P-20 Upgrading of Agro-wastes to Straw Mushroom by Radiation Ngamnit Sermkiattipong Suwimol Jetawattana and Chettachai Banditsing Office of Atoms for Peace, 16 Vibhavadi-rangsit Rd., Chatuchak, Bangkok 10900, Thailand Tel : 02-5795230 Abstract The effect of radiation pasteurization on rice straw, water hyacinth mixed with rice straw, paragrass and trimmed manila grass and fermentation using straw mushroom (Volvariella volvacea) were studied for upgrading of agro-wastes. Total bacterial count and total fungal count in all samples were reduced to 3-5 log cycles and 6 log cycles by 10 kgy irradiation, respectively. At a dose of 10 kgy, coliforms were below the detectable level. To eliminate the pathogens, the pasteurization dose was established at 10 kgy according to the absence of coliforms. The doses used for inactivation of fecal coliforms were ranged from 5 to 9 kgy depended on type of agrowastes. A dose of 5 kgy was sufficient to eliminate Escherichia coli. Comparative productivity of straw mushroom cultivation with various agro-wastes decontaminated by heat and radiation treatments was statistically analyzed. The result revealed that trimmed manila grass showed highly significant productivity from other agro-wastes in both treatments. For the same agro-waste, there were no significant differences of productivity between radiation and heat treatments in all samples. According to our experiments, it was frequently observed that radiation treatment enhanced not only more fruiting bodies but also faster fruiting bodies than heat treatment for 1-2 days. Keywords : Straw mushroom, Radiation pasteurization, Agro- wastes, Heat
Introduction In Thailand, the production of straw mushroom is about 84,000 tons/year (1). Owing to the good taste and high nutritive value, the demand of consumer is more than the supply. At present, the cost of rice straw is increasing due to the progress technology using machine for harvesting rice grain. By this technology, rice straw is not so efficient for cultivation of straw mushroom. Additionally, rice straw is not sufficient in some areas. From these problems, other agro-wastes should be substituted or mixed with rice straw for straw mushroom cultivation. Various kinds of cellulosic wastes can be used in stead of rice straw i.e. water hyacinth, grass, oil palm waste, cassava peel, mung bean seed pod, soybean seed pod, bagasse and substrate residues after mushroom harvesting (1). Water hyacinth and grass are simple, easily found in everywhere in Thailand and both of them are cost-free. On the other hand, both of these wastes are weeds. For grass, it can cause the problem of forest fire in dry season. In the case of water hyacinth, whenever it booms, it can cause the problem of water pollution. To solve these problems, these wastes should be recycled by cultivation of straw mushroom. In our study, water hyacinth and grass were selected in stead of rice straw for three purposes as follows: 1. To reduce the cost for straw mushroom cultivation. 2. To recycle the wastes to substrates for straw mushroom. cultivation. 3. To reduce pollution problem. In this paper, indoor cultivation of straw mushrooms by using irradiated and heated agro-wastes was investigated. Materials and Methods 1. Cellulosic agro-wastes Cellulosic agro-wastes used for this study were rice straw, water hyacinth mixed with rice straw, paragrass and trimmed manila grass. Rice straw was collected from two areas in Pathumtani province. For water hyacinth, paragrass and trimmed manila grass were collected from Kasetsart University and Laksi district. After collection, rice straw was cut into 5-6 cm length. Water hyacinth was cut into small pieces (5-6 cm) and dried. Paragrass was 2
dried and then cut into 5-6 cm length. For trimmed manila grass, it was exposed to air until dry (approximately 10% moisture content). 2. Preparation of samples before disinfection Six hundred grams of each dry agro-waste was soaked in tap water for 3 hours. In the case of water hyacinth mixed with rice straw, the weights of water hyacinth and rice straw were 150 g and 450 g, respectively. Drained the water out and put it in the plastic bag for 1-2 days in room temperature. For microbiological examination, each agro-waste was mixed with tap water and adjusted the moisture content about 70%. Then the mixture was put in the plastic bag for 1-2 days in room temperature. After incubation, each agro-waste was mixed well with 10% commercial supplement and adjusted the final moisture content to be about 70%. Then the