MINIMALLY COMPOSTED SUBSTRATE FOR THE PRODUCTION OF AGARICUS BISPORUS. The Pennsylvania State University. The Graduate School

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1 The Pennsylvania State University The Graduate School Plant Pathology Department MINIMALLY COMPOSTED SUBSTRATE FOR THE PRODUCTION OF AGARICUS BISPORUS A Thesis in Plant Pathology by Stephanie M. Loehr 2010 Stephanie M. Loehr Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science December 2010

2 The thesis of Stephanie M. Loehr was reviewed and approved* by the following: Daniel J. Royse Professor of Plant Pathology Thesis Advisor Donald D. Davis Professor of Plant Pathology Paul H. Heinemann Professor of Agricultural and Biological Engineering C. Peter Romaine Professor of Plant Pathology Frederick E. Gildow Professor of Plant Pathology Head of the Department of Plant Pathology *Signatures on file in the Graduate School ii

3 ABSTRACT Agaricus bisporus is cultivated commercially on a composted substrate that includes raw materials such as straw- bedded horse manure, hay, poultry litter, distiller s grain, corncobs, cottonseed hulls, gypsum, etc. Composting is a two- phase process that may take up to three weeks or more to complete. During that time, a substantial amount of dry matter is lost as carbon dioxide, ammonia, methane, and other volatiles to the environment. Anaerobic zones formed in the compost during phase I composting may result in emissions of malodorous gases such as hydrogen sulfide and various other sulfur- containing compounds. These compounds have a low threshold of detection, so they may become a nuisance to neighborhoods adjacent to mushroom growing regions. In addition to these negative aspects, water runoff from composting may also contaminate surface and groundwater supplies. Shortening or eliminating phase I composting may help reduce some of the environmental problems associated with mushroom composting. In an effort to alleviate some of the drawbacks of composting, we sought to develop a mushroom substrate that would not require phase I composting. We evaluated milled corn stover (MCS), with and without supplements at various phases of the production cycle, for its use as a minimally- composted mushroom substrate. Specific objectives of the study were to: 1) develop a phase II- only composting protocol for MCS, 2) determine suitable particle size of MCS for maximizing mushroom yields and biological efficiencies (BE), 3) evaluate several nutrient supplements added at fill, at spawning and at casing for their effect on mushroom yields, BE and average mushroom size, 4) evaluate use of spent iii

4 mushroom substrate (SMS) added as an ingredient to MCS at fill and 5) evaluate millet- type and synthetic- type spawn and rate of spawn on mushroom yield, BE and average mushroom size. MCS was subjected to a modified phase II- only composting process in minibunkers. Several treatments resulted in a substrate that was selective for A. bisporus growth and mushroom production. Three particle sizes were evaluated for their effect on mushroom yield, BE and mushroom size. Substrate with the smallest particle sizes (89.2% <3 mm) resulted in higher yields (12.28 kg/m 2 ) and BE (59.8%) compared to medium (9.22 kg/m 2, 44.9%) and larger (4.95 kg/m 2, 24.1%) particle size substrates. Supplements (cottonseed meal, ground soybean, distiller s grain), added at fill, increased yields and BE compared to MCS alone. Cottonseed meal addition (5% d/w) at fill resulted in higher yields (20.1 kg/m 2 ) and BE (92.9%) than distiller s grain (5% d/w) and MCS alone. SMS also was evaluated as an ingredient added to MCS at fill into minibunkers. Addition of 48% SMS added at fill to MCS had no effect on yield and BE compared to MCS alone. Delayed- release nutrient supplements, added at spawning and/or casing, were evaluated for their effect on productivity. Substrates supplemented with Lambert T6 at 6% d/w at spawning compared to Remo s All Season at 6% d/w and T6 at 12% d/w produced higher yields. Treatments supplemented with Lambert T6 (4% d/w) at casing, resulted in higher yields and BE compared to Lambert T7 and Remo s All Season supplement used at casing. iv

5 Use of synthetic- type spawn (Sylvan Matrix ) resulted in higher yields (15.8 kg/m 2 ) and BE (73.0%) than millet- based spawn (13.0 kg/m 2, 60.0%). Two spawn rates (0.75% and 1.5% w/w) were evaluated for their effect on mushroom yield and BE. The higher spawn rate resulted in higher yields (13.6 kg/m 2 ) compared to the lower spawn rate (12.7 kg/m 2 ), but BE was not affected. MCS, when subjected to phase II- only composting, may have potential for use as a mushroom substrate, especially with smaller- scale growers. MCS is relatively inexpensive to prepare and dry matter losses are less, relative to traditional compost. In addition, incorporation of SMS into fresh MCS compost may provide a beneficial use for SMS and help reduce environmental impacts of mushroom substrate preparation. v

6 TABLE OF CONTENTS List of Figures List of Tables.. Acknowledgements ix x xii Chapter 1: General introduction Introduction Economic importance of Agaricus bisporus Cultivation history Cultivation methods Nutritional requirements Supplementation Environmental impact Raw materials used for cultivation Shortening or eliminating the composting process Objectives. 13 Chapter 2: Phase II- only composting of milled corn stover for use as substrate Introduction Methods Ingredients Substrate preparation Phase II- only composting Experimental design and data analysis Cropping trial Harvesting, determination of yield, biological efficiency and mushroom size. 25 vi

7 2.3 Results Substrate particle sizes Yield, biological efficiency, and mushroom size Discussion.. 28 Chapter 3: Influence of nutrient supplementation at fill and at casing, temperature zones in minibunkers during phase II composting, and spawn type on yield, biological efficiency and mushroom size Introduction Methods Substrate ingredients Experimental design and data analysis Cropping trials Harvesting and determination of yield, biological efficiency, and size Results Crop Crop Crop Discussion.. 48 Chapter 4: The influence of spawn rate and SMS addition to milled corn stover compost on yield, biological efficiency, and mushroom size Introduction Methods Substrate Experimental design and data analysis Mushroom cropping trial Harvesting and determination of yield, biological efficiency and mushroom size Results.. 56 vii

8 4.3.1 ANOVA Spawn rate SMS additions Discussion.. 59 Chapter 5: Summary and future work Summary Future work. 62 References 64 viii

9 LIST OF FIGURES Fig WIC bale chopper used for pre- chopping of corn stover used for preparation of milled corn stover substrate Fig (A) Milling corn stover with a shredder/chipper. (B) Discharge screens used to prepare mushroom substrate of various particle sizes (from left: 0.64, 1.27, 1.91 cm sieve size). 19 Fig (A) Wetting and mixing of milled corn stover (MCS) by hand for use as substrate. (B) Compacting MCS in minibunker.. 20 Fig (A) HOBO Water Temp Pro v2 data logger. (B) Location of HOBO data loggers within minibunker Fig Milled corn stover (MCS) substrate following phase II- only composting in minibunker. Note white- tinged substrate (Actinomycete fire- fang growth).. 23 Fig (A) Milled corn stover substrate (MCS), subjected to phase II- only composting colonized by mushroom (A. bisporus) mycelium during spawn run. (B) Fragmentation of spawn run substrate using mechanical turner for homogenization and addition of supplement at casing. 25 Fig Mushroom production on milled corn stover substrate subjected to phase II- only composting in a minibunker 26 Fig Assignment of temperature zones in substrate during phase II- only composting in minibunkers. H = high, M = middle, L = low.. 36 Fig Physical appearance of (from left to right) milled corn stover formulas 1, 2, and 3. Formula compositions are listed in Table ix

10 LIST OF TABLES Table 2.1. Particle size range of milled corn stover substrates (MCS) and phase II mushroom compost (Crop 0904) 27 Table 2.2. The effect of substrate particle size on mushroom yield, biological efficiency (BE %) and average mushroom size (Crop 0904).. 28 Table 3.1. Substrate type and moisture and nitrogen content of milled corn stover (MCS) substrates at fill for Crop Table 3.2. Probabilities > F from analysis of variance for substrate type, temperature zone and Remo s added at casing tested for yield and biological efficiency (BE %).. 39 Table 3.3. Effects of distiller s grain added at fill, temperature zone of substrate within mini- bunker and supplement added at casing on yield and biological efficiency (BE %) of A. bisporus Table 3.4. Means and groupings from analysis of variance for temperature zones within substrate in minibunker during composting for yield and biological efficiency (BE %) 41 Table 3.5. Means and groupings from analysis of variance for supplementation at casing for yield and biological efficiency (BE %).. 41 Table 3.6. Substrate type and moisture and nitrogen contents at fill, spawning and casing for milled corn stover (MCS) (Crop 0905).. 42 Table 3.7. Probabilities > F from analysis of variance for supplement at filling and supplement type and rate at casing tested for yield, biological efficiency (BE %), and mushroom size for A. bisporus (Crop 0905). 42 Table 3.8. Effects of ground soybeans and soybean meal added at fill to milled corn stover and supplement type at casing (% d/w) on yield, biological efficiency (BE %), and mushroom size for A. bisporus (Crop 0905) Table 3.9. Means and groupings from analysis of variance for supplementation of milled corn stover substrate at fill using ground soybean and x

11 soybean meal for yield and biological efficiency (BE %) of A. bisporus (Crop 0905) 43 Table Means and grouping from analysis of variance for supplementation type at casing for yield and biological efficiency (BE %) for A. bisporus (Crop 0905) 44 Table 3.11 Substrate type, moisture content and total nitrogen at fill and at spawning for Crop Table Probabilities > F from analysis of variance for supplement at fill, spawn type, and supplement added at casing tested for yield, biological efficiency (BE %), and mushroom size for A. bisporus (Crop 0906).. 45 Table Effects of adding cottonseed meal and distiller s grain at fill, spawn type, and supplement type at casing (%d/w) on yield, biological efficiency (BE %), and mushroom size for A. bisporus (Crop 0906).. 46 Table Means and groupings from analysis of variance for supplementation of milled corn stover substrate at fill using distiller s grain and cottonseed meal for yield and biological efficiency (BE %) for A. bisporus (Crop 0906) 47 Table Means and groupings from analysis of variance of spawn type on mushroom yield and biological efficiency (BE %) for A. bisporus (Crop 0906). 47 Table 4.1. Percentages of various ingredients in three formulas of phase II- only substrates for Crop 1001b. 55 Table 4.2. Probabilities > F from analysis of variance for spawn rate and addition of spent mushroom substrate (SMS) evaluated for yield, biological efficiency (BE %), and mushroom size for A. bisporus (Crop 1001b). 57 Table 4.3. Effects of various levels of spent mushroom substrate (SMS) added to milled corn stover at fill and spawn rate on mushroom yield, biological efficiency (BE %), and mushroom size for A. bisporus (Crop 1001b). 57 xi

