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Production Technology

Auricularia spp., Flamulina velutipes, Grifola frondosa, Hypsizygus marmoreus, Lentinula edodes, Pleurotus spp., Pholiota nameko, Tremella fuciformis, Volvariella spp.

Auricularia spp.

Commonly known as wood ear, Auricularia auricula is the first recorded cultivated mushroom (Chang 1993). Total production of Auricularia spp. in 1997 exceeded 485,000t (fresh weight). This value is an increase of 85,000t or 18% over 1990 levels (Chang 1999). Auricularia spp. production now represents about 7.9% of the total cultivated mushroom supply world-wide.

Auricularia auricula and A. polytricha commonly are produced on a synthetic medium consisting of sawdust, cottonseed hulls, bran and other cereal grains or on natural logs of broad-leaf trees (Quimio 1982, Chang and Quimio 1982, Oei 1996). For cultivation on natural logs, members of the oak family (Fagaceae) are preferred, but many other species of both hard and softwoods may be used.

For synthetic medium production of Auricularias, the substrate may be composted for up to 5 days or used directly after mixing. In either case, the mixed substrate (about 2.5 kg wet wt) is filled into heat resistant polypropylene bags and sterilized (substrate temperature 121C) for 60 min. Composted substrate is prepared by mixing and watering ingredients [sawdust (78%):bran (20%):calcium carbonate (1%):sucrose (1%)] in a large pile. The pile then is covered with plastic and turned (remixed) twice at two-day intervals. For direct use of substrate, a mixture of cottonseed hulls (93%), wheat bran (5%), sucrose (1%), and calcium carbonate (1%) is moistened to about 60% moisture and then filled into polypropylene bags.

After the substrate has cooled, it is inoculated with either grain or sawdust spawn. The spawn then is mixed into the substrate either mechanically or by hand. After the mycelium is allowed to colonize the substrate (spawn run). Temperatures for spawn run are maintained at about 25C +or- 2C for about 28 to 30 days. Light intensity of more than 500 lux during the spawn run may result in premature formation of primordia. Temperature, light intensity and relative humidity all interact to influence the nature and quality of the basidomata.

Flammulina velutipes

Worldwide production of F. velutipes (enokitake, Fig. 1) has increased from about 143,000 metric tons in 1990 to about 285,000t in 1997 (a 2-fold increase, Chang 1999). Japan is the main producer of enokitake (Furukawa 1987). In 1986, Japan produced 74,387t; by 1991, production had risen to 95,123t and, by 1997, Japan produced 174,100t--an increase of about 45% in six years. From these data, it is evident that other countries are enjoying a faster growth rate, in terms of total production. In the United States, for example, enokitake production has increased at an estimated rate of 25% or more per year for the last four years.

Production of most enokitake in Japan is based on synthetic substrate contained in polypropylene bottles. Substrates (primarily sawdust and rice bran; 4:1 ratio) are mechanically mixed and filled into heat resistant bottles with a capacity of 800 to 1,300 ml. Sawdust consisting primarily of Cryptomeria japonica, Chamaecyparis obtusa or aged (9 to 12 months) Pinus spp. appears to offer the best yields. In the United States, a bran-supplemented medium, consisting primarily of corncobs, serves as the primary medium. After filling into bottles, the substrate is sterilized (4 hr. at 95C and 1 hr. at 120C), mechanically inoculated and incubated at 18 to 20C for 20 to 25 days. When the substrate is fully colonized, the original inoculum is removed mechanically from the surface of the substrate and the bottles may be placed upside down for a few days. At the time of original inoculum removal, the air temperature is lowered to 10 to 12C for 10 to 14 days.

To further improve quality during fruiting, temperatures are lowered to 3 to 8C until harvest. As the mushrooms begin to elongate above the lip of the bottle, a plastic collar is placed around the neck and secured with a Velcro® strip. This collar serves to hold the mushrooms in place so that they are long and straight. When the mushrooms are 13 to 14 cm long, the collars are removed and the mushrooms are pulled as a bunch from the substrate. The mushrooms then are vacuum packed and placed into boxes for shipment to market.

Grifola frondosa

Japan is the major producer and consumer of G. frondosa (maitake, Fig. 2.). Commercial production of maitake in Japan (325t) began in 1981 (Takama et al. 1981). By 1986, production was 2,203t and, by 1991, production reached 7,950t (a 261% increase). Japanese production of maitake reached 31,000t in 1997 and was produced primarily in the provinces of Niigata, Nagano, Gunnma, and Shizuoka.

