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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
BIOMASS PRODUCTION OF PLEUROTUSOSTREATUS AND LENTINULA EDODES
ON TEQUILA VINASSES
J. Madrigal, C. Motolinia and A. Arias
Laboratorio de Biotecnología, Departamento de Botánica y Zoología, Universidad de Guadalajara
A. P. 1-139, Zapopan 45101, Jalisco, México. <>
ABSTRACT
Tequila vinasses were evaluated as substrates for the vegetative growth of two strains of Pleurotus ostreatus and two of Lentinula edodes. The vinasses were diluted with distilled water (10, 20, 30, 40 and 50%) and PDA was used as control medium. A six mm diameter mycelial plug was taken from the margin of a ten day old colony and placed in petri dishes containing the tequila vinasses media. The dishes were incubated for seven days at 28°C, and the colony diameter was determined (mm) each 24 h. At the end of the period of incubation, biomass production in dry weight was measured (mg/dish). Growth rate was greater in P. ostreatus than in L. edodes. Only the P1 strain grew in all the vinasses media, (except in the 50% concentration). The growth rate of P1 in PDA and 10% vinasses was 5.2±0.7 mm and 4.7±0.2 mm. For L. edodes, the L1 strain in 10% vinasses medium, the growth rate was 3.3±0.1 mm/day and for L2 of 2.9±0.4 mm/day. The average biomass production of P. ostreatus was 14.0 mg/dish for the P1 and 10.2 mg/dish for P2. Biomass production in L. edodes was 2.8 mg/dish for L2 and 2.6 mg/dish for L1 strain. Diluted tequila vinasses can be used for vegetative culture as a medium for propagation of edible mushrooms strains.
INTRODUCTION
The tequila industry is still growing in Mexico. Jalisco was recently designed by the European Union as the place of origin for this beverage. This means that the tequila produced in Jalisco can be legally sold under that name. Tequila is a beverage obtained by distillation of the agave plant juice, specifically Agave tequilana Weber var. azul, in particular (Secofi 1993).
Throughout the distillation process the alcohol from fermentation wort is separated and concentrated. The discharges from distillation columns are known as vinasses. Tequila vinasses have a biological oxygen demand (BOD) of 25 to 60 g/l, low pH (< 3.9) and must be treated before they are discharged. In a tequila distillery, seven to ten liters of effluent are produced per liter of tequila. Fermented wort contains solid agave particles with cellulose and pectin, yeast cells, proteins, mineral salts and some organic acids (Cedeño 1995).
Vinasses are discharged as an effluent, and due to the high concentration of dissolved matter they have become a source of environmental pollution (Linerio et al. 1999). Some utilization schemes for the vinasses have been proposed: evaporation or combustion (Sheenan and Greenfield 1980), the production of biomass and biochemicals (Quin and Marchant 1980), food supplement for cattle, recycling of the initial wort, direct land application as irrigation water and as fertilizers. Biological, aerobic, or anaerobic treatments have been proposed as means of disposal (Speece 1983). Tequila vinasses can be a raw material rather than a waste when employed in mushroom production (Cedeño 1995).
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
P. ostreatus and L. edodes are the most important edible mushrooms in the world market, led by the buttom mushroom Agaricus bisporus. In Mexico, the estimated production of Pleurotus spp was 356 tons and 1,825 tons in 1990 and 1997, respectively (Sobal et al. 1997).
Production of mycelium of edible mushroom can be used as spawn for the production of fruiting bodies. Various studies have been conducted on mycelial propagation in solid and submerged cultures evaluating agro-industrial wastes and effluents. The most economical and easily accessible materials were evaluated as culture media for P. ostreatus (Soto-Velazco et al. 1993). Wheat flour (HIT), wheat bran (FT) and wheat fiber (F) were found to be the best materials because abundant aerial and cottony mycelia were associated with them. For L. edodes , the growth rate on the same media was from 0.64 to 0.9 mm/day in HIT (Soto et al. 1994).
The mycelial growth of P. ostreatus in submerged cultures in different agro-industrial effluent was studied by Nieto-Lopez and Sanchez-Vazquez (1997). After seven days of culture, the best effluent source of nutrients for biomass production was cheese whey (0.26 to 3.96 g/l). With cooking honey from the tequila industry, greater growth was observed in P. ostreatus than L. edodes (Fausto and Arias 1999).