12 Table 4.4. Means and grouping from analysis of variance for spawn rate on mushroom yield and biological efficiency (BE %) for A. bisporus (Crop 1001b).. 58 Table 4.5. Means and grouping from analysis of variance for spent mushroom substrate (SMS) addition at fill to milled corn stover on yield, biological efficiency, and mushroom size for A. bisporus (Crop 1001b). 58 xii

13 ACKNOWLEDGEMENTS First, and foremost, I would like to thank my advisor, Dr. Daniel Royse, for his guidance, helpful suggestions, and positive reinforcement and support of my work. I am sincerely grateful for his help in writing and professional development. Much thanks goes to my committee members Drs. Peter Romaine, Donald Davis, and Paul Heinemann for their advice and suggestions. I am very grateful to Henry Shawley, Doug Keith, Joey Martain, and John Pecchia for their help and direction at the Mushroom Research Center. I would also like to express my thanks to the Mushroom Industry for their generous support of my research. I appreciate the help and guidance from fellow graduate students and faculty in the Plant Pathology Department. To all my friends, especially Vasileios, thank you. I would like to thank my family, my parents Michael and Cheryl, for their love, care, understanding and moral support and Grandma O for making this all possible. And last, but not least, I would like to thank my brothers, Sean and Taylor, for being the best little brothers I could have. xiii

14 Chapter 1: General introduction 1.1 Introduction Mushrooms have been a valued food since the earliest human civilizations, including the Greeks, Romans, Chinese, and Mayans. This is for good reason, since mushrooms are a low calorie, high protein food source with a low nucleic acid content that allows for daily ingestion (Chang, 2006). Mushrooms also are highly prized for their medicinal properties. The first mushrooms (Auricularia auricula, the wood ear) were successfully cultivated in 600 A.D. by Chinese growers (Chang, 2006). Several other species were cultivated first in China, such as Flammulina velutipes, Lentinula edodes, Volvariella volvacea, and Tremella fuciformis (Chang, 2006). Agaricus bisporus, the most widely cultivated mushroom in the western world, was first cultivated in France around 1650 A.D. Pleurotus ostreatus, the oyster mushroom, was first produced in the U.S., but not until By 2002, world mushroom production was 12,250,000 tons, with approximately 70.6% of that contributed by China (Chang, 2006). Modern mushroom production has experienced many advances, leading to increased yields and quality, due to technologies such as computerized controls, automated harvesting machinery, and bulk handling of raw materials, i.e. use of tunnels (Sanchez, 2004). 1

15 1.2 Economic importance of Agaricus bisporus The mushroom industry is vital to the economy of the Commonwealth of Pennsylvania. Agaricus bisporus was valued at $885.8 million for the season in the U.S. (USDA, 2010). Pennsylvania produced 64.4% of the mushrooms grown in the U.S. (USDA, 2010) valued at $438.9 million in the season. California ranks second (14%) in the U.S. in terms of mushroom production, with a value of $184 million in Production of A. bisporus in was approximately 360 million kg, with an average value per kg of $2.57. Production was down 1% from 2009, in addition, mushroom value decreased about 3%. 1.3 Cultivation history Agaricus bisporus was first cultivated around 1650 A.D. by French growers. It was the first mushroom cultivated before 1900 that did not originate in China (Chang, 2006). Farmers noticed that the mushroom fruited on freshly prepared horse manure inoculated with colonized manure. It was originally grown under greenhouse benches until farmers realized they could profit much more from the mushrooms than their other crops and converted their hothouses to mushroom houses (Atkins, 1979). A complete description of the cultivation of A. bisporus was written in 1707 by the French botanist Tournefort (Rettew, 1948). Mines in France that had once been used for mining building stones became ideal locations to cultivate mushrooms. These mines offered several advantages over open- air cultivation including more constant 2

16 temperature and humidity and fewer animal pests. Mines were not without some disadvantages, however. The flake spawn used in France at the time was not a pure culture and often contained fly and nematode eggs, competitor molds and pathogens. These pests multiplied in the mines and, after a few years, a mine had to be abandoned for production. England too, had tried growing mushrooms underground, using brick spawn to inoculate their beds. The brick spawn was similar to the French flake spawn in the sense that both types were often highly contaminated and vigor was often questionable. With the development of pure culture spawn in 1903 in the United States, the mushroom industry started to see increased harvests and less contamination and fewer pests. In 1915, pasteurization during phase II composting was introduced as a way to prevent mushroom pests from becoming established (Van Griensven & Roestel, 2004). Mushroom cultivation reached the United States in 1865 via England. One hot spot in the U.S. mushroom industry was southeastern Pennsylvania in the Kennett Square area. This region today continues to produce a large portion of the mushrooms grown in the U.S. The Pennsylvania State University has a long background of mushroom research starting in the 1920s and continuing today. Much of this research has focused on increasing yield and profits for farmers by improved composting practices, eliminating and controlling diseases, developing better strains and increasing shelf life and nutritional aspects of the mushroom. In 1930, average mushroom yield was 4.9 kg/m 2 of bed surface, in 1964, this increased to about 10.8 kg/m 2 of bed surface (Snetsinger, 1970). Today, farmers can expect average yields of 29 kg/m 2 of bed surface. Mushroom Research at Penn State continues to provide solutions to industry problems. 3

17 1.4 Cultivation methods Mushroom cultivation is performed in six main steps: phase I composting, phase II composting, spawning, casing, pinning, and harvesting (Royse & Beelman, 2007). Prior to phase I composting, the materials undergo pre- wetting, i.e., the wetting and mixing of raw materials. Phase I composting is the forming of moistened materials into stacks, windrows, or bunkers. The piles are turned at 2-3 day intervals and water is added to ensure consistent and even wetting and mixing of raw materials. Turning, usually conducted outdoors, allows for aeration and more uniform degradation of materials. Aerobic breakdown of organic matter by microorganisms occurs in phase I composting (Fordyce, 1970). However, anaerobic microenvironments may occur in the compost. Indications of anaerobic conditions include high methane levels and lack of oxygen in air surrounding compost piles (Derikx et al., 1990b). As microorganisms proliferate, they produce more heat and pile temperature increases. Higher temperatures promote growth of mesophilic and thermophilic microorganisms that play a major role in degrading complex substrates. Temperatures within a compost pile may reach an average 63 C, although, they can range between 25 and 80 C, depending on the location within the pile. This temperature gradient causes airflow through the pile, releasing offensive odors, produced by bacteria that use sulfur for energy, to the environment. The final result of phase I composting is a softened, dark brown product that smells strongly of ammonia. After phase I composting is complete, phase II composting begins. There are two main goals of phase II composting: 1) pasteurization to kill any harmful or competitive 4

18 microorganisms that may affect growth of A. bisporus and 2) conditioning of the substrate so that it becomes selective for the growth of A. bisporus and free of gaseous ammonia. After composting is complete, the substrate is inoculated with spawn. Spawn is mixed into the substrate as it is filled into trays or beds. Mycelium from spawn quickly fuzzes out and the mycelium starts to colonize the substrate. Complete substrate colonization usually takes about two weeks. Following this, a casing layer (roughly 3-4 cm thick) is added to promote the growth of mushrooms. The casing layer is composed of neutralized peat moss mixed with casing inoculum (fine organic material colonized by A. bisporus) that is used to shorten production time and produce a cleaner product. Pinning occurs approximately 10 days after the casing layer is applied. Mushroom mycelium is visible throughout the casing layer and mushroom primordia, resembling pins, begin to emerge from the surface of the casing layer. These pins mature and enlarge to form mushrooms. Mushrooms are produced in flushes lasting about 5-7 days followed by a 2-3 day period where no mushrooms are harvested. Typically, an average mushroom crop will have three distinct flushes, with mushroom production decreasing with each successive flush. 1.5 Nutritional requirements A. bisporus produces two ligninolytic enzymes, laccase and manganese peroxidase (MnP) (Bonnen et al., 1994). These enzymes are produced mainly during the colonization of the substrate, prior to pinning. The MnP, in association with laccase, 5

19 catalyze the breakdown of lignin into simpler forms, providing food for growing mycelium (Tuomela et al., 2000; Iiyama et al., 1994). Weil et al. (2006) showed that the addition of 184 mg kg - 1 Mn to conventional compost increased yields by about 10%. Optimal Mn 2+ level in mushroom compost is about 400 mg kg - 1. Nitrogen is also an important growth factor for A. bisporus. Typical finished compost has a nitrogen level of about 2.5%, however, this nitrogen must not be in the form of ammonia, a nitrogenous compound that greatly inhibits mycelial growth. While high nitrogen levels in the raw materials are important for good mushroom yield, phase II composting may need to be extended for conditioning due to excess ammonia production (Noble & Gaze, 1995). A. bisporus grows best at a carbon : nitrogen ratio of about 17:1 and most conventional compost has a C:N ratio of 14-19:1 (Wuest, 1977). 1.6 Supplementation Most mushroom growers use supplements to increase production. Any nutrient added after phase I composting is termed a supplement (Gerrits, 1988). The goal of supplementation is to provide A. bisporus with all the nutrients it needs to produce the highest yields, while remaining cost effective. Supplementing can be done at most steps of production including prior to phase II, at spawning, at casing (Sinden and Schisler, 1962) and after first, second, or third break (Royse et al., 2008; Royse and Sanchez, 2008a,b). Prior to composting, distiller s grain (24% protein, 0.8% fat) is often added to stimulate microbial communities to initiate organic material breakdown. 6