Commercial production of most G. frondosa is on synthetic substrate contained in polypropylene bottles or bags. A common substrate used for production is composed of sawdust supplemented with rice bran or wheat bran in a 5:1 ratio, respectively (Takama et al. 1981). In the United States, researchers (Shen and Royse 2001, 2002) have developed a formula consisting of oak sawdust (70%), wheat bran (10%), millet (10%), and rye (10%). This formula gave the highest yields, best quality and shortest crop cycle time (12 wk). For bottle production, the containers are filled with moistened substrate and sterilized or pasteurized prior to inoculation. Most growers use automated inoculation equipment thereby saving on labor costs. For production in bags, the moistened substrate (2.5 kg) is filled into microfiltered polypropylene bags and sterilized to kill unwanted competitive microorganisms. After cooling (16 to 20 hr), the substrate is inoculated and the bags are heat sealed and shaken to uniformly distribute the spawn throughout the substrate. Spawn run lasts about 30 to 60 days depending on strain and substrate formulation. After primordia formation, two holes usually are cut in the bags exposing the developing primordia that tend to develop around the outside perimeter of the substrate surface. The darkest colored and largest primordia usually are selected because these primordia are the most consistent in development of mature basidiomata. The top of the bag is then folded over, exposing only the developing primordia to the fruiting environment.

Most maitake is marketed as food. However, maitake has been shown to have both anti-tumor and anti-viral properties (Jong and Birmingham 1990, Mizuno and Zhuang 1995, Stanets 2000, Wasser 2002). Powdered fruitbodies are used in the production of many health foods such as maitake tea, whole powder, granules, drinks, and tablets.

Hypsizygus marmoreus

Japanese are the main producers and consumers of H. marmoreus (Fig. 3). Also known as the beech murhsoom, bunashimeji production has increased steadily over the last few years although not as fast as some other types of mushrooms (Royse 1997). In 1990, production of Bunashimeji was 22,600t worldwide; by 1997 production reached 74,200t-an increase of more than 3-fold.

Bunashimeji usually is produced in polypropylene bottles contained in plastic trays. After the completion of vegetative mycelial growth, bottle lids are removed and the colonized substrate subjected to environmental conditions known to stimulate fruiting. When the mushrooms are mature, the entire cluster of fruiting bodies are removed from the bottles. The mushrooms are packaged by placing an entire cluster (or multiple clusters) into each over-wrapped package. Only one flush of mushrooms is harvested prior to mechanical removal of the "spent" substrate from the bottles. The bottles then are refilled with fresh substrate and the process is repeated.

Lentinula edodes

The cultivation of L. edodes (shiitake, Fig. 4) first began in China about AD 1100 (Nakamura, 1983, Royse et al. 1985, Chang and Miles 1987, 1989). It is believed that shiitake cultivation techniques developed in China were introduced to the Japanese by Chinese growers (Ito 1978). Please see http://pubs.cas.psu.edu/FreePubs.ul203.html for additional information.

Cultivation on natural logs

Various species of trees have been used for the cultivation of shiitake (San Antonio 1981). One of the primary species used in one area of Japan in past years was the shii tree--thus the derivation of the name shii-take (Singer 1961). Most production today, however, is on various species of oak (Harris 1986, Stamets and Chilton 1982, Przybylowicz and Donoghue 1988).

Natural logs usually are cut in the fall (after leaf drop) and may be inoculated within 15 to 30 days of felling. Trees that are cut in the fall also may be left intact through winter and, just before inoculation, cut into lengths of about one meter. Trees that are cut in the summer tend to have bark that is more loosely bound and sugar contents usually are lowest during this time. If trees are cut during the summer, the bark may strip off more easily, increasing the chances of contamination of the wood by competitive organisms. The most efficient log diameter appears to be in the 7 to 15-cm range (Ito 1978). Logs greater than 25 cm in diameter often are cut in half prior to inoculation (Royse et al. 1985).

Growers who inoculate the logs with wood-piece spawn drill holes in the logs with high speed drills to correspond to the diameter and length of the wood-piece spawn. Enough holes are drilled in the log to provide spacing of about one hole per 500 cm sq. The wood spawn then is driven into the holes with a hammer and then usually covered with hot wax to prevent excessive drying of the spawn. Sawdust spawn sometimes is used instead of wood-piece spawn.