The aim of this study was to evaluate tequila vinasses as a medium for the mycelial growth of P. ostreatus and L. edodes. This work will determine the best concentration of tequila vinasses media for biomass production of these edible mushrooms.
MATERIALS AND METHODS
Fungal strains
Two strains of P. ostreatus (P1 and P2) and two of L. edodes (L1 and L2) were used. They are deposited in the mycological culture collection of the Department of Botany and Zoology, University of Guadalajara.
Culture media
Vinasses medium: Tequila vinasses were obtained from “Tequilas del Señor” company, Tlaquepaque, Jalisco. Vinasses were diluted to 10, 20, 30, 40 and 50% with distilled water. Potato dextrose agar (PDA) was used as control medium. Culture media were autoclaved at 121 °C for 15 min and 20 ml were put in each petri dish of 90 mm of diameter. The pH of the media was 4.0 before sterilization.
Condition for mycelial growth
Inocula preparation
For preparation of inocula, the fungus was grown on PDA plates at 20 °C (for L. edodes ) and 28°C (for P. ostreatus ) for seven days. Mycelium agar plugs of 6 mm in diameter (cut along the edge of an actively growing colony) were used as inocula.
Inoculation
Three plates, per media and strain, containing 20 ml of medium were inoculated with a mycelium agar plug, and were kept at 20 °C for 7 days.
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
Mycelial growth rate
Mycelial growth rate was determinated (mm/day) by measuring the diameter of culture growth in the petri dish every day from days 3 to 7.
Biomass production
Fungal biomass was recovered by diluted agar and filtered through tared Whatman No. 1 filter paper, washed with boiling water and quantified by gravimetry on dry mass at 100°C to a constant weight (mg/dish) after seven days of incubation.
RESULTS AND DISCUSSION
Mycelial growth rate
We observed that tequila vinasses diluted with distilled water are a good source of nutrients for the mycelial growth of P. ostreatus and L. edodes in solid culture. Mycelial growth of P. ostreatus (strain P1) was observed on all tequila vinasses media from 10 to 40%, only in the 50% media no growth was obtained for any strains evaluated. The content of residual alcohol or the Maillard products in the vinasses media could explain this (Table 1).
Great differences in growth rate have been reported for L. edodes and P. ostreatus in other media. Levanon et al. in 1993 reported that 21 strains of L. edodes were classified in five groups according to esterase isoenzymes and growth rates in cotton straw (CS) and wheat straw media (WS). The growth rate in vinasses media was slower than CS and WS probably due to content of residual alcohol or the products of Maillard’s reaction.
Table 1. Diameter of cultures of Pleurotus ostreatus (P1 and P2) and Lentinula edodes (L1 and L2) on PDA and 10% tequila vinasses diluted with water.
Strain
/ Diameter of culture (mm)PDA / Concentration of tequila vinasses on culture media
10 % / 20 % / 30 % / 40 % / 50 %
L1 / 21±1.3 / 23±0.5 / - - / - - / - - / - -
L2 / 26±2.5 / 20±3.1 / - - / - - / - - / - -
P1 / 37±4.7 / 33±1.4 / 20±3.1 / 23±1.7 / 23±2.0 / - -
P2 / 25±2.0 / 26±4.8 / - - / - - / - - / - -
Mycelial growth rate of P. ostreatus and L. edodes on PDA and 10% tequila vinasses medium is shown in Figure 1 and 2. The greater growth rate on 10% for P. ostreatus was from 3.7±0.68 mm/day (P1) to 4.7±0.02 mm/day (P2) in comparison to L. edodes from 2.86±0.44 mm/day (L1) to 3.3±0.07 mm/day (L2). Similar results were obtained for P. ostreatus and L. edodes by Fausto and Arias (1999), but with cooking honey from the tequila industry. The best mycelial growth rate for L. edodes in 12 days, 7.5 mm/day, reported with coffee husk extract medium were comparable with vinasses (Leifa et al. 2000).