20 Additions of vegetable oil at spawning and at casing are known to increase mushroom yields (Schisler and Patton Jr., 1970, 1971). In addition, seed oils and animal fats added to A. bisporus grown on basal media all increased mycelium growth rate (Wardle & Schisler, 1969). By adding vegetable oil at the beginning of phase II composting, additional lipids are available for the microorganisms to grow and degrade the substrate, making it more selective for A. bisporus (Schisler & Sinden, 1962). Schisler and Sinden (1966) also found that yields were greater when supplement was added at casing with whole ground seeds compared to seed oil meals, when added at similar nitrogen concentrations. Many commercial mushroom supplements are available to growers today. These supplements are usually treated to delay the release of nutrients over time. Some of the common ingredients in commercial supplements include feather meal, newspaper, corn gluten, soybean meal and many other agricultural by- products. Mineral supplements, such as Micromax, have been tested for use in mushroom production (Weil et al., 2006), but most compost contains sufficient mineral levels for optimum mushroom growth and development. 1.7 Environmental impact Agaricus bisporus is cultivated commercially on a composted substrate commonly consisting of agricultural by- products. During the composting process, offensive odors may be generated, prompting nuisance complaints from residents in nearby communities. Various sulfur compounds, generated by bacteria during phase I 7

21 composting, are the principal malodorous emissions produced. Some of these emissions have very low human detection threshold levels, so minute quantities may have a large impact (Miller & Macauley, 1988; Noble et al., 2001). Another negative environmental consequence of composting may be nutrient- rich run- off from compost piles that may contaminate surface and groundwater. Wheat straw has a very waxy cuticle that inhibits rapid water uptake (Atkey & Wood, 1983), and may lead to large quantities of nutrient- rich runoff. Additionally, at crop termination, a large amount of spent mushroom substrate (SMS) must be moved off the farm for reprocessing or disposal. 1.8 Raw materials used for cultivation Some agricultural waste products that have been used for mushroom production include wheat straw, hay, sugarcane bagasse, cotton wastes (seed hulls) and corn stover (CS). Raw materials used for mushroom cultivation should be readily available and inexpensive. Currently, most commercial mushroom producers use a blend of straw, hay, cottonseed hulls, poultry manure, brewer s grains and gypsum (John D Amico, personal communication). Raw materials account for about 25-40% of the costs associated with production. Therefore, less expensive raw materials are of interest. Corn stover (stalks, leaves, shucks, and cobs of corn (Zea mays)) is a suitable raw material ingredient for mushroom compost (Beyer et al., 2010). Quantities of corn stover, up to 50% of the dry weight, may be added as a compost ingredient without a significant effect on mushroom yield (Beyer et al., 2010). Use of corn grain as a source 8

22 of biofuels has resulted in an increase of corn production across the U.S., creating an abundance of CS. CS is one of the most underutilized agricultural by- products in the U.S.; however, CS is often left on the field to protect soil from erosion. Using one- pass harvesting, in which the grain and CS is harvested at the same time, farmers can expect to see returns from CS of up to $112 per acre on a no- till field (Atchinson & Hettinhaus, 2003). Currently, CS is being used for production of hemi- cellulosic ethanol and as a raw material in mushroom compost on at least one mushroom farm in Pennsylvania. 1.9 Shortening or eliminating the composting process The objectives of abbreviated composting are to overcome the emission of objectionable odors and to conserve dry matter, making more efficient use of raw materials. Bypassing phase I composting would drastically decrease anaerobic fermentation, the cause of the majority of unpleasant odors emitted from compost. In addition, decreasing composting duration would conserve raw materials, thus improving mushroom yield per kg of raw material, and increasing grower profits. Since the development of the two- phase, short method of composting by Sinden and Hauser (1950), composting time has been significantly reduced. However, the shortened two- phase method of composting still requires at least two to three weeks to complete. During this time, a substantial amount of dry weight of raw materials (up to 40%) is lost to the environment in the form of gases such as ammonia and CO2. The 9

23 remaining dry matter and proteins, fats, and carbohydrates associated with microorganisms in the compost will later become nutrition for A. bisporus. Following the development of two- phase composting, many researchers (Till, 1962; Murphy, 1973; Laborde, 1980; Randle & Flegg, 1985; Nair & Price, 1993) have attempted to shorten the composting process even further. In 1962, Till (1962) developed a non- composted substrate by autoclaving chopped raw materials and subsequently spawning the sterile substrate. However, he had little success when he pasteurized the material. Six years later, Hunke and Sengbusch (1968) modified the Till- method. The Huhnke procedure used the Till substrate, but immediately following sterilization, the substrate was treated in a fermentation process until it was spawned. This allowed spawning to be done under non- sterile conditions, eliminating the need for special equipment. In 1972, Murphy demonstrated that phase I composting was not needed in order to produce sufficient mushroom yield. He used a synthetic substrate consisting of corncobs, cottonseed meal, timothy hay and used compost. Following a modified phase II, consisting of a peak- heating then a gradual conditioning phase, suitable mushroom substrate was produced. One of Murphy s (1972) formulas consisting of 50% used compost, 40% corncobs, and 10% cottonseed meal, at 72-76% moisture at the start of phase II, produced the highest yield of 25.1 kg/m 2. Sawdust addition to the mixture did not alter the yield of the compost significantly. This method of eliminating phase I has promise due to its reuse of spent substrate and the elimination of phase I composting. However, if farms were to eliminate phase I composting and switch to entirely synthetic composts, the availability of used substrate would dwindle rapidly. Laborde (1980) 10

24 discussed two methods of rapid composting named L.C.T. and H.C.T. (Low/High Controlled Temperature). These methods were introduced in attempt to shorten the composting process by controlling compost temperatures to efficiently compost the mushroom substrate. Derikx et al. (1990b) suggested a minimal phase I composting of 3.3 days would be required to provide a suitable substrate for A. bisporus using conventional materials. However, this method was never adopted commercially. Another avenue to minimize composting duration is to exploit microorganisms in the substrate that can help impart selectivity. The presence of thermophilic fungi in mushroom compost is important in promoting growth of mushroom mycelium and mushroom yield (Straatsma & Samson, 1993; Straatsma et al., 1994). Thermophilic fungi are capable of growing at temperatures exceeding 40 C (Crisan, 1973) and are particularly important in the later stages of phase II composting by helping to clear the compost of ammonia (Ross & Harris, 1983) and make it selective for A. bisporus growth. Growth of microorganisms, specifically thermophilic fungi imparts selectivity to the substrate and inhibits the growth of microorganisms that may compete with A. bisporus. One particular thermophilic fungus commonly isolated from mushroom compost is Scytalidium thermophilum (synonyms, Humicola grisea var. thermoidea, Humicola insolens, and Torula thermophila) (Kleyn & Wetzler, 1981). S. thermophilum is present in phase II compost in very high populations and the density is positively correlated to mushroom yield (Straatsma et al., 1989). S. thermophilum breaks down cellulose rapidly and immobilizes those nutrients for later use by A. bisporus. Another closely related species is Myriococcum thermophilum which has a similar affect on mushroom compost and subsequent mushroom yield (Straatsma et al., 1989). The extension rate 11

25 of A. bisporus mycelium is positively affected when thermophiles are present in the compost (Straatsma et al., 1994; Sanchez & Royse, 2009; Coello- Castillo et al, 2009). Increased CO2 may be responsible for this observation since there have been no growth promoting compounds isolated from these thermophilic fungi aside from increased CO2. However, A. bisporus mycelium has been shown to replace disintegrated S. thermophylum hyphae on agar media (Op den Camp et al., 1990), suggesting that S. thermophylum may be a nutrient source for A. bisporus. Several alternatives to the shortened two- phase method of composting have been proposed. Sanchez and Royse (2001) adapted a shiitake substrate for use as a pasteurized, non- composted substrate for A. bisporus. They achieved the highest biological efficiencies (77.1%) using a basal mixture, consisting of oak sawdust (28%), millet (29%), rye (8%), peat (8%), alfalfa meal (4%), soybean flour (4%), wheat bran (9%), and CaCO3 (10%), yielding 31.4 kg/m 2. Several other researchers (Mamiro & Royse, 2007; Bechara et al., 2006ab; Bechara et al., 2007) have experimentally used a non- composted substrate. This may be sufficient for small- scale specialty growers to produce Portobello mushrooms, although, at present, it may not be economically viable for large- scale operations due to materials costs. Reusing spent mushroom substrate in combination with a non- composted substrate has been shown to produce high yields, up to 27.2 kg/m 2 (Mamiro & Royse, 2007), while having the potential to recycle large amounts of waste. However, the economic viability of this type of substrate has yet to be determined. Another method to eliminate composting is to first inoculate the substrate with S. thermophylum and allow it to colonize for four days to impart selectivity to the 12

26 substrate. Then the substrate may be spawned normally as demonstrated by Sanchez and Royse (2009, Sanchez et al., 2008). The mechanisms of S. thermophylum s ability to impart selectivity are not fully known (Sanchez & Royse, 2009). Yields of 21.6 kg/m 2 and biological efficiencies of 99% were obtained on S. thermophylum- inoculated wheat straw supplemented with 9% Lambert T6 both at spawning and at casing (Sanchez & Royse, 2009) Objectives The overarching goal of this study was to reduce the environmental impacts of mushroom composting by eliminating phase I composting. This should, in turn, reduce loss of raw materials, decrease time required for composting, and increase grower profits. Using milled corn stover amended with lime and MnSO4, several cropping trials were conducted to determine the usefulness of corn stover as a main ingredient in minimally composted mushroom substrate. This thesis is divided into five chapters, the first consisting of the general introduction to the project. The second chapter is dedicated to the methods of phase II- only composting of MCS substrate. The third chapter discusses the effects of particle size of milled corn stover on mushroom yield, BE, and mushroom size. The fourth chapter discusses the effect of spawn rate, temperature zones of MCS in minibunkers during phase II composting, and the addition of nutrient supplements added at fill, 13

27 spawning, and casing on mushroom yield, BE, and mushroom size. The final chapter reports on the effects of spawn type and SMS addition to MCS at fill on mushroom yield, BE and mushroom size. 14