mixture was divided into three parts and each part was packed tightly in plastic bag. So each bag contained 200 g of dried weight of agro-waste. Afterwards, each set of three plastic bags was disinfected by radiation and heat. 3. Gamma irradiation A Co-60 irradiator (Gammabeam 650) at Office of Atoms for Peace was used for this study. The dose rate determined with radiochromic film (FWT-60-00) dosimeter was 3.14 kgy/hr (kilogray per hour). The doses used for inactivation of microorganisms were 0, 5, 6, 7, 8, 9 and 10 kgy. For comparative productivity of straw mushroom, various agro-wastes in plastic bags were irradiated at 10 kgy. 4. Heat treatment The samples in plastic bags were sterilized by autoclave. 5. Enumeration of microorganisms Enumeration of total bacterial count, total fungal count and indicator microoganisms (coliforms, fecal coliforms and E. coli) were determined mainly as described in the Standard Methods for Examination of Water and Wastewater (3). 5.1 Total bacterial count Twenty grams of each irradiated sample were serially diluted in sterilized 0.85% sodium chloride. Then 0.1 ml of each appropriated dilution was plated in duplicate on the surface of nutrient agar and incubated at 30 0 C for 3 days. 3
5.2 Total fungal count The method was almost the same as mentioned in item 5.1 except using potato dextrose agar in stead of nutrient agar. The plates were incubated at 30 0 C for 5 days. 5.3 Enumeration of coliforms Coliforms were enumerated by the muliple - tube fermentation procedure with a series of five tubes per dilution as a Most Probable Number (MPN) index. Lauryl tryptose broth was used for the primary fermentation and brilliant green lactose bile broth fermentation tube were used for the confirmed test. Incubated the inoculated brilliant green lactose bile broth tube at 35 ± 0.5 0 C for 48 ± 3 hr. 5.4 Enumeration of fecal coliforms Fecal coliforms were determined by the multiple-tube fermentation procedure. All positive presumptive tubes from the total coliform MPN test were transferred to EC medium. Inoculated tube were incubated in a water bath at 44.5 ± 0.2 0 C for 24 ± 2 hr. 5.5 Enumeration of E. coli The MPN for E. coli was determined by tranferring the numbers of all positive tubes from fecal coliforms to Eosin methylene blue agar plate to isolate colonies. Incubation was done at 37 0 C for 18-24 hr and a suspicious colony was subsequently picked to test for biochemical characteristics. 6. Sowing After disinfection by radiation and heat, each plastic bag was sowed with 16 g of spawn on the top of the substrate. Then the plastic bag was tied with rubber ring not so tightly and put it in a chamber (darkness, 32-38 0 C, little ventilation) for 3-4 days or until the mycelia covered on the surface of substrate. 7. Management for buttoning period 7.1 Temperature The indoor temperature should be controlled within 28-35 0 C, never less than 25 0 C. 7.2 Moisture The air relative humidity is 85-95%. To increase the humidity, spray water into the air, but not directly onto the fruiting bodies. 4
7.3 Light If the light intensity of the cultivation can reach to 50-100 lux in daytime, it is unnecessary to use lights. 7.4 Ventilation Keep the air fresh by occasionally opening the entrance in the daytime. But be careful to avoid large temperature difference and high wind speed. 8. Harvesting of straw mushroom The buttons will come out after sowing 10-12 days. For harvesting, keep one hand on the rice straw around the button of the straw mushroom, then use the other hand to rotate lightly and pick it up. Every fruiting period is about 5-6 days. The second flush mushroom will appear in 4-5 days. In this experiment, the whole cultivation for straw mushroom (sowing to harvesting) is about 20 days. 9. Statistical analysis Data were analyzed by using the SPSS software package. An Analysis of Variance and paired t-test were applied to this study. Results and Discussion 1. Effect of gamma radiation on total bacterial count As shown in Fig.1, the initial loads of all samples were 1.35 x 10 9 3.65 x 10 9 CFU/g (colony forming unit per gram). A dose of 5 kgy, total bacteria were reduced approximately 2 log cycles in all samples. After 10 kgy irradiation, total bacteria of all samples decreased to the range of 2.40 x 10 3 1.15 x 10 6 CFU/g. The results showed that bacteria contaminated in trimmed manila grass were the most resistance to radiation in comparison with other agro-wastes. On the contrary, total bacteria in rice straw were the most sensitive to radiation. In other report, a dose of 10 kgy decreased total bacterial count to 10 4 CFU/g from an initial load of 1.3 x 10 9 CFU/g in sugar cane bagasse. In the case of rice straw, aerobic bacterial load decreased to 2.5 x 10 4 CFU/g from an initial load of 2.3 x 10 7 CFU/g by a dose of 12 kgy (4). Kume, T. et al. also reported that bacteria in cellulosic oil palm wastes were radioresistance and the dose required for elimination below the detectable level was more than 15 kgy (5). 5