Biomass production
Biomass production average of P. ostreatus and L. edodes strains on PDA and vinasses 10 % medium is shown in Figure 3. The greatest biomass production was for P. ostreatus strains with
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
14.0±8.74 mg/dish for P1 and 10.2±2.32 mg/dish for strain P2. In L. edodes biomass production was from 2.8±4.26 mg/dish to 2.6±3.93 mg/dish.
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
Figure 1. Growth rate of Pleurotus ostreatus (P1 and P2 strain) and Lentinula edodes (L1 and L2 strain) on PDA medium.
Figure 2. Growth rate of Pleurotus ostreatus (P1 and P2 strain) and Lentinula edodes (L1 and L2 strain) on 10% tequila vinasses medium.
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
Figure 3. Biomass production average of Pleurotus ostreatus (P1 and P2 strain) and Lentinula edodes (L1 and L2 strain) on PDA and 10% tequila vinasses medium.
Biomass production of strains P1 and P2 of P. ostreatus and L1 and L2 of L. edodes on PDA and each of the tequila vinasses media studied is shown in Table 2. The greatest biomass production was for P. ostreatus (strain P2) on the 10% medium with 46.2±10.3 mg/dish. Biomass production for P1 was from 12.0±4.7 mg/dish to 21.9±6.6 mg/dish on 10% and 40% media, respectively, and 24.2±2.9 on PDA. The greatest biomass production in 10% media is similar to cheese whey reported by Nieto-Lopez and Sanchez-Vazquez for P. ostreatus (1997).
Table 2. Biomass production of Pleurotus ostreatus (P1 and P2) and Lentinula edodes (L1 and L2) on tequila vinasses media.
Strain / Culture media / Biomass production (mg/dish)P2 / 10 % / 46.2±10.3 a*
P1 / PDA / 24.2±2.9 b
P1 / 40 % / 21.9±6.6 b
P2 / PDA / 21.4±2.4 b
P1 / 30% / 13.4±2.4 b
P1 / 20% / 12.2±3.2 b
P1 / 10% / 12.0±4.7 b
L2 / 10% / 9.6±0.7 b
L1 / PDA / 9.3±1.0 b
L2 / PDA / 7.4±2.1 c
L1 / 10% / 6.2±1.4 c
* Same letters indicate no significant difference in value at a level a=0.05, according to the Duncan test.
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
For L. edodes biomass production was from 6.2±1.4 to 9.6±0.7 mg/dish, and the greatest biomass production was observed with the L2 strain. A strain of L. edodes produced 48.7 mg/dish in coffee
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
husk extract medium (Leifa et al. 2000). Studies need to continue for better biomass production of L. edodes in vinasses media. A biomass production of 46.2 mg/dish obtained with P2 strain in 10%
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Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN968-878-105-3
vinasses medium was similar with coffee husk extract medium, 48.7 mg/dish, with L. edodes strains studied during 12 days by Leifa et al. (2000).
We propose 10% tequila vinasses as a good medium for the edible mushroom P. ostreatus and L. edodes. Some utilization of vinasses could be a media for production of enzymes such as xylanasa (Mishra et al. 1990), laccase (Buswell et al. 1995), exopolysaccharides (Burns et al. 1994), other fungal metabolites, or decolorizing or removing the chemical oxygen demand (Kahraman and Yesilada 1999). The effect of different carbon and nitrogen sources on the amount of biomass and production of polysaccharides of Pleurotus sp floridawas studied by Burns, et al. (1994) and is interesting to know the effect of different carbon and nitrogen source on the biomass production in vinasses media.
ACKNOWLEDGMENTS
This was supported by a research grant from the University of Guadalajara (Grant: 99/SA-CI/DBZ/4.1/29). We thank Professor M. Harker and L. Hernandez from Department of Botany and Zoology, University of Guadalajara for the critical English review.
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Soto-Velazco, C., A. Arias-García y L. Ortiz-Cano. 1994. Evaluación de medios de cultivo sólido para cepas de Lentinus spp In: Memoirs V Congreso Nacional de Micología, Guanajuato, México.
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UAEM. ISBN968-878-105-3
Speece, R.E. 1983. Anaerobic biotechnology for industrial wastewater treatment. Environ. Sci. Technol:17:416-420.
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