28 Chapter 2: Phase II-only composting of milled corn stover for use as substrate 2.1 Introduction A. bisporus is produced on a composted substrate consisting mainly of hay, straw- bedded horse manure, poultry manure, gypsum, cottonseed hulls, and distiller s grain. The composting process is time- consuming and energy- intensive, but when compost is prepared properly, it is a selective substrate for the growth and development of mushroom mycelium. Mushroom (A. bisporus) production in the U.S. is a $886 million industry (USDA, 2010) Composting and substrate preparation may account for up to 40% of costs involved in mushroom production (Wuest, 1983). Composting for mushroom substrate production has been met with much criticism. Mushroom growers deal with increasing nuisance complaints from neighborhood residents regarding compost preparation. These complaints include emissions of malodorous compounds, surface and groundwater water run- off in composting yards, and unsightly compost piles. Continued nuisance complaints have forced some growers to relocate their composting operations to less populated areas. The composting process is lengthy and a considerable amount of dry matter (up to 40% or more) is lost during composting (Randle and Hayes, 1972). Compost dry (organic) matter is the nutrient source of the mushroom, greatly affecting mushroom production. Minimizing composting time may lessen the extent of dry matter losses that occur during the composting process, possibly resulting in increased grower 15

29 profits. Therefore, alternative substrates, prepared in a way that conserves raw materials and that requires less composting, are of particular interest to growers who strive to reduce their inputs. Since mushroom cultivation began, numerous attempts have been made to shorten the composting process. In 1950, Sinden and Hauser developed the short method of composting. This method, consisting of separate and distinct phase I and phase II composting, reduced the duration of composting by three weeks, but the process still requires 3-4 weeks for completion. Several attempts have been made to shorten the composting process even further (Till, 1962; Murphy, 1972; Laborde, 1980; Randle & Flegg, 1985; Nair & Price, 1993). However, none of these developments have been adopted by the mushroom industry. Murphy (1972) demonstrated that phase I composting could be eliminated from substrate preparation, while still producing high mushroom yields. He used a substrate formula consisting of 50% spent mushroom substrate (SMS), 40% corn cobs, and 10% cottonseed meal. This formulation was subjected to phase II- only composting, following a traditional peak- heating and conditioning phase. Milled corn stover (MCS) is a material that may be used for mushroom production. Subjecting MCS to phase II- only composting may result in a selective substrate for A. bisporus (see Chapter 2). MCS contains approximately 0.85% nitrogen and with supplementation at filling and spawning, nitrogen content of MCS substrate may approach that of conventional mushroom compost ( % N at spawning, Daniel J. Royse, personal communication). Corn stover also is rich in lignin, cellulose, and hemicellulose, all utilizable nutrient sources for A. bisporus. However, MCS, 16

30 depending on the degree of milling, may have low bulk density compared to commercial mushroom compost. Sufficient bulk density is important in order to obtain high mushroom yields on a given surface area (per m 2 )(expressed as kg/m 2 ). In order to increase bulk density to achieve a higher yield of mushrooms and biological efficiency (yield based on compost dry matter and expressed as a percentage), corn stover requires milling to reduce particle size of the finished substrate. The objective of the research presented in this chapter is to expand upon Murphy s results using milled corn stover (MCS) as a main ingredient for mushroom substrate preparation. Methods of preparing MCS and the phase II- only composting of MCS substrate for mushroom production are discussed herein. Also, influence of MCS particle size on mushroom yield (kg/m 2 ), biological efficiency (%) and mushroom size (g/mushroom) is presented and discussed. 2.2 Methods Ingredients Baled (15-18 kg/bale) corn stover was obtained in November 2008 and 2009 from Russell E. Larson Agricultural Research Center, The Pennsylvania State University, Rock Springs, PA and stored indoors until it was used (up to one year). Pulverized limestone (Graymont, Ltd.), hydrated lime (National Gypsum Co.), and manganese sulfate (Man- Gro DF, Tetra Micronutrients, Inc.) were added to adjust the ph and supply substrate- deficient manganese. 17

31 2.2.2 Substrate preparation Bales of corn stover were chopped using a WIC bale chopper (Fig 2.1). The chopped material (245 kg) then was milled using a shredder/chipper (McKissic Mighty Mac) fitted with a 1.91 cm discharge screen (Fig. 2.2). One third (81 kg) of the chopped material was set aside. The remaining material was further milled using the shredder/chipper fitted with a 1.27 cm discharge screen. Finally, one half of this finer material (81 kg) was milled further using the shredder/chipper fitted with a 0.64 cm discharge screen. Samples of each of the three substrates, in addition to phase II mushroom compost, were collected and analyzed at Agricultural Analytical Services Laboratory, The Pennsylvania State University using a series of sieves to determine particle sizes according to the Test Methods for the Evaluation of Compost and Composting, U.S. Composting Council (2010). Figure 2.1. WIC bale chopper used for pre- chopping of corn stover used for preparation of milled corn stover substrate. 18

32 A B Figure 2.2. A. Milling corn stover with a shredder/chipper. B. Discharge screens used to prepare mushroom substrate of various particle sizes (from left: 0.64, 1.27, 1.91 cm sieve size). In order to raise the ph of the milled corn stover substrate (from 7.2 to 7.4), lime mix (2:1 pulverized lime:hydrated lime) was added at 1.5% dry weight basis (d/w) to each of the three separate piles of corn stover. Corn stover is deficient in manganese (45 mg/kg d/w) compared to commercial mushroom compost ( mg/kg d/w; D.J. Royse personal communication). Both the titre and time of appearance of manganese peroxidase, an important enzyme in Agaricus responsible for lignin degradation, is affected by the amount of manganese (Mn 2+ ) present in the substrate. Therefore, manganese sulfate (MnSO4) was added at a rate of 0.96 g/kg of substrate to each of the three substrate piles to bring Mn 2+ concentrations to approximately 300 mg/kg. After mixing all dry ingredients, water was applied and mixed into the substrate (Fig. 2.3). The piles were turned once and more water was applied to achieve approximately 72-75% moisture in all three substrates. The substrates then were filled into separate mini- bunkers (Pecchia, 2000). 19

33 A B Figure 2.3. (A) Wetting and mixing of milled corn stover (MCS) by hand for use as substrate. (B) Compacting MCS in minibunker. Substrate within the minibunkers was compacted by hand (Fig. 2.3) two- three times during fill to ensure sufficient mass of substrate within the minibunker. Headspace (10-15 cm) was left between the surface of the substrate and the ceiling of the minibunker to allow for adequate air circulation. HOBO temperature probes (Fig. 2.4) were placed throughout the substrate to monitor compost temperatures during phase II- only composting (Fig. 2.4). Chameleon temperature probes connected to a data logger were also placed throughout the substrate in the minibunkers for real- time monitoring of compost temperatures. Temperature profiles were graphed using data collected from the temperature loggers following completion of phase II- only composting. 20

34 A Figure 2.4. (A) HOBO Water Temp Pro v2 data logger. (B) Location of HOBO data loggers within minibunker. B Phase II-only composting Minibunkers were placed into the phase II room at the Mushroom Test Demonstration Facility (MTDF), The Pennsylvania State University, University Park, PA with air temperatures set to 35 C. Airflow was controlled automatically using a fan at the base of the minibunker. At filling, the fan was set to run 8 min/h, one min per run, at 7-8 min intervals. At the beginning of phase II, compost temperatures were approximately C. Air temperatures were set at 35 C and airflow was maintained at approximately 1 min every 10 min. Peak heating occurred approximately 36 h after filling the minibunkers. At this time, air temperatures were raised to 60 C for pasteurization by injecting full steam into the phase II room. Airflow was increased by operating fans min/h (2-3 min intervals every 10 min) to ensure the entire contents of the minibunker reached 60 C. Once all Chameleon temperature probes registered 60 C or 21

35 higher, timing began for pasteurization and continued for 2 h. At the end of the pasteurization cycle, air temperatures were lowered to 53 C and airflow was decreased to 8 min/h (3 min intervals every 10 min) to allow slow cooling of substrate. Airflow was adjusted as needed to obtain compost temperatures of C after pasteurization. Compost temperatures were decreased by decreasing air temperature and allowing the fan to run gradually, but did not drop below 46 C. Conditioning of the substrate at these temperatures allowed for ammonia clearing and growth of thermophilic organisms thought to be important for producing a selective substrate. On day 6, steam was turned off and fresh air was injected into the room. Fans were disconnected from the timer and allowed to run full time to cool the substrate to below 26C for spawning the following day. After phase II- only composting, actinomycete growth ( fire- fang ) was visible throughout the substrate (Fig. 2.5) Substrates were removed from minibunkers and processed through a mechanical turner to homogenize and further cool the material. 22

36 Figure 2.5. Milled corn stover (MCS) substrate following phase II- only composting in minibunker. Note white- tinged substrate Actinomycete ( fire- fang ) growth. 23

37 2.2.4 Experimental design and data analysis The experiment had three treatments of different substrate particle sizes arranged in a completely randomized design. Each particle size treatment had eight replicates, for a total of 24 bins. Analysis of variance was completed using JMP 8 (SAS Institute, Cary, NC) and treatment means were separated using Tukey- Kramer least significant difference test at p= Cropping trial Substrates were through- spawned in bins (57 x 44.5 x 22.8 cm) and placed in a production room at the MRC for 16 days for spawn run. After spawn run, substrates contained in bins of the same treatment were processed through a compost turner to fragment and homogenize the spawn run compost before the addition of a nutrient supplement (Fig. 2.6). Bins (57 x 44.5 x 22.8 cm) were refilled with 18.2 kg of substrate supplemented with Remo s All Season mushroom supplement (Remo s Mushroom Service) at a rate of 95 g/kg substrate (d/w basis). Bins were mechanically pressed to compact the colonized substrate. A 3 cm casing layer (with Lambert 901 CAC) was applied on top of the substrate and bins were placed in the production room at 90-95% relative humidity and C until mushroom primordia began to appear. Casing soil was hand watered daily with a rose- face hose attachment to maintain adequate moisture for mushroom production. 24

