Mushroom Cultivation on the Substrates with Different Animal Feces and Supplementary Hay Combinations

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1 Original Paper Mushroom Cultivation on the Substrates with Different Animal Feces and Supplementary Hay Combinations Hideo Minagawa 1, Tetsuya Doi 2, Hiroshi Sakata 3 and Masaru Nagai 3 1 School of Veterinary Medicine, Kitasato University Higashi, Towada, Aomori , Japan 2 Field Production Science Center, Graduate School of Agricultural and Life Sciences, University of Tokyo 111 Midori, NishiTokyo, Tokyo , Japan 3 Institute for Environmental Sciences 17 Ienomae, Obuchi, Rokkasyo, Aomori, , Japan Received March 23, 2010; Accepted April 15, 2010 ABSTRACT Oyster mushroom (Pleurotus ostreatus) and Shiitake mushroom (Lentinus edodes) were examined to cultivate on the substrate of animal feces and supplementary hay in a chamber controlled for thermal, humid, CO 2 and illumination conditions. The animals were goat, cattle, pig, hen and horse. After the fresh animal feces were dried and crushed into powder, the chemical of the animal feces and the hay used for cattle and goat was analyzed and compared with that of typical bed materials such as cedar saw dust and rice bran. To investigate an optimal fraction of the hay supplemented with the feces, mushroom s spawn growth test on a plate culture with each kind of the feces was examined. The three beds made of the pure feces, the feces 90% and hay 10%, and the feces 80% and hay 20% were examined. After the beds mounted on a glass plate were controlled in moisture of 65% and autoclaved, Oyster fungus was inoculated. A bottle culture using a glass bottle was experimented for Oyster and Shiitake fungi on the two beds of the pure feces and the feces 90% and hay 10%. The animal feces were rich in protein and scarce in fiber except those from cattle and horse. The optimal combination was the feces 90% and the hay 10%. In the test of the bottle culture, Oyster mushroom was obtained on the beds of animal feces except that of hen. Pig s feces showed the greatest yield. Shiitake mushroom was failed to harvest on the all beds of the animal feces, even though of enough spawn growth excepting the hen. Key words : Animal feces, Hay, Oyster mushroom, Shiitake mushroom, Sustainable agriculture 1. Introduction Livestock and poultry farms in Japan produced their wastes of 90 million tons in 2001, including the solid manure of 60 million tons (MAFF, 2008). The animal solid manure which contains feces, some urine, or bedding materials is almost composted as an organic fertilizer for land application. However, the application of the compost in the paddy fields, which account for 54% of the whole area of 5.0 million ha, has been reduced due to an increase of rice farmers with a side job and an advance in their age, resulted in an issue of surplus compost (Kameoka, 2004). When handling animal solid manure, the ways of composting, burning, and carbonization are available in Japan (Kameoka, 2004). To find more practical way of reusing animal solid manure, mushrooms cultivation on the beds made of feces from goats which reared in a closed ecosystem was examined, resulted in fruiting Oyster mushroom (Minagawa et al., 2005). In this study, we examined the feces from cattle, swine, goat, hen and horse to cultivate mushrooms as an alternative *Corresponding author : Phone: , Facsimile: , minagawa@vmas.kitasatou.ac.jp The Society of EcoEngineering

2 utilization of animal solid manure for a sustainable agriculture. In addition, mushroom cultivation need a special chamber controlled for thermal, humid, CO 2 and illumination conditions (Akabane, 2001). We developed an experimental chamber for growing mushroom. 2. Materials and Method 2.1 Mushroom growth chamber An experimental mushroom growth chamber was made in an airconditioned room as shown in Fig.1. The walls except the front wall had a double layer of the 30 mm thick wooden board and the 10 mm thick insulation inside. The front wall was the slide open window of clear glass in a thickness of 0.3 mm combined with the 10 mm thick wooden board outside. Illumination in a range of 10 lx to 500 lx is needed for growing Oyster mushroom (Akabane, 2001). At the front outside wall the two clear plastic windows in a square of 400 mm were created for illuminating mushroom in a range of 50 lx to 500 lx with daylight through the window of the room or artificial light from fluorescent lamps in the room. When pining or fruiting stage of mushroom began, the