Spawn run may last from 6 to 9 months, depending on the tree species, log size, spawn cultivar, moisture, temperature, and other variables (Leatham 1982). After the spawn run period the logs often are transferred to a "raising" yard. Raising yards usually are cooler and more moist than the spawn run area. The change in conditions provides an optimum environment for the growth and development of mushrooms. In the raising yard, the logs are arranged to provide for convenient harvesting of the mushrooms. Most production occurs in the spring and fall when conditions are most favorable. However, prices received by the growers usually are lowest during these periods.

Growers may use greenhouses for winter production of mushrooms (Przybylowicz and Donoghue 1988). More overall production is possible, and prices for fresh mushrooms are considerably higher, in winter than during the rest of the year. In the greenhouse method, logs usually are soaked in water (usually less than 48 hr) and vibrated mechanically for various periods prior to placement in the greenhouse. After the mushrooms are harvested, the logs are incubated further (up to three months) and the process is repeated (up to five times).

Synthetic log production

Sawdust is the most popular basal ingredient used in synthetic formulations of substrate used to produce shiitake (Miller and Jong 1987). Other basal ingredients that may be used include straw and corn cobs or mixtures thereof. Regardless of the main ingredient used, starch-based supplements such as wheat bran, rice bran, millet, rye, corn, etc are added to the mix in a 10 to 40% ratio (dry wt) to the main ingredient. These supplements serve as nutrients to provide an optimum growing medium (Royse et al. 1990, Royse 1996).

Once the proper ratio of ingredients are selected, they are combined in a mixer and water is added to raise the moisture content of the mix to around 60%. On large farms, the mix then is augured to a machine that fills and weighs the substrate so that a uniform amount is filled into each bag. The filled bags are stacked on racks, loaded into a industrial-sized autoclave, sterilized for 2 hours at 121C, cooled and inoculated with shiitake spawn.

After a 20 to 25 day spawn run, the bags are removed and the substrate blocks are exposed to an environment conducive for browning of the exterior log surfaces. As the browning process nears completion (4 weeks), primordia begin to form about 2 mm under the surface of the log indicating that the log is ready to produce mushrooms.

Primordium maturation is stimulated by soaking the substrate in water (12C) for 3 to 4 hours (or 3 to 4 min if vacuum soaking is used; see Royse et al. 2002). Soaking allows water rapidly to displace carbon dioxide contained in air spaces, providing enough moisture for one flush of mushrooms. Approximately 9 to 11 days after soaking, mushrooms are ready to harvest.

The main advantages of using synthetic medium over natural logs are time and efficiency. The cycle for synthetic medium cultivation lasts approximately 4 months from time of inoculation to cleanout. Biological efficiencies for this method may average from 75% to 125%. In contrast, the natural log cultivation cycle usually lasts about 6 years with maximum efficiencies around 33%. The time required on synthetic substrate, therefore, only is about 1/15th that of the natural system with about 3 times the yield efficiency. As a result of these developments, shiitake production in the United States has increased dramatically in the last nine years.

Shiitake is one of the best known and best characterized mushrooms used for medicinal purposes. Several medicinal properties have been attributed to shiitake in recent years. These properties include antitumor polysaccharides activity (Breene, 1990; Mizuno, 1995a) and glycoproteins, antiviral nucleic acids, platelet agglutination inhibitive substances, and anti-cholesterol active substances (Tokuda et al. 1974, Fujii et al. 1978, Suzuki et al. 1979, Tokuda and Kaneda 1978, Mizuno 1995a, Wasser 2002).

Pleurotus spp.

Oyster mushroom production has decreased world-wide during the last few years (Chang, 1999). From 1990 to 1997, oyster mushroom production decreased from 900,000t to 876,000t (3% decrease). China was responsible for 86% of the world's supply. In the United States, production of oyster mushrooms was 1,939t in 2002, up 11% from the previous year (USDA 2000). Pleurotus spp. (P. ostreatus and P. cornucopiae) production in Japan peaked in 1989 at about 36,000t. Production was 24,000t in 1993, a decrease of 33% in four years. Recently, however, Japanese growers have begun producing large quantities of P. eryngii (Fig. 5) and total production in Japan may now equal or exceed 1989 production levels.

Substrate preparation. In the United States, the primary ingredients used for Pleurotus spp. production is chopped wheat straw (Triticum aestivum L) or cottonseed hulls (Gossypium hirsutum L) or mixtures thereof. For production on wheat straw, the material is milled to a length of about 2 to 6 cm. Production of Pleurotus spp. on cottonseed hulls has some advantages over straw-based production systems in that chopping of the hulls is not required. One of the most common substrates used on modern mushrooms is a mixture of 75% cottonseed hulls, 24% wheat straw and 1% ground limestone. This mixture of cottonseed hulls and wheat straw has a higher water holding capacity than cottonseed hulls used along. At the University's MRC, a large capacity, scale-mounted feed mixer is used to simultaneously grind and mix the material as water is added to increase the moisture content to 67-69%.