38 A B Figure 2.6. (A) Milled corn stover substrate (MCS), subjected to phase II- only composting, colonized by mushroom (A. bisporus) mycelium during spawn run. (B) Fragmentation of spawn run substrate using mechanical turner for homogenization and addition of supplement at casing Harvesting, determination of yield, biological efficiency and mushroom size Mushrooms were harvested closed (just prior to exposure of the lamellae) (Fig. 2.7), counted and weighed daily. The first flush was harvested beginning 19 days after the casing layer was applied. Harvesting continued for three flushes. Yield was determined as fresh mushroom weight divided by total production surface area and expressed as kg/m 2. Biological efficiency (BE) was calculated as the ratio of fresh mushroom weight (g) divided by the dry substrate weight and expressed as a percentage. Average mushroom size was calculated as fresh mushroom weight (g) divided by number of mushrooms per bin and expressed as g/mushroom. 25

39 Figure 2.7. Mushroom production (1st break) on milled corn stover substrate subjected to phase II- only composting in a minibunker. 2.3 Results Substrate particle sizes Particle size was directly correlated to screen size used for substrate milling (Table 2.1). Based on the sieves used by the Agricultural Analytical Services Laboratory to separate the substrate, particle sizes of all three MCS substrates fell into two size classes only: <3 mm and mm. Particle size distribution of all MCS substrates was smaller than particle sizes of conventional phase II mushroom compost (Table 2.1). Phase II mushroom compost is highly variable (visibly) in terms of particle size, due to the variety of ingredients used in its formulation. As expected, the smallest screen size (0.64 cm) produced the highest percentage (89.2%) of particles <3 mm. The largest screen size (1.91 cm) produced the lowest percentage (58.4%) of particles in the <3 26

40 mm particle size class. 79.9% of particles produced using the medium screen (1.27 cm) were <3 mm. Table 2.1. Particle size range of milled corn stover substrates (MCS) and phase II mushroom compost (Crop 0904). Particle Size Range (mm) B Substrate Type < > % Phase II compost MCS (small) 0.64 cm A MCS (medium) 1.27 cm A MCS (large) 1.91 cm A A Discharge screen on hammermill shredder/chipper. B Particle sizes were determined according to U.S. Composting Council (2010) Yield, biological efficiency (BE), and average mushroom size The highest mushroom yields (12.28 kg/m 2 ) and BEs (59.8%) were obtained from substrate prepared from the smallest screen size (0.64 cm), while the lowest mushroom yields (4.95 kg/m 2 ) and BEs (24.1%) were obtained from substrate prepared using the largest screen (1.91 cm) (Table 2.2). Mushroom size did not differ significantly among the three particle size treatments (Table 2.2). 27

41 Table 2.2. The effect of substrate particle size on mushroom yield, biological efficiency (BE %), and mushroom size (Crop 0904). Substrate particle size (screen size) Yield (kg/m 2 ) A BE (%) A Avg. Size (g) Small (0.64) a 59.8 a 11.8 a Medium (1.27) 9.22 b 44.9 b 12.1 a Large (1.91) 4.95 c 24.1 c 11.6 a A Yields, BE, and average mushroom size followed by the same letter within the same crop and column are not significantly different according to the Tukey- Kramer honestly significant difference (P= 0.05). 2.4 Discussion Highest mushroom yields depend on a fully colonized substrate with the ability of the mushroom mycelium to extract nutrients from the substrate. The ability of A. bisporus to use nutrients in the substrate may be dependent on the particle size of the substrate. Smaller particle sizes may allow for more extensive mycelial colonization due to greater overall particle surface area. Smaller substrate particle sizes, in turn lead to a more compact substrate so that higher dry weights can be filled into containers of similar size. Compared to commercial mushroom compost, MCS has a much smaller particle size distribution, when chopped with a bale chopper and milled through a 1.9 cm discharge screen. Particle size, in part, determines bulk density. Smaller particles allow for more dry weight to fit in a given volume, thereby increasing bulk density. However, particle size is not solely responsible for bulk density. The physical nature of substrate ingredients and length of composting also determine bulk density (Noble & Gaze, 1996). Bulk density in commercial mushroom composts may range from

42 kg/m 3 (Noble and Gaze, 1996). Bulk densities of the substrates used in this study were not measured; however, maximum substrate that could be filled into the mini- bunker (1 m 3 ) was about 320 kg, approximately one- half the bulk density of conventional mushroom composts. MCS did not adhere to tools, hands, and containers to the same extent as commercial mushroom compost. Therefore, it was cleaner to handle during filling, spawning, and emptying. One disadvantage of preparing MCS substrate is dust production, especially when milling through small discharge screens. Since the corn stover is dry- milled, the process produces a substantial amount of dust. This may lead to decreased air quality and a safety hazard in areas closely adjacent to the mill. Milling of corn stover should be done in ventilated areas or with a cyclone mill to prevent dust from accumulating in the mill area. Dust production may also create a flammable hazard when dust particle density in the air is high. The substrate produced from milling corn stover through the smallest discharge screen (0.64 cm) resulted in the highest yields and BE. As particle size increased, yields and BEs decreased. A second cropping trial would be helpful to confirm results presented here, as well as accurately measuring bulk density of the MCS substrates. Particle size of substrates prepared for other species of mushrooms affects mushroom yield (Royse & Sanchez, 2001). Size reduction methods (chopping vs. grinding) and particle size of rice straw were evaluated for oyster mushroom production (Zhang et al., 2002). Results of their study confirmed the results presented by Royse and Sanchez (2001). Yields and BE increased as particle size decreased to a certain point where particle size became too small and over- compaction occurred, 29

43 resulting in decreased air flow throughout the substrate. Zhang et al. (2002) also reported that oyster mushroom mycelium grew faster, approximately 5 days faster, on ground straw compared to chopped straw. In order to obtain bulk densities that compare favorably with commercial composts, corn stover may have to be milled using a screen smaller than 0.64 cm, keeping in mind that mushroom yield and BE may decrease as particle size becomes too small. Further experiments would be helpful to determine optimal particle size and bulk density for MCS substrate. 30

44 Chapter 3: Influence of nutrient supplementation at fill and at casing, temperature zones in minibunkers during phase II composting, and spawn type on yield, biological efficiency and mushroom size B 3.1 Introduction We have demonstrated that milled corn stover (MCS) subjected to phase II- only composting may yield a high quality crop of mushrooms (see chapter 2, 3, 5). However, yields from MCS do not yet equal yields from conventional mushroom compost. It is known that higher nitrogen content, up to a point, in mushroom substrate results in increased yields (Royse et al., 1982). Since MCS has relatively low nitrogen content (approximately % d/w fresh), supplementation of MCS with nitrogen- rich ingredients may be required in order to obtain yields and BEs comparable to conventional mushroom compost. Nutrient- rich supplements may be added at fill to MCS to increase substrate nitrogen content. Yield increases of up to 4.9 kg/m 2 have been reported when cottonseed oil was sprayed onto compost prior to phase II (Schisler & Patton Jr., 1970). Supplement addition before phase II composting may increase available nutrients, thereby increasing biological activity in the compost. Schisler and Patton Jr. (1970) observed increased temperatures during phase II composting and more rapid ammonia clearing in composts treated with cottonseed oil prior to phase II. In addition, mycelial growth was increased, leading to shorter spawn run (Schisler & Patton Jr., 1970) 31

45 Previous research on supplementing conventional compost at casing has lead to improvements in mushroom yields (Sinden & Schisler, 1962, 1966). However, similar work has not been done for MCS. Typically supplements are not added at casing due to the economically infeasible nature of through- mixing supplement into colonized substrate. However, supplements added to colonized substrate at casing may increase yields. Previous research has shown that kg carbohydrate supplements containing lipids gave the best results (Vedder, 1978). However, adding nutrient- rich supplements at casing can be a source of contamination. Supplements treated with certain fungicides have been used to decrease the probability of contamination by competitor fungi. Delayed- release nutrient supplements added at spawning are an alternative to adding supplement at casing, although this was not tested with regard to MCS substrate. Since our method of phase II- only composting using minibunkers is novel, not well tested, and not well understood, we hypothesized that temperature zones within the substrate contained in the minibunker during phase II- only composting may influence mushroom yield. During previous experiments, we noted inconsistent temperatures in the minibunker during phase II composting. The bottom of the minibunker, constructed of wire mesh covered with window screen, is open to the air, possibly making the substrate near the bottom cooler than the remainder of the substrate within the minibunker. Also, headspace between the top of the substrate and the minibunker ceiling may hold warm air emanating from the substrate. Spawn type is another factor that may affect mushroom yields. Recent developments in the spawn- making industry have helped to reduce the amount of time 32

46 required for spawn run. Synthetic spawns (Sylvan Matrix, Lambert Speedspawn ) are recent introductions into the mushroom industry. They contain no grain such as rye or millet; instead, they use various mixtures of agricultural byproducts (such as oat hulls and paper) and vermiculite to produce spawn. Synthetic spawns provide an advantage for mushroom cultivation in two ways: they eliminate a potential food source (free carbohydrates) for competitor fungi such as Trichoderma spp., and the much smaller pieces present more points of inoculation (POI) when mixed into mushroom compost. Increased POI lead to more rapid colonization of the substrate, reducing time needed for spawn run and also decreasing the time window for Trichoderma spp. and other competitor fungi to become established (Mark Spear, personal communication). The work presented herein attempts to increase mushroom yields and biological efficiencies (BE) from MCS by adding nutrients (based on nitrogen concentrations) at various phases of the mushroom production process. We evaluated several nutrient supplements added at fill, at spawning (data not presented), and at casing for their effects on mushroom yield, BE, and average mushroom size. Also, we evaluated the productivity of substrate contained within three temperatures zones of the minibunker. In addition, we evaluated synthetic- based and millet- type spawn for their effects on yield, BE and mushroom size. 33