illumination was manually controlled by opening the front wall of the chamber for three hours a day. Air temperature and relative humidity suitable for spawn or mushroom growth are reported to range from 18 to 20 and from 80% to 90%, respectively (Taniguchi, 1986). For air conditioning the chamber was equipped with an electric heat wire of 100 W in power and 100 m in length, an airconditioner of 980 W in refrigeration power, and an ultrasonic humidifier. The electric heat wire was used to round uniformly the cultivation benches of mushroom and was controlled with a voltage transformer and an air temperature controller. The airconditioner was installed beside the chamber and was controlled by manual operation. Thus the wire heater and the airconditioner provided the optimal air temperature for mushroom cultivation in the chamber when the outside air temperature was ranged less than 0 to more than 30. Relative humidity was controlled manually and kept in 80% or more with the ultrasonic humidifier placed in the chamber. Ventilation was achieved in a rate of 30 times hr 1 with an exhaust fan, providing the chamber with fresh air through the filter inlet as well as the airconditioner and keeping the CO 2 concentration in the chamber in a suitable range of less than 0.1%, that was reported by Akabane (2001). A compound sensor of air temperature and relative humidity was set at three places inside and at one place outside. At every ten minutes, the electric signal of the sensor was transmitted, received by the receiver of the device outside, and recorded in the computer. 2.2 Collection of feces and analysis of the chemical compositions Fresh feces from three or four heads and five kinds of animals such as goats, cattle, pigs, hens and horses were collected in a few hours after their excretion. The goats were matured, female and Japanese native Shiba variety. The horses were matured, female and racing type. The pigs were finishing and two crossbreeds of Landrace by Duroc. The cattle were matured, female, beef type and Japanese Shorthorn variety. The hens were matured, laying and White Leghorn variety. The goats, cattle, pigs and horses were raised at a farm of Kitasato University, and the hens were bred at a farm around the university. To sterilize the collected fresh feces, the feces were dried at 120 and for 48 hours with an electric drying chamber. After drying, the feces in the chamber were laid in ambient temperature for 24 hours and were crushed to make uniformity with a mill. In addition, the crushed feces were filtered with a sieve in a mesh of 0.5 mm. The remained feces on the sieve were crushed again with the mill. Thus we made the five samples of powdery animal feces. Chemical compositions of the powdered animal feces were analyzed with a traditional method (Schaible, 1970). The chemical composition contains crude protein, crude fat, crude fiber, crude ash, mineral, soluble carbohydrate and moisture. In addition, moisture of the fresh animal feces was also measured by the weight loss after drying. EcoEngineering

3 Shortage of crude fiber and air permeability in the powdered goat feces was reported and hay for the goats was cut into pieces less than 3 cm and was supplemented to mushroom cultivation on the bed of goat feces (Minagawa et al., 2005). In this experiment, the hay fed for goats and cattle was also used to improve the lack of crude fiber and air permeability. The hay contained rice straw 50%. Italian rye grass 30% and Timothy hay 20% in dry material ratio. Nutrition and moisture content of the hay was also investigated as like to that of the powdered animal feces. 2.3 Mushroom species and spawns Oyster mushroom (Pleurotus ostreatus) and Shiitake mushroom (Lentinus edodes) were examined to cultivation. They are whiterot fungi (Kuwahara, 2002), being expected to decay lignin and cellulose in the goat feces. In addition, Oyster mushroom is produced worldwide and Shiitake mushroom are cultivated in Japan and East Asia (Taniguchi, 1986). In the experiment, we used the two kinds of strains, H67 and S29 (Kinokkusu Co., Sendai), for the Oyster and Shiitake mushrooms respectively. The strains were provided in a form of sawdust spawn that the grown, divided, and fused hyphae germinated from the haploid spores of a variety of mushroom, called mycelia or diploid mycelia, were cultured in the substrate of sawdust. 