Pasteurization. On some commercial mushroom farms, ingredients are filled into revolving mixers, water is added to the desired level and live steam is injected into the mixer while it is in operation. At the MRC, moistened, mixed substrate is filled into galvanized metal boxes with a perforated floor. The substrate is pasteurized with aerated steam at 65 C for 1 hr by passing the air-steam mixture through the substrate from top to bottom. After pasteurization is complete, filtered air (HEPA filter; 99.9% efficiency) is passed through the substrate for cooling (approximately 1.5 hr).

Spawning and spawn rate. Growers have sought, in the past, to optimize the amount of spawn used to inoculate their substrate. Increasing the amount of spawn used (up to 5% of the wet weight of the substrate) has resulted in increased yields (Royse 2002). Increasing spawn rates from 1.25% (substrate wet weight) to 5% may result in yield of increases of nearly 50%. Yield increases may be due to several factors. First, the increased level of nutrient available in higher levels of spawn used would provide more energy for mycelial growth and development. Second, more inoculum points, available from increased spawn levels, would provide faster substrate colonization and thus, more rapid completion of the production cycle. Finally, a more rapid spawn run would reduce the time non-colonized substrate is exposed to competitors such as weed molds and bacteria.

For increasing levels of spawn used (up to 5%), there is a negative correlation between spawn rate and days to production. As the spawn rate increases, the number of days to production decreases. By using a spawn rate of 5% of the wet substrate wt it is possible to reduce the time to production by more than 7 days compared to a spawn rate of 1.25%. Thus, growers could complete the crop cycle faster, minimizing the exposure of the production substrate to pest infestations especially sciarid [Lycoriella mali (Fitch)] flies. It has been shown that the sciarid fly may complete its life cycle in 25 days at 21 C while 35-38 days are required at 18C. Timely disposal of spent substrate may help to minimize the buildup of fly populations on a mushroom farm.

Use of delayed release supplements. At time of spawning, a commercial delayed release supplement consisting of paraffin coated whole soybean or formaldehyde-denatured soybean and feather meal may be added (rates of 3 to 6% of dry substrate wt) to stimulate yield of the mushroom (Royse and Schisler 1987, Royse 2002). Yield increases of up to 90% have been observed when 6% (dry wt) is added to substrate at time of spawning. Delayed release nutrient supplements have also been shown to decrease the number of days to harvest. The addition of 3% nutrient at time of spawning may reduce time to production by 2 to 3 days. Thus, growers wishing to hasten the production process may do so by supplementing with only small quantities of supplement. Use of supplements, however, may cause overheating of the substrate if growers are unable to anticipate and control air temperatures to maintain a steady substrate temperature. Additional cooling capacity is required when higher levels of supplement are used.

Filling plastic bags with substrate. The pasteurized, supplemented hull/straw mixture is spawned and filled (25 to 30 lbs) into clear or black perforated polyethylene bags and then incubated at 23 to 25C (substrate temperature) for 12 to 14 days. Mushrooms then begin to form around the edges of bag perforations and they are harvested from the substrate approximately 3 to 4 weeks after spawning depending on strain, amount of supplement used and temperature of spawn run. In Japan, bottle production of oyster mushrooms is most common. Substrate is filled into bottles, sterilized and inoculated with Pleurotus spawn. Upon completion of spawn run, bottle lids are removed and mushroom emerge from the surface of the substrate. After the mushrooms are harvested they are weighed and packaged for shipment to market.

The pH of the material is adjusted with limestone to about 7.5 or higher to provide selectivity against Trichoderma green mold (Stolzer and Grabbe 1991).

After completion of pasteurization (60C for 1 to 2 hr.) the substrate is cooled an spawned with the desired strain. At time of spawning, a delayed release supplement (rates of 3 to 10% of dry substrate wt) may be added to increase yield and size of the mushroom (Royse and Schisler 1987, Royse et al. 1991, Royse and Zaki 1991). Use of supplements, however, may cause overheating of the substrate if growers are not able to anticipate and control air temperatures to maintain a steady substrate temperature.