47 3.2 Methods Substrate ingredients Baled corn stover, obtained from the Russell E. Larson Research and Education Center at Rock Springs, The Pennsylvania State University, was chopped using a bale chopper then milled using a shredder/chipper (McKissic Mighty Mac) fitted with a 1.91 cm discharge screen and then subsequently ground through a 0.64 discharge screen (see Fig 2.2B, Chapter 2). In order to raise substrate ph, a mixture of lime (2:1 ratio of pulverized limestone to hydrated lime and added at 1.5% (d/w of CS)) was added to the raw materials. Manganese sulfate (MnSO4) (Man- Gro DF, Tetra Micronutrients, Inc.) was added to the substrates to increase manganese levels to approximately 300 mg/kg. Distiller s grain, whole soybeans, soybean meal and cottonseed meal were obtained from Martin s Feed and Fertilizer, Coburn, PA. Whole soybeans were processed through a hammer mill at the Mushroom Research Center to provide ground soybean Experimental design and data analysis Three cropping experiments (0903, 0905, 0906) were conducted at the Mushroom Research Center (MRC), The Pennsylvania State University, University Park, PA to evaluate nutrient supplementation at fill, productivity of substrate in three temperature zones in the minibunker, and spawn type on mushroom yield and BE. Crop 0903 was a completely randomized 2 x 3 x 2 factorial design with nine replicates 34

48 per treatment. Crop 0903 contained two substrate types, three temperature zones in the substrate within a minibunker during phase II composting, and two supplement levels at casing. Crop 0905 was a completely randomized 3 x 3 factorial design with four replicates per treatment. There were three substrate types and three supplements at casing. Crop 0906 was a completely randomized 3 x 2 x 3 factorial design with nine replicates per treatment. There were three substrate types, two spawn types, and three supplements at casing. An analysis of variance was conducted to determine level of significance and means were separated using Tukey- Kramer Honestly significant difference (p<0.05) (JMP, 2009) Cropping trials For Crop 0903, distiller s grain (10% d/w) was added at fill to MCS and then subjected to phase II- only composting in a minibunker. MCS alone was subjected to phase II- only composting in a separate minibunker. In preliminary experiments, we determined that substrate temperatures in minibunkers were not consistent throughout the minibunker during phase II composting. Therefore, we assigned temperature zones, each consisting of 1/3 of the height of the substrate in the minibunker (Fig. 4.1). Zones were labeled high - (top 1/3 of substrate in the minibunker), middle - (middle 1/3), and low - (bottom 1/3). At the completion of phase II, substrate from each temperature zone was processed separately through a mechanical turner to homogenize the substrate prior to 35

49 spawning. Substrate was through- spawned (Lambert 901, 1.6% w/w), supplemented (Lambert T6, 12% d/w) and placed into large (22.5 x 17.5 x 22.8 cm) bins. Substrates were compacted with a hydraulic press in large bins then placed in a production room at the MRC for 14- day spawn run. Following spawn run, substrates of the same treatment were bulk processed through a mechanical turner for fragmentation and to allow for the addition of supplement at casing. Each treatment was supplemented with 5% or 10% (d/w) Remo s All Season supplement, placed into small (22.8 x 18 x 23.5 cm) bins and hand- pressed. A casing layer (3 cm) consisting of neutralized peat moss and casing inoculum (Lambert 901 ) was applied and bins were placed in a production room at 90-95% relative humidity. The casing layer was watered daily with a rose- face hose attachment to maintain adequate moisture for mushroom production. H M L Figure 3.1. Assignment of temperature zones in substrate during phase II- only composting in minibunkers. H = high, M = middle, L = low. For Crop 0905, three substrates were formulated and consisted of 1) MCS only, 2) MCS + ground soybean (10% d/w), and 3) MCS + soybean meal (10% d/w). All three 36

50 substrates were subjected to phase II- only composting. Substrates were processed through a mechanical turner for homogenization and through- spawned (Lambert 901, 1.6% w/w) and supplemented (Lambert T6, 6% d/w). Large bins were placed in a production room at the MRC for 14- day spawn run. Following spawn run, substrates of the same treatment were processed through a mechanical turner. Each treatment was supplemented with 4% (d/w) Remo s All Season, Lambert T6, or a 50:50 mixture of Remo s All Season and Lambert T6, placed into small bins and hand- pressed. A casing layer (3 cm) consisting of neutralized peat moss and casing inoculum (Lambert 901) was applied and bins were placed in a production room at 90-95% relative humidity. The casing layer was watered daily with a rose- face hose attachment to maintain adequate moisture for mushroom production. For Crop 0906, three substrates were formulated and consisted of 1) MCS only, 2) MCS + distiller s grain (5% d/w), and 3) MCS + cottonseed meal (5% d/w). All three were subjected to phase II- only composting in minibunkers. Substrates were processed through a mechanical turner separately for homogenization. Treatments were through- spawned using millet- type or synthetic Matrix (Sylvan A15, 1.6% w/w), supplemented (Lambert T6, 6% d/w), placed into large bins and mechanically compacted using a hydraulic press. Bins were placed in a production room at the MRC for 14- day spawn run. Following spawn run, substrates of the same treatment were processed through a mechanical turner. Each treatment was supplemented with Remo s All Season, Lambert T6, or Lambert T7, 5% (d/w), placed into small bins and hand- pressed. A casing layer (3 cm) consisting of neutralized peat moss and casing inoculum (Lambert 901 ) was applied and bins were placed in a production room at 90-95% relative 37

51 humidity. The casing layer was watered daily with a rose- face hose attachment to maintain adequate moisture for mushroom production Harvesting and determination of yield, biological efficiency and size Mushrooms (with closed veil) were harvested, counted and weighed daily. At the end of 3 th flush, yield, biological efficiency (BE), and average mushroom size were determined. Yield was expressed as kg/m 2 and BE was determined by dividing the weight of fresh mushrooms harvested (g) by the dry weight of the substrate (g) and expressed as a percentage. Average mushroom size (g/mushroom) was calculated as fresh mushroom weight divided by the number of fresh mushrooms harvested. 3.3 Results Crop 0903 MCS- only substrate contained less nitrogen than MCS with distiller s grain (10% d/w) (Table 3.1). However, distiller s grain addition alone did not have a significant effect on mushroom yield or BE (Table 3.2). Significant sources of variation for yield and BE included temperature zones in substrate within minibunkers during phase II composting and amount of Remo s All Season supplement added at casing (Table 3.2). Significant interactions were observed between and among all factors. (Table 3.2). Yields ranged from 0.1 kg/m 2 to 17.1 kg/m 2, while BEs ranged from 0.3% to 70.3% (Table 3.3). The highest yields (17.1 kg/m 2 ) and BE (70.3%) were obtained from MCS 38

52 supplemented with distiller s grain (10% d/w) at fill and selected from the middle zone in the minibunker and supplemented with 5% (d/w) Remo s All Season at casing (Table 3.3). Lowest yields (0.1 kg/m 2 ) and BE (0.3%) were obtained from MCS supplemented with distiller s grain (10% d/w) at fill, selected from the lower zone in the minibunker and supplemented with 10% (d/w) Remo s All Season at casing. Table 3.1. Substrate type and moisture and nitrogen content of milled corn stover (MCS) substrates at fill for Crop Substrate Moisture (%) Total Nitrogen (% d/w) MCS MCS + DG A A DG = Distiller s grain (10% d/w) Table 3.2. Probabilities > F from analysis of variance for substrate type, temperature zone and Remo s added at casing tested for yield and biological efficiency (BE %). Source df Yield A BE (%) A Substrate (S) Temperature zone (TZ) 2 <.0001 <.0001 S x TZ 2 <.0001 <.0001 Remo casing (RAC) 1 <.0001 <.0001 S x RAC TZ x RAC S x TZ x RAC A Values of less than 0.05 were considered significant according to Fisher s LSD. 39

53 Table 3.3. Effects of distiller s grain added at fill, temperature zone of substrate within mini- bunker and supplement added at casing on yield and biological efficiency (BE %) of A. bisporus. Trt. No. Supplement Temperature Zone B Remo Yield (kg/m 2 ) C BE (%) C casing (% d/w) 1 None H bc 38.9 b 2 None H de 7.2 cd 3 None M a 64.3 a 4 None M bc 33.9 b 5 None L c 29.4 b 6 None L e 1.5 d 7 DG A H ab 59.7 a 8 DG H cd 26.6 bc 9 DG M a 70.3 a 10 DG M de 6.6 cd 11 DG L cd 22.7 bc 12 DG L e 0.3 d A DG = distiller s grain, added at fill. B H = high, M = middle, L = low. C Means followed by the same letter within the same column are not significantly different according to Tukey s honestly significant difference (HSD) (P = 0.05). Yields from the different temperature zones ranged from a low of 3.3 kg/m 2 (lower zone) to a high of 10.5 kg/m 2 from the middle zone (Table 3.4). The highest yield differences occurred during the first and second breaks, where the middle zone produced 6.7 kg/m 2 compared to 4.8 kg/m 2 from the high zone and 1.2 kg/m 2 from the low zone in the first break alone (Table 3.4). No significant differences occurred in the third break. However, the middle zone yielded significantly higher than the lower zone in the fourth break (Table 3.4). BE from the different temperature zones ranged from a low of 13.5% from the lower zone to a high of 43.8% from the middle zone (Table 3.4). BE of substrate in the middle zone did not differ significantly from that of the high zone (Table 3.4). Treatments that were supplemented with 5% (d/w) Remo s All Season at 40

