/2014/07/Pokhrel-et-al.pdf. Access on: 7 Sep. 2020.
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). Moreover, rapid mycelium growth lowers the possibility of contamination and growth of competing fungi and bacteria (JONATHAN et al., 2008JONATHAN, S.G.; FASIDI, I.O.; AJAYI, A.O.; ADEGEYE, O. Biodegradation of Nigerian wood wastes by Pleurotus tuber-regium (Fries) Singer. Bioresource Technology, Lucknow, v.99, n.4, p.807-811, 2008. https://doi.org/10.1016/j.biortech.2007.01.005
https://doi.org/10.1016/j.biortech.2007....
). Substrates S1, S2, S3 and S4 promoted similar mycelium growth rates (0.8 cm·d-1) and reached total colonization of the substrate mixtures in the tubes after 15 days of inoculation. The substrate S5 supported a lower daily growth rate of 0.73 cm·d-1 and 15 days after inoculation, colonization by P. ostreatus reached 10.95 cm of the substrate (Fig. 1). In banana wastes, the growth rate of P. ostreatus mycelium was 1.08 cm·d-1 (CARVALHO et al., 2013CARVALHO, C.S.M.; AGUIAR, L.V.B.; SALES-CAMPOS, C.; MINHONI, M.T.A.; ANDRADE, M.C.N. Cultivo in vitro de Pleurotus ostreatus em resíduos de bananeira. Ambiência, Guarapuava, v.9, n.3, p.651-660, 2013. Available from: https://core.ac.uk/download/pdf/230458268.pdf. Access on: 7 Sep. 2020.
https://core.ac.uk/download/pdf/23045826...
). In sawdust and coconut shells, the growth rates of different isolates of P. ostreatus were 0.63 and 0.67 cm·d-1, respectively (MARINO et al., 2008MARINO, R.H.; ABREU, L.D.; MESQUITA, J.B.; RIBEIRO, G.T. Crescimento e cultivo de diferentes isolados de Pleurotus ostreatus (Jacq.: fr.) Kummer em serragem da casca de coco. Arquivos do Instituto Biológico, São Paulo, v.75, n.1, p.29-36, 2008. Available from: http://201.55.42.151/uploads/docs/arq/v75_1/marino.pdf. Access on: 7 Sep. 2020.
http://201.55.42.151/uploads/docs/arq/v7...
).
After the 15th day of inoculation, mycelium vigor was evaluated and classified (PEDRA, 2006PEDRA, W.N.; MARINO, R.H. Cultivo axênico de Pleurotus spp. em serragem da casca de coco (Cocos nucifera Linn.) suplementada com farelo de arroz e/ou de trigo. Arquivos do Instituto Biológico, São Paulo, v.73, n.2, p.219-225, 2006. Available from: http://www.biologico.sp.gov.br/uploads/docs/arq/V73_2/pedra.PDF. Access on: 8 Feb. 2020
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). Substrates S1, S2, and S3 supported a highly dense mycelium, whereas substrates S4 and S5 presented medium dense mycelium (Fig. 1B). Medium dense mycelium was reported when P. ostreatus was grown in sawdust from coconut shells (MARINO et al., 2008MARINO, R.H.; ABREU, L.D.; MESQUITA, J.B.; RIBEIRO, G.T. Crescimento e cultivo de diferentes isolados de Pleurotus ostreatus (Jacq.: fr.) Kummer em serragem da casca de coco. Arquivos do Instituto Biológico, São Paulo, v.75, n.1, p.29-36, 2008. Available from: http://201.55.42.151/uploads/docs/arq/v75_1/marino.pdf. Access on: 7 Sep. 2020.
http://201.55.42.151/uploads/docs/arq/v7...
). These authors pointed out the importance of fast substrate colonization by a vigorous mycelium, which was observed in this study with the substrates S1, S2, and S3 (Fig 1B).
Vertical mycelium growth of P. ostreatus in the substrates formulated with wastes from palm oil fruit mesocarp (POFM) and cocoa almond peels (CAP) processing and in vitro growth.