2.4 Optimal combination of manure and supplemented bedding for spawn growth of Oyster mushroom on plate culture To know the optimum ratio of the supplement of the dried hay to the basic material of the powdered animal feces, a spawn growth test on plate culture was examined. The fungus of Oyster mushroom was used for spawn cultivation. There were three types of bedding materials in combination with basic and supplement for spawn culture; pure feces (100%), feces (90%) and hay (10%), and feces (80%) and hay (20%) in a total dry weight of 3.5 g for goat, cattle and horse or 7.0 g for pig and hen, as shown in Table 1. The difference in the total weight depends on the feces that contain lot of fiber or less. The dried hay was cut into pieces less than 3 cm. Each type of bedding materials was filled in the three Petri glass dishes of 91 mm in diameter and 21 mm in depth and was added with distilled water to keep in a moisture content of 65%. The dishes were sterilized at 120 and for 90 min in an autoclave. After being cooled at 25 in a clean bench, the dishes were inoculated over the bed surface with the fungus of Oyster mushroom in a weight of 0.1 g. After being wrapped with glass cover, the inoculated dishes were cultivated at an air temperature ranged from 18 to 20 and at a relative humidity from 80% to 90% relative in the mushroom growth chamber, as shown in Fig. 1. After four days, spawn growth in each dish was observed every other day. 2.5 Mushroom cultivation on bottle culture From the results of the spawn growth test as mentioned before, two types of bedding materials, pure feces (100%) and feces (90%) and hay (10%) in weight fraction, were selected to growth mushroom on bottle culture. Two kinds of fungi, Oyster and Shiitake mushrooms, were used. In a glass bottle of 450 ml in volume, the bedding material for each type and each animal was filled in a height of 8 cm from the bottle base and added with distilled water to keep in a moisture content of 65%. The bottle was sterilized at 120 and for 90 min in the autoclave. After being cooled at 25 in the clean bench, the bottle was inoculated over the bed surface with the fungi of Oyster mushroom or Shiitake mushroom in a weight of 0.2 g. After being wrapped with absorbent cotton, the inoculated bottle was cultivated at an air temperature ranged from 18 to 20 and at a relative humidity from 80% to 90% relative in the mushroom growth chamber. When the spawn covered around the inside surface of the bottle was observed, a process of cutting spawn and dipping the bed material into distilled water was done to form fruit. While forming the fruit, the air temperature in the chamber was kept at 15 and the relative humidity at more than 90%. After the fruit cap was grown in a width of two or three cm, the fruit was harvested. Mister content and dry mater weight was measured Goat Cattle Pig Hen Horse A B C D E F G H I J K L M N O Feces (g)* Hay (g)** Total (g) Hay rate * Feces were dried and grounded in a grain of powder. ** Hay was dried and cut within a length of 3 cm. EcoEngineering

4 with the electric drying chamber. The cultivation of Oyster and Shiitake mushrooms began on Aug. 18, Results and Discussion 3.1 Air temperature and relative humidity in the mushroom growth chamber In contrast with the outside air temperature and the outside relative humidity, an example of diurnal changes of average air temperature and average relative humidity inside was shown in Fig. 2. Both the inside air temperature and the inside relative Air Temperature ( ) Relative Humidity Inside Average Temp. Outside Temp Time of Day (hr) Inside Average Humidity Outside Humidity Time of Day (hr) humidity did not depend on the diurnal change of the outside environments; the outside air temperature was the highest at midday and the outside relative humidity the lowest. Those were due to the manual operation of the airconditioner and the ultrasonic humidifier. When the outside air temperature changed from 23 to 26, the inside air temperature kept in a small range of 18 to 20. Maximum difference between inside and outside air temperature was 7. However mean standard deviation of the inside air temperatures at three places was within 0.4, meaning relatively uniformity of air temperature in the chamber. Although the outside relative humidity ranged from 40% at daytime and 65% at nigh, the inside relative humidity kept more than 80% even at midday. Mean standard deviation of the inside relative humidity at three places was approximately within 3%. The air temperature and the relative humidity in the chamber were controlled in the optimal environmental conditions of mushroom cultivation. 