54 casing resulted in higher yields (10.9 kg/m 2 ) and BEs (47.5%) than those supplemented with 10% (d/w) Remo s at casing (3.0 kg/m 2, 12.7%, respectively) (Table 3.5). Table 3.4. Means and groupings from analysis of variance for temperature zones within substrate in mini- bunker during composting for yield and biological efficiency (BE %). Temperature No. Reps Yield (kg/m 2 ) A BE zone Total 1 st Break 2 nd Break 3 rd Break 4 th break (%) A H b 4.8 a 1.5 b 0.2 a 0.5 b 33.1 a M a 6.7 a 2.4 a 0.5 a 1.2 a 43.8 a L c 1.2 b 0.9 b 0.2 a 0.7 ab 13.5 b A Yields and BE (%) followed by the same letter within the same column are not significantly different according to the Tukey- Kramer honestly significant difference (P = 0.05). Table 3.5. Means and groupings from analysis of variance for supplementation at casing for yield and biological efficiency (BE %). Remo No. Reps Yield (kg/m 2 ) A BE casing (%) Total 1 st Break 2 nd Break 3 rd Break 4 th break (%) A a 5.7 a 2.6 a 0.6 a 1.3 a 47.5 a b 2.0 b 0.7 b 0.04 b 0.2 b 12.7 b A Yields and BE (%) followed by the same letter within the same column are not significantly different according to the Tukey- Kramer honestly significant difference (P = 0.05) Crop 0905 MCS- only substrate contained less nitrogen (0.9%) at spawning than MCS supplemented with ground soybean or soybean meal (10% d/w) (1.5% N for both) (Table 3.6). Moisture contents of the substrates ranged from %. Adding supplement at fill and at casing significantly increased yield and BE (Table 3.7). Also, there was a significant interaction between the two factors (Table 3.7). 41

55 Table 3.6. Substrate type and moisture and nitrogen contents at fill, spawning and casing for milled corn stover (MCS) (Crop 0905). Substrate A Moisture (%) B Total Nitrogen Casing MCS MCS + GS MCS + SM A GS = ground soybean (10% d/w), SM = soybean meal (10% d/w). B At spawning. Table 3.7. Probabilities > F from analysis of variance for supplement at filling and supplement type and rate at casing tested for yield, biological efficiency (BE %), and mushroom size for A. bisporus (Crop 0905). Source df Yield B (kg/m 2 ) BE (%) B Substrate (S) 2 < < casing (SAC) A 2 < < S x SAC 4 < < ARemo s All Season, Lambert T6 with Mertect, Lambert T7 with Mertect (4% d/w). B Values less than 0.05 were considered significant according to Fisher s LSD. The highest yields (14.0 kg/m 2 ) and BE (71.6%) were observed from MCS with 10% ground soybean added at fill and supplemented with Lambert T6 (4% d/w) at casing (Table 3.8). Across all treatments, yield did not differ with regard to substrate type, however we observed yield differences for individual breaks (Table 3.9). Substrate type did not affect BE (Table 3.9). Lambert T6 gave the best yields (11.9 kg/m 2 ) and BE (60.9%) when used alone at casing (Table 3.10). Remo s All Season supplement, when used alone at casing, resulted in lower yields and BE compared to Lambert T6 and a 50:50 mixture of T6 and Remo s. Yields from substrates supplemented with a 50:50 mixture of Remo s and T6 at casing were not significantly different from either of the supplements when used alone (Table 3.10). Lambert T6, 42

56 when used alone, resulted in higher yields than Remo s alone for total yield and all breaks except the third break (Table 3.10). Table 3.8. Effects of ground soybeans and soybean meal added at fill to milled corn stover and supplement type at casing (% d/w) on yield, biological efficiency (BE %), and mushroom size for A. bisporus (Crop 0905). BE (%) C Size (g) C Trt. Supplement Remo s at T6 at casing B Yield No. at filling A casing B (kg/m 2 ) C 1 None bc 57.1 bc 13.3 a 2 None c 48.7 c 12.4 ab 3 None bc 53.9 bc 12.7 ab 4 GS c 47.8 c 12.8 ab 5 GS a 71.6 a 12.4 ab 6 GS bc 56.9 bc 13.7 a 7 SM d 33.3 d 11.7 b 8 SM ab 62.3 ab 12.6 ab 9 SM bc 52.3 bc 13.1 ab A GS = Ground Soybean, 8.16 kg or SM = Soybean Meal, 8.16 kg added at fill. B Remo s All Season with Topsin (% d/w), Lambert T6 with Mertect (% d/w). C Means followed by the same letter within the same column are not significantly different according to Tukey s honestly significant difference (HSD) (P = 0.05). Table 3.9. Means and groupings from analysis of variance for supplementation of milled corn stover substrate at fill using ground soybean and soybean meal for yield and biological efficiency (BE %) for A. bisporus (Crop 0905). Supplement A No. Reps Yield (kg/m 2 ) B BE (%) Total 1 st Break 2 nd Break 3 rd Break None a 7.7 a 2.3 a 0.6 a 53.2 a GS a 6.6 b 4.2 b 0.7 a 58.8 a SM a 5.3 c 3.6 ab 0.7 a 49.3 a A GS = Ground soybean (10% d/w) or SM = Soybean meal (10% d/w), added at fill. B Means followed by the same letter within the same column are not significantly different according to Tukey s honestly significant difference (HSD) (P = 0.05). 43

57 Table Means and grouping from analysis of variance for supplementation type at casing for yield and biological efficiency (BE %) for A. bisporus (Crop 0905). Supplement A No. Yield (kg/m 2 ) B BE (%) B Reps Total 1 st Break 2 nd Break 3 rd Break Lambert T a 7.5 a 3.8 a 0.6 a 60.9 a Remo s b 5.4 b 3.0 b 0.6 a 46.1 b T6 + Remo s ab 6.6 ab 3.2 b 0.8 a 54.3 ab A Supplement added at 4 % d/w. T6 + Remo s = 50:50 mixture. B Means followed by the same letter within the same column are not significantly different according to Tukey s honestly significant difference (HSD) (P = 0.05) Crop 0906 Supplements (distiller s grain, cottonseed meal) added to MCS at fill increased total nitrogen content from 0.8% to 1.1% or 1.4% (Table 3.11). MCS + DG (10% d/w) from Crop 0904 had 1.37% N (Table 3.11), while MCS +DG (5% d/w) contained 1.95% N. Sampling or analysis error may have occurred in MCS + DG (5% d/w), since calculations indicate that N content should be less than 1.5% for this substrate. Supplement added at fill significantly increased mushroom yield, BE and average mushroom size (Table 3.12). Synthetic- type spawn significantly increased mushroom yield and BE compared to millet- based spawn (Table 3.12). Supplement type used at casing significantly increased average mushroom size (Table 3.12). Significant interactions occurred between supplement added at fill and spawn type and supplement added at fill and supplement used at casing with regard to mushroom yield and BE (Table 3.12). A significant interaction effect was observed between spawn type and supplement added at casing with regard to mushroom size (Table 3.12). 44

58 Table 3.11 Substrate type, moisture content and total nitrogen at fill and at spawning for Crop Substrate A Moisture B Total Nitrogen Content Spawning MCS MCS + DG MCS + CM A MCS = milled corn stover, DG = distiller s grain (5% d/w), CM = cottonseed meal (5% d/w) B At spawning Table Probabilities > F from analysis of variance for supplement at fill, spawn type, and supplement added at casing tested for yield, biological efficiency (BE %), and mushroom size for A. bisporus (Crop 0906). Source df Yield (kg/m 2 ) A BE (%) A Size A fill (SAF) 2 < < Spawn type (ST) 1 < < SAF x ST casing (SAC) SAF x SAC ST x SAC SAF x ST x SAC A Values of less than 0.05 were considered significant according to Fisher s LSD. Highest mushroom yields (23.6 kg/m 2 ) were obtained from MCS + cottonseed meal (CM) (5% d/w) spawned with synthetic- type Matrix spawn and Remo s added at casing (Table 3.13). However, this treatment did not differ significantly from a majority of the MCS + CM substrate treatments (Table 3.13). BEs in excess of 100% were observed in Crop 0906 when cottonseed meal was added at fill, Matrix was used for spawn, and Lambert T7 or Remo s All Season was added at casing (Table 3.13). The poorest treatments with regard to yield and BE were MCS alone combined with 45

59 millet spawn (Table 3.13). Average size did not differ significantly for most of the treatments (Table 3.13). Table Effects of adding cottonseed meal and distiller s grain at fill, spawn type, and supplement type at casing (%d/w) on yield, biological efficiency (BE %), and mushroom size for A. bisporus (Crop 0906). Yield (kg/m 2 ) D BE (%) D Size Trt. No. Supplement Spawn Supplement at Fill A type B at casing C (g) D 1 None Millet T6 5.5 gh 25.2 gh 11.9 ab 2 None Millet T7 4.6 h 21.3 h 9.5 b 3 None Millet Remo s 5.3 gh 24.6 gh 12.6 ab 4 None Matrix T6 8.7 fgh 40.2 fgh 11.5 ab 5 None Matrix T7 9.7 efgh 44.8 efgh 10.9 ab 6 None Matrix Remo s 10.5 efg 48.6 efg 11.0 ab 7 DG Millet T bcd 78.8 bcd 10.9 ab 8 DG Millet T bcd 77.9 bcd 10.7 ab 9 DG Millet Remo s 13.7 def 63.4 def 11.7 ab 10 DG Matrix T cde 71.4 cde 12.8 ab 11 DG Matrix T bcd 80.4 bcd 12.0 ab 12 DG Matrix Remo s 14.5 def 67.0 def 10.0 ab 13 CM Millet T bcd 81.1 bcd 13.4 a 14 CM Millet T abcd 83.9 abcd 12.0 ab 15 CM Millet Remo s 18.6 abcd 86.1 abcd 12.9 a 16 CM Matrix T abc 94.7 abc 12.5 ab 17 CM Matrix T ab ab 12.2 ab 18 CM Matrix Remo s 23.6 a a 12.8 ab A DG = Distiller s grain (5% d/w) or CM = Cottonseed meal (5% d/w), added at fill. B Sylvan A15 Millet Spawn, Sylvan A15 Matrix Spawn. C Lambert T6 and T7 supplement treated with Mertect. Remo s All Season Supplement D Means followed by the same letter within the same column are not significantly different according to Tukey s honestly significant difference (HSD) (P = 0.05). Cottonseed meal (5% d/w) addition at fill resulted in the highest yields (20.1 kg/m 2 ) across all treatments, followed by distiller s grain (5% d/w) addition (15.8 kg/m 2 ) and MCS alone (7.4 kg/m 2 ) (Table 3.14). BE followed a similar pattern (Table 3.14). 46