Mycelium growth rate and substrate colonization are directly related to the nutritional composition and granulometry of the substrate, as well as O2 concentration, temperature, and fungal genome (SOFI et al., 2014SOFI, B.; AHMAD, M.; KHAN, M. Effect of different grains and alternate substrates on oyster mushroom (Pleurotus ostreatus) production. African Journal of Microbiology Research, Africa, v.8, n.14, p.1474-1479, 2014. https://doi.org/10.5897/AJMR2014.6697
https://doi.org/10.5897/AJMR2014.6697...
; HOA; WANG, 2015HOA, H.T.; WANG, C.-L. The Effects of Temperature and Nutritional Conditions on Mycelium Growth of Two Oyster Mushrooms (Pleurotus ostreatus and Pleurotus cystidiosus). Mycobiology, Jinju, v.43, n.1, p.14-23, 2015. https://doi.org/10.5941/MYCO.2015.43.1.14
https://doi.org/10.5941/MYCO.2015.43.1.1...
). The porosity of the POFM waste allows the translocation of nutrients, water, and rapid fungal colonization (ZHU et al., 2017ZHU, X.; CHEN, B.; ZHU, L.; XING, B. Effects and mechanisms of biochar-microbe interactions in soil improvement and pollution remediation: a review. Environmental Pollution, Roskilde, v.227, p.98-115, 2017. https://doi.org/10.1016/j.envpol.2017.04.032
https://doi.org/10.1016/j.envpol.2017.04...
). The substrates formulated with POFM and CAP were not tested for granulometry and aeration, but allowed for fast and dense mycelium growth of P. ostreatus. Wastes with smaller particles can affect gas exchange and the colonization rate of the substrate (PEDRA; MARINO, 2006PEDRA, W.N.; MARINO, R.H. Cultivo axênico de Pleurotus spp. em serragem da casca de coco (Cocos nucifera Linn.) suplementada com farelo de arroz e/ou de trigo. Arquivos do Instituto Biológico, São Paulo, v.73, n.2, p.219-225, 2006. Available from: http://www.biologico.sp.gov.br/uploads/docs/arq/V73_2/pedra.PDF. Access on: 8 Feb. 2020
http://www.biologico.sp.gov.br/uploads/d...
).
The substrates tested herein promoted rapid mycelium growth of P. ostreatus, reaching complete colonization on the 15th day after inoculation. MARLINA et al. (2015)MARLINA, L.; SUKOTJO, S.; MARSUDI, S. Potential of Oil Palm Empty Fruit Bunch (EFB) as Media for Oyster Mushroom, Pleurotus ostreatus Cultivation. Procedia Chemistry, Bandung, v.16, p.427-431, 2015. https://doi.org/10.1016/j.proche.2015.12.074
https://doi.org/10.1016/j.proche.2015.12...
reported that complete colonization of P. ostreatus in bags with 1 kg of substrate formulated with palm oil fruit waste, rice bran, and mineral fertilization took 28-35 days. CONDÉ et al. (2017)CONDÉ, V.F.; OLIVEIRA, J.E.Z.; OLIVEIRA, D.M.F. Farinha de Pleurotus ostreatus (Jacq.) P. Kumm. (cogumelo Hiratake) enriquecido em ferro. Ciência e Natura, Santa Maria, v.39. n.1, p.1-6, 2017. https://doi.org/10.5902/2179460X21437
https://doi.org/10.5902/2179460X21437...
observed complete substrate colonization 20 days after inoculation with P. ostreatus by using substrates formulated with sugar cane (Saccharum officinarum) processing waste enriched with different concentrations of Fe. Pleurotus ostreatoroseus and Pleurotus florida grown in substrates formulated with cotton residues or sawdust and supplemented with rice bran showed total substrate colonization within 24 to 30 days after inoculation, and started the emission of primordia within 5 to 10 days after colonization (REIS et al., 2010REIS, M.F.; DUCCA, F.; FERDINANDI, D.M.; ZONETTI, P.C.; ROSADO, F.R. Análise de substratos alternativos para o cultivo de Pleurotus ostreatoroseus e Pleurotus florida. Revista em Agronegócio e Meio Ambiente, Maringá, v.3, n.2, p.79-91, 2010. Available from: https://periodicos.unicesumar.edu.br/index.php/rama/article/view/582. Access on: 5 Sep. 2020.
https://periodicos.unicesumar.edu.br/ind...
).