2. Effect of gamma radiation on total fungal count Figure 2 shows the decrease in number of fungi in various agro-wastes by gamma irradiation. Total fungal count of all samples were determined to be 3.52 x 10 8 2.52 x 10 9 CFU/g in non-irradiated agro-wastes. After 5 kgy irradiation, total fungi were reduced 3-4 log cycles which was higher reduction than total bacteria. A dose of 10 kgy resulted in approximately a 6 log cycle reduction in total fungal count. In other literature search, an initial load of fungi were 10 4 10 8 CFU/g and 10 5 10 8 CFU/g in Malaysian oil palm wastes and Japanese cellulosic wastes, respectively (5,4). From our investigation, all samples were highly contaminated with fungi more than the others. However, irradiation at 10 kgy, total fungi still survived to the range of 1.15 x 10 2 1.06 x 10 3 CFU/g. The dose required for elimination fungi below detectable level was more than 10 kgy which was higher than other reports. Three probable reasons for explanation were as follows: 1. The initial loads of fungi might be one of the factors concerned. 2. The samples contaminated with radioresistant fungi. 3.,Type of agro-wastes were not the same. In our experiment, the yeasts still survived in rather high doses (7-10 kgy) whereas molds were hardly detected in the same dose. Ito, H. reported that one of the famous fungi is Aureobasidium pullulans (black yeast) which radiation-resistance is similar to Deinococcus radiodurans (6). 3. Effect of gamma radiation on indicator bacteria Table 1 shows inactivation of indicator microorganisms in various agro-wastes by gamma irradiation. Coliforms in all agro-wastes were ranged from >1.32 x 10 8 to 9.6 x 10 8 MPN/g. At a dose of 9 kgy, coliforms were below detectable level in rice straw and trimmed manila grass. In the case of paragrass and water hyacinth mixed with rice straw, coliforms were below detectable level by 10 kgy. In other report, coliforms in most of sludge samples were inactivated by the doses ranged from 1.2 to 6.0 kgy except in dried sludge. At a dose of 6 kgy, coliforms remained to be 3.5 x 10 2 MPN/g in dried sludge (7). 6
The initial load of fecal coliforms in all agro-wastes were 2.54 x 10 7 1.72 x 10 8 MPN/g. Fecal coliforms were below detectable level in paragrass, trimmed manila grass, rice straw and water hyacinth mixed with rice straw by irradiation at 5, 7, 9 and 9 kgy, respectively. Sermkiattipong, N. et al also reported that fecal coliforms in sludge samples were below detectable level by doses range of 1.2-6.0 kgy except in dried sludge. After 6.0 kgy irradiation, fecal coliforms decreased to be 54 MPN/g (7) which was higher survivors than samples in this study. The contamination of E. coli in all agro-wastes were ranged from 2.05 x 10 5 to 3.47 x 10 7 MPN/g. At a dose of 5 kgy, E. coli organisms were below detectable level in all samples. It was possible that the dose for inactivation of E. coli might be lower than 5 kgy because there was no data in lower doses. In other report, E. coli organisms were below detectable level by 6 kgy irradiation in all type of sludge samples (7). In our investigation, it can be concluded that the study on radiation pasteurization dose of substrates for cultivation of straw mushroom was carried out at 10 kgy because coliforms were below detectable level in all agro-wastes. 4. Indoor cultivation of straw mushroom by using irradiated and heated agro-wastes Comparative productivity of straw mushroom cultivation with different agro-wastes decontaminated by heat and radiation treatment was shown in Fig.3 and Table 2. In this study, trimmed manila grass showed significant difference (p < 0.05) among other agro-wastes for each treatment by analysis of variance. On the other hand, there were no significant differences among water hyacinth mixed with rice straw, rice straw and paragrass in the same treatment. This result indicated that trimmed manila grass was the best substrate for straw mushroom cultivation in comparison with other agro-wastes in both radiation and heat treatments. For the same agro-waste, there was no significant difference of productivity between radiation and heat treatment in all samples by using paired-t test. In our experiment, the dose used for radiation treatment was pasteurization whereas heat treatment was sterilization. If agro-waste was irradiated with sterilization dose, straw mushroom might have probability to be higher productivity than heat treatment by statistical analysis. However, higher dose caused more expensive cost 7