Production of Pleurotus spp. on cotton seed hulls has some advantages over straw-based production systems in that chopping of the hulls is not required (Royse 1995). The pasteurized, supplemented hulls are spawned and filled (12 to 15 kg) into clear or black perforated polyethylene bags and then incubated at 23 to 25C for 12 to 14 days.

In Japan, bottle production of oyster mushrooms is most common. Substrate is filled into bottles, sterilized and inoculated with Pleurotus spawn. Upon completion of spawn run, bottle lids are removed and mushroom emerge from the surface of the substrate. After the mushrooms are harvested they are weighed and packaged for shipment to market.

Pholiota nameko

Japan produced 24,500t of P. nameko in 1997--an increase of only 3,700t (15% increase) from 1990. World-wide production increases averaged 27% over the same time period. In 1997, Japan produced about 44% of the total world production (Chang, 1999).

Nameko means "viscid mushroom" in Japanese. This mushroom is prized for its gelatinous viscosity and for its flavor and is generally used in miso soup, cooked fresh with grated radish, and steamed in pipkin.

Preparation of the medium for nameko production is similar to that for enokitake except that a higher moisture content of the substrate is desirable. A substrate of broad leaf tree sawdust is preferred but research has shown that sawdusts from conifers such Pinus spp. and Cryptomeria japonica are suitable for growth. Rice bran usually is added as a supplement in the ratio of 15% for conifer sawdust and 10% for broad-leaf sawdust.

Mushrooms are harvested from the substrate by cutting the stems near the base with scissors. The harvested mushrooms are washed and packed for shipment to market.

Tremella fuciformis

Known as the white jelly fungus or silver ear, T. fuciformis has been used as a delicacy food in China for many years. This mushroom can be cultivated on natural logs or on synthetic medium (Quimio et al. 1990). Cultivation techniques used to produce the mushroom on natural logs is similar to that used for shiitake production. In recent years, most production of T. fuciformis has been on synthetic substrate using a mixed culture inoculum technique first developed in Fujian, China (Huang 1982).

The mixed culture technique involves the use of "helper" mycelium of Hypoxylon archeri, an ascomycete commonly associated in nature with decaying wood. Hypoxylon archeri increases the ability of T. fuciformis to digest the substrate thereby increasing mushroom yields. Exploitation of this mycelial association is accomplished through use of dual cultures to make mother spawn (Quimio et al. 1990).

Substrate used for mushroom production is the same as that used for spawn production. The supplemented substrate is packed into plastic bags (50 cm long; 9 cm diameter) and ends of the bags are tied with cotton string. Six holes (1 cm diam) then are punched in the filled bags and covered with a breathable fabric. The substrate is sterilized for 6 to 8 h, cooled and inoculated with the mother culture.

After about 30 days of vegetative mycelial growth, the hole covers are removed and the exposed substrate is exposed to conditions favorable for primordia formation (Huang 1982). If optimum conditions are maintained in the growing houses, clusters of jelly fungus should be ready for harvest within 12 to 15 days. Yield for each bag of substrate is in the range of 350 to 500 g fresh weight (35 to 50 g dry weight).

Volvariella spp.

The straw mushroom derives its name from the substrate on which it originally was grown (San Antonio and Fordyce 1972). Cultivation of Volvariella was believed to have begun in China as early as 1822 (Chang 1977). In the 1930's, straw mushroom cultivation began in the Philippines, Malaysia, and other Southeast Asian countries (Chang 1982). Production of the straw mushroom decreased from 207,000t in 1990 to about 181,000t in 1997-a 13% decrease. Volvariella accounts for approximately 3% of the total world-wide production of edible mushrooms.

Many agricultural by-products and waste materials have been used to produce the straw mushroom. These include paddy straw, water hyacinth, oil palm bunch, oil palm pericarp waste, banana leaves and sawdust, cotton waste and sugarcane waste (Chang 1982, Ho 1985). Volvariella is well suited for cultivation in the tropics because of its requirement for higher production temperatures. In addition, the mushroom can be grown on nonpasterized substrate-more desirable for low input agricultural practices.

In recent years, cotton wastes (discarded after sorting in textile mills) have become popular as substrates for straw mushroom production (Chang 1982). Cotton wastes give higher and more stable biological efficiencies (30 to 45%), earlier fructification (four days after spawning) and harvesting (first nine days after spawning) than that obtained using straw as a substratum. Semi-industrialization of paddy straw cultivation on cotton wastes has occurred in Hong Kong, Taiwan and Indonesia as a result of the introduction of this method (Chang 1979).