In the present study, primordia emission occurred 17 days after inoculation, which is a shorter period than that described previously for P. ostreatus in other substrates. Formation of fruiting bodies of P. florida and Pleurotus flabellatus cultivated in substrate composed of anaerobically digested biomass waste, mostly banana leaf biomass from a biogas plant, took 20-30 days after inoculation (CHANAKYA et al., 2015CHANAKYA, H.N.; MALAYIL, S.; VIJAYALAKSHMI, C. Cultivation of Pleurotus spp. on a combination of anaerobically digested plant material and various agro-residues. Energy for Sustainable Development, Portland, v.27, p.84-92, 2015. https://doi.org/10.1016/j.esd.2015.04.007
https://doi.org/10.1016/j.esd.2015.04.00...
).
Biological efficiency (BE) and mushroom yield
The BE and mushroom yield for P. ostreatus cultivated with all the formulated substrates described herein were higher than the values reported in the literature. Indeed, BE ranged from 92.7 to 148.8% and yield from 349.3 to 560.5 g·kg-1 (Fig. 2). For BE and mushroom yield, S1 was the most efficient substrate, followed by S2, and S3, while S4 and S5 were the least efficient substrates (Fig. 2). The greater the proportion of palm oil fruit mesocarp processing waste in the substrates (Table 1), the more efficient they were in terms of BE and yield. Lower values were reported for P. ostreatus produced in substrates composed of corn cobs and wheat straw, with BEs of 52.82 and 66.93% and yields of 119 and 165.7 g·kg-1, respectively (KOUTROTSIOS et al., 2014KOUTROTSIOS, G.; MOUNTZOURIS, K.C.; CHATZIPAVLIDIS, I.; ZERVAKIS, G.I. Bioconversion of lignocellulosic residues by Agrocybe cylindracea and Pleurotus ostreatus mushroom fungi- Assessment of their effect on the final product and spent substrate properties. Food Chemistry, Norwich, v.161, p.127-135, 2014. https://doi.org/10.1016/j.foodchem.2014.03.121
https://doi.org/10.1016/j.foodchem.2014....
). In addition, P. ostreatus cultivated in substrates containing wastes of sisal (Agave sisalana) leaves and stems, enriched with caw manure presented values for BE and yield of 62.9% and 188.64 g·kg-1, respectively (RAYMOND et al., 2013RAYMOND, P.; MSHANDETE, A.M.; KIVAISI, A.K. Cultivation of oyster mushroom (Pleurotus HK-37) on solid sisal waste fractions supplemented with cow dung manure. Journal of Biology and Life Science, Las Vegas, v.4, n.1, p.273-286, 2013. https://doi.org/10.5296/jbls.v4i1.2975
https://doi.org/10.5296/jbls.v4i1.2975...
).
The higher BE and mushroom yield presented here in relation to those previously reported could be due to the medium to high mycelium vigor supported by our substrate mixtures (Fig. 2). Accordingly, colonization and production of fruiting bodies in lignocellulosic substrates are directly related to the fungus mycelium vigor (FIGUEIRÊDO; DIAS, 2014FIGUEIRÊDO, V.R.; DIAS, E.S. Cultivo do champignon em função da temperatura. Ciência Rural, Santa Maria, v.44, n.2, p.241-246, 2014. https://doi.org/10.1590/S0103-84782013005000155
https://doi.org/10.1590/S0103-8478201300...
). Another reason is the nutritional characteristics of the substrate and the environmental conditions of the cultivation process, as reviewed by KUMLA et al. (2020)KUMLA, J.; SUWANNARACH, N.; SUJARIT, K.; PENKHRUE, W.; KAKUMYAN, P.; JATUWONG, K.; VADTHANARAT, S.; LUMYONG, S. Cultivation of Mushrooms and Their Lignocellulosic Enzyme Production Through the Utilization of Agro-Industrial Waste. Molecules, Emeryville, v.25, n.12, p.2811, 2020. https://doi.org/10.3390/molecules25122811
https://doi.org/10.3390/molecules2512281...