which was not suitable for application to the farm. Therefore, radiation could be applied as an alternative to the use of heat. According to the experiments, it should be noted that radiation treatment enhanced faster fruiting bodies than heat treatment for 1-2 days. Therefore, radiation treatment will be benefit for harvesting mushrooms earlier than conventional method (heat treatment). Furthermore, it was frequently found that there were more fruiting bodies on irradiated agro-wastes than heated agro-wastes. Acknowledgement This research work was carried out under UNDP/IAEA/RCA sub-project on Upgrading of Cellulosic Agro-wastes to Useful Products. The authors wish to express our sincere thanks to IAEA for supporting training course on edible mushrooms in China. Special thanks are given to Mr. Arag Vititheeranon for dose rate determination and dose measurement in process control. Lastly, we are grateful to Mr. Charnyut Parnutat to contribute our work more completely. References 1. Department of Agricultural Extension, 2000. "Technique for cultivation of Straw Mushroom". p.1-36. Bangkok, Thailand (in Thai). 2. Anonymous. 1998. In the textbook for International Training Class on "Jun-Cao Technology" Fuzhou. China. 3. American Public Health Association, American Water Works Association and Water Pollution Control Federation. 1980. Standard Methods for the Examination of Water and Wastewater, 15th Ed., p.747-829. American Public Health Association; Washington, D.C. 4. Malek, M. A. et al.; 1994. Radiation and fermentation of cellulosic wastes. Mycoscience 35 : 95-98. 5. Kume, T. et al.; 1998. Study on Upgrading of oil palm wastes to animal feeds by radiation and fermentation processing. JAERI Research 98-013. 6. Ito, H. 1993. Radiation Microbiology. In UNDP/IAEA/RCA Regional Training Course on Radiation Technology for Environmental Conservation, September 27 October 8, 1993. Takasaki Radiation Chemistry Research Establishment, Japan. 8
7. Sermkiattipong, N. and Pongpat, S. 1995. Sludge pasteurization and upgrading by radiation. JAERI Research 95-047. Table1 Inactivation of indicator microorganisms in various agrowastes by gamma irradiation Agro-waste Manila grass Rice straw Water hyacinth mixed with rice straw Paragrass Dose (kgy) Coliforms (MPN/g) Fecal coliforms (MPN/g) E. coli (MPN/g) 0 9.6 10 8 1.72 10 8 3.47 10 7 5 4.8 0.4 < 0.2 6 2 0.2 < 0.2 7 0.8 < 0.2 < 0.2 8 0.2 < 0.2 < 0.2 9 < 0.2 < 0.2 < 0.2 10 < 0.2 < 0.2 < 0.2 0 1.32 10 8 4.75 10 7 6.0 10 5 5 30 0.4 < 0.2 6 1.17 0.27 < 0.2 7 0.55 0.2 < 0.2 8 0.3 0.1 < 0.2 9 < 0.2 < 0.2 < 0.2 10 < 0.2 < 0.2 < 0.2 0 1.6 10 8 2.6 10 7 3.65 10 6 5 30 2.3 < 0.2 6 6 2.3 < 0.2 7 3 1.1 < 0.2 8 0.5 0.2 < 0.2 9 0.3 < 0.2 < 0.2 10 < 0.2 < 0.2 < 0.2 0 2.6 10 8 2.54 10 7 2.05 10 5 5 0.7 < 0.2 < 0.2 6 0.2 < 0.2 < 0.2 8 0.2 < 0.2 < 0.2 9 0.1 < 0.2 < 0.2 10 < 0.2 < 0.2 < 0.2 9
Table 2 The productivity of straw mushroom from various agro-wastes treated by gamma radiation and heat Agro-waste Weight of straw mushroom*(g) Radiation Heat Water hyacinth +Rice 148.35 + 13.18 133.32 + 14.95 straw Rice straw 141.48 + 16.85 132.84 + 20.27 Paragrass 129.29 + 12.54 150.33 + 15.59 Manila grass 201.76 + 11.00 220.92 + 26.25 *The values represent the mean + standard error (mean of 5 experiments) of straw mushrooms per 600 g of dry agro-waste. 10 log of total bacterial count 8 6 4 2 0 0 2 4 6 8 10 Dose (kgy) Manila grass Rice straw Water hyacinth + Rice straw Paragrass Fig.1 Effect of gamma irradiation on total bacterial count in various agro-wastes. 10
10 log of total fungal count 8 6 4 2 0 0 2 4 6 8 10 Dose (kgy) Manila grass Rice straw Water hyacinth + Rice straw Paragrass Fig. 2 Effect of gamma irradiation on total fungal count in various agro-wastes. % Biological efficiency (%B.E.) 40 30 20 10 0 Water hyacinth + Rice straw Radiation Heat Rice straw Paragrass Manila grass Type of agro-wastes Fig.3 Percent biological efficiency of straw mushroom in various kind of agro wastes %B.E. = Fresh weight of mushrooms x 100 Dry weight of substrate 11
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