60 Synthetic- type Matrix spawn resulted in higher yields (15.8 kg/m 2 ) across all treatments compared to millet- type spawn (13.0 kg/m 2 ) (Table 3.15). BE of substrates spawned with Matrix were higher (73.0%) than those spawned with millet- type spawn (60.0%) (Table 3.15). Yield differences with regard to spawn type occurred only during first and second break (Table 3.15). Table Means and groupings from analysis of variance for supplementation of milled corn stover substrate at fill using distiller s grain and cottonseed meal for yield and biological efficiency (BE %) for A. bisporus (Crop 0906). Supplement A No. Reps Yield (kg/m 2 ) B BE (%) B Total 1 st Break 2 nd Break 3 rd Break 4 th Break None a 4.3 a 1.7 a 1.0 a 0.4 a 31.1 a DG b 10.5 b 4.1 b 0.8 a 0.4 a 73.1 b CM c 14.5 c 4.1 b 0.7 a 0.7 a 92.9 c A DG = Distiller s grain (5% d/w) or CM = Cottonseed meal (5% d/w), added at fill. B Means followed by the same letter within the same column are not significantly different according to Tukey s honestly significant difference (HSD) (P = 0.05). Table Means and groupings from analysis of variance of spawn type on mushroom yield and biological efficiency (BE %) for A. bisporus (Crop 0906). Spawn Type No. Reps Yield (kg/m 2 ) A BE (%) A Total 1 st Break 2 nd Break 3 rd Break 4 th Break Millet a 7.9 a 3.9 a 0.8 a 0.4 a 60.0 a Matrix b 11.7 b 2.8 b 0.8 a 0.5 a 73.0 b A Means followed by the same letter within the same column are not significantly different according to Tukey s honestly significant difference (HSD) (P = 0.05). 47

61 4.4 Discussion The addition of distiller s grain, cottonseed meal and ground soybean to MCS at fill increased yields. This is most likely due to increased N content of the substrate. Microorganisms that help to break down complex carbohydrates in the substrate require nitrogen for growth. It was noted that substrates supplemented at fill produced more ammonia during composting and took more time to clear ammonia during the conditioning phase. Some ammonia could still be detected at spawning when soybean meal was added (10% d/w). Excess ammonia in this substrate at spawning may have been the cause of lower yields observed for this substrate treatment compared to MCS alone. Preliminary results (not presented) suggested that 6% Lambert T6 gave higher yield when compared to 12% T6 or Remo s used at spawning. Therefore, we used that level for all subsequent crops. Results from Crop 0905 suggested that Lambert T6 (4% d/w) was best suited for supplementation at casing. Most mushroom growers do not add additional supplement at casing. Current research is being conducted on delayed- release nutrient supplements that may be applied to MCS substrate, possibly eliminating the need for supplementing at casing. Fragmenting the substrate following spawn run has also been shown to increase yields, so this may have influenced yields and BE aside from adding additional supplement. Currently, the process of fragmentation is also not economically feasible for commercial mushroom operations. So these results may be more applicable to small- scale specialty growers seeking to produce white button mushrooms. 48

62 Temperature zones existed in substrate within minibunkers subjected to phase II- only composting. We attempted to eliminate these zones in subsequent experiments by aiming for consistent temperatures (by adjusting airflow) throughout the substrate during phase II composting. Also, all substrates were processed through a mechanical turner after phase II- only composting to ensure that substrate was homogenized. These temperature zones exist primarily because our minibunker set- up was not designed specifically for phase II composting, but rather a modified phase I composting to eliminate odors (Pecchia, 2000). Upon scaling up in a tunnel designed specifically for phase II composting, these zones may be insignificant or non- existent. Synthetic spawn resulted in higher yields and BE compared to millet- type spawn. This is most likely due to increased POI in the substrate. Substrate appeared more rapidly colonized by the synthetic spawn compared to millet- type spawn. We did not observe contamination in either treatment, but that is not to say that the synthetic spawn did not offer more protection against such diseases as green mold. Influence of synthetic spawn rate is reported in Chapter 4. Spawn and supplement type and rate used for mushroom production are crucial to the performance and resulting yields and BE of the substrate. Upon developing a new substrate such as MCS, these factors should be addressed to determine their effects on yield. MCS may offer growers and hobbyists that may have been unable to generate typical mushroom compost, a new method of producing white button mushrooms. At least one grower is currently using corn stover as a raw material ingredient in compost formulations. Commercial mushroom production has always relied on a composted substrate. Until yields are as good or greater than those produced on conventional 49

63 mushroom substrate, the use of MCS for mushroom production may be limited. Consequences of conventional mushroom compost production, such as odor emissions, costly ingredients, and time required for composting will continue to drive the industry to develop more efficient composting processes. The work reported here may be one step in that direction. 50

64 Chapter 4: The influence of spent mushroom substrate addition to milled corn stover compost and spawn rate on mushroom yield, biological efficiency, and size 4.1 Introduction Growers have sought beneficial uses for spent mushroom substrate (SMS) for more than two decades. Currently, SMS is being used as an ingredient in mulches to prevent plant diseases and artillery fungi and as a soil amendment to increase nitrogen and organic matter. However, in Southeastern Pennsylvania more SMS is produced than can be used beneficially. Commercial SMS has about % nitrogen (dry weight basis) (Fidanza and Beyer, 2009), so it is a potential, no- cost source of this nutrient that may be used in compost. It is estimated that at least 36 million m 3 of SMS has to be moved off mushroom farms in the United States each year (AMI, 2005). If nitrogen and other nutrients in SMS could be reclaimed by re- using SMS as an ingredient in fresh compost, it may reduce the need to dispose of large amounts of this material. In experiments conducted at the Campbell Institute for Agricultural Research, Murphy (1972) was able to add up to 50% SMS, along with 40% corn cobs and 10% cottonseed meal in a synthetic substrate that was subjected to phase II- only composting. Additions of up to 20% SMS to regular compost at fill yielded no differently than phase II compost (D.J. Royse, personal communication). Based on 51

65 Murphy s (1972) successes, we were interested in determining if SMS could be used as an ingredient in milled corn stover (MCS) substrate. Growers also are continuing to seek ways to shorten the crop cycle. A shortened crop cycle increases the number of crops a grower can complete each year. Recent developments in the spawn- making industry have helped to reduce the amount of time required for spawn run. Synthetic spawns (Sylvan Matrix, Lambert Speedspawn ) are recent introductions into the mushroom industry. They contain no grain such as rye or millet; instead they use various mixtures of agricultural byproducts (such as oat hulls and paper) and vermiculite to create spawn that looks like a mixture of casing inoculum (CI) and grain spawn. Synthetic spawns provide an advantage in mushroom cultivation in two ways: they eliminate a food source (free carbohydrates) for competitor fungi such as Trichoderma spp., and the much smaller particles create more points of inoculation (POI) when mixed into mushroom compost. Increased POI leads to more rapid colonization of the substrate, reducing time needed for spawn run and also decreasing the time window for Trichoderma spp. and other competitor fungi to become established (Mark Spear, personal communication). In this work we sought to: 1) assess the use of SMS as an ingredient in MCS substrate prepared by phase II- only composting, and 2) evaluate the effect of various rates of synthetic (Sylvan Matrix ) spawn used in MCS on yield, BE and mushroom size. 52

66 4.2 Methods Substrate Baled corn stover (CS), obtained from the Russell E. Larson Research and Education Center at Rock Springs, The Pennsylvania State University, was chopped using a bale chopper then milled using a shredder/chipper (McKissic Mighty Mac) fitted with and 1.91 cm discharge screen and then subsequently ground through a 0.64 discharge screen (see Fig 2.2). Pasteurized (60C for 8 h) SMS including the casing layer was obtained from the Mushroom Test Demonstration Facility, The Pennsylvania State University. A mixture of lime (2:1 ratio of pulverized limestone to hydrated lime and added at 1.5% (d/w of CS)) was added to the raw materials to raise ph. Distiller s grain, added to adjust nitrogen content of the different substrates to the same level, was obtained from Martin s Feed and Fertilizer, Coburn, PA. Manganese sulfate (MnSO4) (Man- Gro DF, Tetra Micronutrients, Inc.) was added to the substrates to increase manganese levels to approximately 300 mg/kg. A white hybrid strain (Sylvan Matrix A15) of A. bisporus was selected as the cultivar for these experiments Experimental design and data analysis Phase II- only composting was done at the Mushroom Test Demonstration Facility while the cropping trial was conducted at the Mushroom Research Center at 53

67 The Pennsylvania State University. This crop was designed as a 3 x 2 factorial in a completely randomized design with six replicates per treatment. The experiment contained three substrate formulas and two spawn rates. Mushrooms were harvested for four flushes (17-40 days from day of casing). JMP (SAS Institute, 2009) was used to conduct an analysis of variance (ANOVA) and mean separations were completed using the Tukey- Kramer Honestly Significant Difference (HSD) Mushroom cropping trial Three substrate formulas were developed using various mixtures of CS, SMS, distiller s grain, and lime (Table 5.1) (Fig 5.1). Distiller s grain was added to each substrate separately in differing amounts to adjust nitrogen content of all substrates to 1.3%. The three formulas were moistened to approximately 75% moisture. The substrates were filled into separate mini- bunkers and subjected to a modified phase II composting process (see chapter 2). Mushrooms were produced in plastic bins (56 x 44 x 24 cm) filled with 20.4 kg of substrate. Two spawn rates (w/w) were used: 0.75% and 1.5% for each formula. Lambert T6 supplement (306 g, 6% d/w) was mixed into the substrate at spawning prior to filling bins. After a 14- day spawn run, compost was cased with a layer (3 cm) of neutralized peat moss containing Sylvan A15 CI. During case- hold, water was applied daily to maintain moisture contents near field capacity and relative humidity was maintained above 90%. 54

68 Table 4.1. Percentages of various ingredients in three formulas of phase II- only substrates for Crop 1001b. Ingredient A Formula CS SMS DG Lime B % A CS = corn stover, SMS = spent mushroom substrate, DG = distiller s grain, Lime = 2:1 pulverized limestone:hydrated lime B Oven dry weight basis Figure 4.1. Physical appearance of (from left to right) milled corn stover formulas 1, 2, and 3. Formula compositions are listed in Table