. Indeed, the C/N ratio of the wastes used to formulate the substrates to cultivate Pleurotus spp. is particularly important since it interferes directly with substrate colonization and the production of fruiting bodies (EIRA, 2004EIRA, A.F. Fungos comestíveis. In: ESPÓSITO, E.; AZEVEDO, J.L. (ed). Fungos uma introdução à biologia, bioquímica e biotecnologia. Caxias do Sul: Educs, 2004. p.379-448.). The C/N ratios of the POFM (42.5/1) and CAP (12.5/1) used in the substrates formulated herein were adequate for the successful production of P. ostreatus, as suggested by EIRA (2004)EIRA, A.F. Fungos comestíveis. In: ESPÓSITO, E.; AZEVEDO, J.L. (ed). Fungos uma introdução à biologia, bioquímica e biotecnologia. Caxias do Sul: Educs, 2004. p.379-448. who indicated that the ideal C/N ratio for early colonization should be between 20/1 and 50/1.
Biological efficiency (a) and yield (b) of P. ostreatus produced in substrates formulated with different concentrations of wastes derived from the processing of palm oil fruit mesocarp (POFM) and cocoa almond peels (CAP). Harvesting period lasted 31 days.
Taken together, the good results for BE and yield presented herein indicate that the tested substrate formulations using POFM and CAP are adequate to cultivate P. ostreatus. Substrate S1, composed of 86.4% POFM and 9.6% CAP, presented the best BE and yield results and is, therefore, recommended for the production of P. ostreatus.
Mushroom nutritional composition
Several factors affect the nutritional composition of mushrooms, such as fungal genetics, substrate origin and nutritional composition, growth conditions, and the developmental stage of the fruiting bodies (WANG et al., 2015WANG, S.; XU, F.; LI, Z.; ZHAO, S.; RONG, C.; GENG, X.; LIU, Y. The spent mushroom substrates of Hypsizigus marmoreus can be an effective component for growing the oyster mushroom Pleurotus ostreatus. Scientia Horticulturae, Amsterdam, v.186, p.217-222, 2015. https://doi.org/10.1016/j.scienta.2015.02.028
https://doi.org/10.1016/j.scienta.2015.0...
). In this study, the nutritional composition of P. ostreatus fruiting bodies did not significantly vary according to the different substrate formulations for the parameters: humidity (92.36%), ashes (8.19%), fat (1.24%), fiber (13.4%), and carbohydrates (51.64%). The energy value of the fruiting bodies varied from 311.37 to 333.17 kcal·g-1 (data not shown). However, the protein content was affected by the substrate composition. Mushrooms produced in the S2 substrate were the richest, followed by those cultivated in the substrates S1 and S3, while mushrooms cultured in the substrates S4 and S5 had lower crude protein content (Table 3). Overall, the nutritional composition of P. ostreatus fruiting bodies produced in this study is similar to that reported for P. ostreatus produced in substrates derived from several agro-industrial wastes (OYETAYO; ARIYO, 2013OYETAYO, V.O.; ARIYO, O.O. Micro and macronutrient properties of Pleurotus ostreatus (Jacq: Fries) cultivated on different wood substrates. Jordan Journal of Biological Sciences, Zarqa, v.6, n.3, p.223-226, 2013. https://doi.org/10.12816/0001537
https://doi.org/10.12816/0001537...
; KOUTROTSIOS et al., 2014KOUTROTSIOS, G.; MOUNTZOURIS, K.C.; CHATZIPAVLIDIS, I.; ZERVAKIS, G.I. Bioconversion of lignocellulosic residues by Agrocybe cylindracea and Pleurotus ostreatus mushroom fungi- Assessment of their effect on the final product and spent substrate properties. Food Chemistry, Norwich, v.161, p.127-135, 2014. https://doi.org/10.1016/j.foodchem.2014.03.121
https://doi.org/10.1016/j.foodchem.2014....
; FERNANDES et al., 2015ERNANDES, Â.; BARROS, L.; MARTINS, A.; HERBERT, P.; FERREIRA, I.C.F.R. Nutritional characterization of Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm. produced using paper scraps as substrate. Food Chemistry, Norwich, v.169, p.396-400, 2015. https://doi.org/10.1016/j.foodchem.2014.08.027
https://doi.org/10.1016/j.foodchem.2014....
).
Content of humidity (H), mineral matter (MM), fat, crude protein (CP), fiber, and carbohydrates (C) (g in 100 g of dry matter, means ± SD, n = 5) of P. ostreatus fruiting bodies grown in substrates with different concentrations of the palm oil fruit mesocarp (POFM) and cocoa almond peel (CAP) processing wastes.