3.2 Chemical components of the bedding materials Average fraction of chemicals in dry mass base in the animal feces and the hay was shown in Table 2. It was compared with that of typical bedding materials for mushroom cultivation, such as cedar saw dust 60% and rice bran 40% reported by Kitamoto (1978). For understanding the differences among the substrates, the chemicals were modified to dry mass base by eliminating moisture from wet mass base. Chemical component was large discrepancy among the animals. In contrast with the typical bedding materials of cedar saw dust and rice bran, the chemicals in the pigs and hens were poor in crude fiber and rich in crude protein, due to having a single stomach. However the horses are a monogastric animal, but they included crude fiber more than that of other ruminant animals. The horse has a long and large intestine and Animal Crude Protein Crude Fat Crude Fiber Crude Ash Soluble Carbohydrate Goat 24.3 c* 7.1 b 39.5 c 1.4 e 27.7 b Cattle 31.2 b 4.8 c 58.7 b 2.9 c 2.4 d Pig 39.7 a 16.5 a 16.3 e 2.1 d 25.4 b Hen 42.7 a 4.0 d 23.1 d 8.5 b 21.7 c Horse 13.6 e 5.2 c 77.3 a 2.3 d 1.6 e Hay 16.4 d 1.9 e 22.6 d 12.9 a 46.2 a Popular substrates in dry base for mushroom cultivation (cedar saw dust 60%+ rice bran 40%)** * Mean S.D. (n=3). Value followed by the same letter in column is not significantly different (P<0.01 or P<0.05) by the Fisher's LSD method. ** The original data are reported by Kitamoto (1978) and those are modified from wet mass base to dry one in this table. Total 100 EcoEngineering

5 needs fibrous material (Ponting, 1986). The hay showed both rich in soluble carbohydrate and crude fiber. From the results, the bedding material of the animal feces needs the hay as a carbon resource. 3.3 Spawn growth of Oyster mushroom on the plate culture To investigate an effect of the supplemental hay in the basic fecal bed on the mushroom growth, covering area of spawn growth on the plate culture after four days from the inoculation was shown in Table 3. The covering area percentage of spawn over the surface of the plate was measured visually. On the plates of the goat s feces, the bedding material B, feces 90% and hay 10%, was best and reached a coverage of 100% after the 12 days from the inoculation. On the cattle plates, E was failed to grow spawn for some reasons. For example a contamination might be happened when inoculating. However D and F showed the 85% coverage after 10 days. On the pig plates, H and I that contained the hay was better than G, 100% feces. G was failed to grow spawn. This means that the feces of the pigs were insufficient in crude fiber and the supplemental hay helped to grow spawn. The best spawn growth was the horse plates and the worst one to the hen plates. On the hose plates, there was no difference of spawn growth among M, N and O. They showed the 100% coverage after 12 days. This is due to that the feces of the horses contain approximately 80% of crude fiber, as shown in Table2. On the other hand, the hen plates did not attain to the 85% coverage after 14 days even though the hay supplements, K and L. The manure of the hen consists of solid and liquid. The liquid contains urine, which might play a negative effect to grow spawn. When considering the optimum bedding material from all the plates, it was given to the combination of the feces 90% and the hay 10%. 3.4 Mushroom cultivation on the bottle culture 1) Oyster mushroom Oyster mushroom cultivation in the glass bottle of 450 ml was shown in Table 4. The fruiting of Oyster mushroom begins on the 40 days to 45 days after the inoculation on bottle culture using sawdust and rice bran, as reported by Akabane (1982). On the bottles of the goat s bedding materials, only the combination of the bed of feces 90% and hay 10% resulted in fruiting. However the time of fruiting was 86 days after inoculating. It was late and could not be shortened. This can be due to the contamination of the bottle when inoculating. On the cattle bottles, all the beds provided fruits of Oyster mushroom. The time of fruiting was similar to that of the traditional bed material. On the pig bottles, the time of fruiting ranged from 38 days to 45 days. They could be shortened by the 12 days in maximum compared with that of the traditional bed material. Fruiting of Oyster mushrooms on the bed of the pig feces was shown in Fig. 3. On the horse bottles, the hay beds brought a shortage of fruiting. On the hen bottles, the two beds of the 100% feces and the 90% feces and 10% hay gave rise to fruiting, but to no harvesting. Second harvesting was attained on the bottles of the horse and the pig, resulting in the optimal bedding materials. 2) Shiitake mushroom Spawn growth of Shiitake mushroom on the bedding material of feces 90% and hay 10% on the bottle culture was shown in Table 5. The covering area percentage of spawn over the surface of the bottle was measured visually. The time The day after the inouculation (days) A* 25% Goat B C Feces 80% + Hay 20% D Cattle E F Feces 80% + Hay 20% G Pig H I Feces 80% + Hay 20% J Hen K L Feces 80% + Hay 20% M Horse N O Feces 80% + Hay 20% * Weight of dry materials is given in Table 1. EcoEngineering