In the present study, the composition of the substrates was the only varying factor, since only one isolate of P. ostreatus was tested and all other growth conditions were kept the same. Substrate S1 promoted the highest yield and BE, which was 22.7% higher than the BE obtained for the S2. Considering a large-scale production system, this difference in BE can represent an increase of approximately 220 kg in mushroom production for each ton of dried substrate. Therefore, although S2 allowed the production of mushrooms with higher protein content, it is not the most viable or profitable formulation when compared to the yield that can be obtained with S1.
Mycelium vigor is another factor that must be considered for mushroom production. Besides promoting less incidence of competitive microorganisms, rapid substrate colonization, which in this study occurred 15 days after inoculation, allows the mushroom producer to run more production cycles. In turn, profit margins can potentially increase since more mushrooms will be produced within a fixed period and allocated space and resources.
The principal component analysis displayed in Figure 3 indicates that BE, yield, and crude protein were directly and positively correlated, and formed one cluster. These parameters are of special interest for mushroom producers to evaluate the quality of the mushrooms, and they are affected by the quality of the growth substrate. Thus, S1, which had the highest content of the palm oil processing waste, stimulated a high production of mushrooms with high protein content. On the other hand, BE, yield, and crude protein were inversely correlated with fat content, which formed another cluster with crude fiber and mineral matter. Furthermore, carbohydrate content and energy were directly and positively correlated and formed a third cluster.
Principal Component Analysis (PCA), with distribution of the variables studied in the dimensions 1 and 2 (Dim 1 and Dim 2)
Accordingly, the correlogram (Fig. 4) indicates a strong positive correlation between BE and yield, and between carbohydrate and energy. It also shows the strong positive correlation between BE, yield, and crude protein content as well as between crude fiber and mineral matter. A weaker, but positive correlation is observed between energy, BE, yield, and crude protein, as well as between fat and crude fiber. On the other hand, energy and carbohydrate have a strong negative correlation with crude fiber and mineral matter. Negative weaker correlations are observed between fat and crude protein, and between crude protein, crude fiber, and mineral matter (Fig. 4). In fact, BELLETTINI et al. (2019)BELLETTINI, M.B.; FIORDA, F.A.; MAIEVES, H.A.; TEIXEIRA, G.L.; ÁVILA, S.; HORNUNG, P.S.; MACCARI JÚNIOR, A.; RIBANI, R.H. Factors affecting mushroom Pleurotus spp. Saudi Journal of Biological Sciences, Riyadh, v.26, n.4, p.633-646, 2019. https://doi.org/10.1016/j.sjbs.2016.12.005
https://doi.org/10.1016/j.sjbs.2016.12.0...
discussed that fruiting bodies with high protein contents tend to have low fat content.
Correlogram of biological efficiency, yield, and the nutritional values of the fruiting bodies of P. ostreatus, grown in substrates formulated with palm oil fruit mesocarp (POFM) and cocoa almond peels (CAP) processing wastes.
CONCLUSIONS
Substrates S1, S2, and S3 supported good mycelium growth and vigor, with highly dense substrate colonization by P. ostreatus. Since complete substrate colonization was reduced to 15 days, which is shorter than most of the reported periods in the literature, substrates composed of palm oil fruit mesocarp and cocoa almond peel processing wastes have the potential to allow more production cycles of P. ostreatus. The recommended substrate is formulated with 86.4% of POFM and 9.4% of CAP (substrate S1), as it results in higher yields of nutrient rich mushrooms. The bioconversion of these waste products into high protein and nutritious mushrooms is suggested as an excellent recycling alternative with potentially significant ecological and economic benefits to the regions where palm oil and cocoa are produced.
ACKNOWLEDGEMENTS
The authors thank FINEP and FAPESB for supporting the laboratory research infrastructure.
-
FUNDING
This study was financed in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) (Finance Code 001 and postdoctoral scholarship), by Conselho Nacional de Desenvolvimento Científico e Tecnológico (nº 308771/2017-6) and by Secretaria de Ciência, Tecnologia e Inovação da Bahia ( nº 001/2013). -
ETHICAL APPROVAL
Not applicable -
AVAILABILITY OF DATA AND MATERIAL:
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Publication Dates
-
Publication in this collection
04 Dec 2020 -
Date of issue
2020
History
-
Received
23 July 2018 -
Accepted
18 Oct 2020