6 Animal Goat Cattle Pig Hen Horse * The day after the inoculation. ** data. Bed material Artificial pinning (day)* ** Fruiting (day)* Harvesting period (days) Mushroom formation Mushroom weight (g) in wet mass base Second fruiting 65 mm (A) (B) 70 mm of fruiting of Shiitake mushroom on bottle culture needs 80 days to 100 days after inoculating (Ohmori, 2001). Except the hen s bed, spawn growth on the animals beds showed a coverage of 100% over the bottle surface on 40 days after inoculating. However there was no Shiitake mushroom formation on 60 days or more after pining. A reason of no Shiitake formation is due to the difficulty of cultivation rather than Oyster mushroom (Nakamura, 1982). Spawn growth rate increased in order of the goat, cattle, pig and horse bed. But the difference between the rates was small. Visual density of the spawn increased in order of the goat, pig, horse, and cattle bed. 4. Conclusions The air temperature and the relative humidity in the chamber were controlled in the optimal environmental conditions of mushroom cultivation. On the plate culture of spawn growth of Oyster mushroom, the optinum bedding material was the combination of the feces 90% and the hay 10% in dry mass base. On bottle culture of Oyster and Shiitake mushroom cultivation, Oyster mushroom was obtained on the beds of animal feces except that of hen. Urine included in hen manure might play a negative effect to grow spawn of Oyster mushroom on the hen beds. Among the animal feces, pig s feces showed the greatest cultivation. Shiitake mushroom was failed to harvest on the all beds of the animal feces on the bottle culture, even though of enough spawn growth excepting the hen. Animal Bed material 10th day* Mushroom formation Goat 10% Cattle Pig Hen Horse * The day after the inoculation. EcoEngineering

7 Acknowledgements The author, H. Minagawa, received a grantinaid for scientific research (C, ) from the Ministry of Education, Science, Sports, and Culture of Japan. Thanks are also due to T. Nakayama and C. Furumi, students at Kitasato University, for their help with the experiments. References Akabane, H., 2001: Oyster mushroom cultivation. In Practical handbook of mushroom cultivation (eds. by Ohmori, S. and Koide, H.). Rural Culture Association (bunkyo), Tokyo, pp : pp Kameoka, T., 2004: Burning and carbonization of animal manure and applications of their ash and carbide waste. In Handling, Recycling, and Reusing in energy of animal waste (ed. Bookers). NTS Pub. Co., Tokyo, pp : pp Kitamoto, Y Nutrition of mushrooms (1). Kinjin, 24(8), : (1) 24(8) Kuwahara, M., 2002: Lignocellulose. In Handbook of Plant Metabolic Engineering (eds. Shinmyo, A. and Yoshida, K.). NTS Pub. Co., Tokyo, pp : pp MAFF, 2008: A summary of emission of animal waste and its management in Ministry of Agriculture, Forestry, and Fisheries of Japan. Available At: 1.manure.html. Accessed 20 March, Minagawa, H., Sato, M. and Sano, K., 2005: Cultivate mushrooms in the beds of animal feces (in Japanese with English abstract). In Proc. the Annual Meeting of the Society of EcoEngineering in The Society of EcoEngineering, Tokyo, pp Nakamura, K., 1982: Shiitake mushroom. In Handbook of Mushrooms (ed. by Nakamura, K.). Asakura Pub. Co., Tokyo, pp : pp Ohmori, S., 2001: Shiitake mushroom cultivation on bottle cultivation. In Practical handbook of mushroom cultivation (eds. by Ohmori, S. and Koide, H.). Rural Culture Association (bunkyo), Tokyo, pp : pp Ponting, M., 1986: Horses. In The Agricultural tebook (ed. Hally, R., J.). WileyBlackwell, London, UK, pp Schaible, P. J., 1970: Measuring values of feedstuffs. In Poultry: Feeds and Nutrition. The AVI Pub. Co., Inc., Westport, Connecticut, USA, pp Taniguchi, T., 1986: Cultivation of mushrooms. In Plant Biotechnology (eds. by Yamada, Y. and Okada, Y.). Tokyo Kagaku Dozin Pub. Co., Tokyo, pp : pp EcoEngineering