Loehr Thesis

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

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

Paul H. Heinemann
Professor of Agricultural and Biological Engineering

C. Peter Romaine

Frederick E. Gildow
Head of the Department of Plant Pathology

*Signatures on file in the Graduate School

ii
ABSTRACT
Agaricus bisporus is cultivated commercially on a composted substrate that

includes raw materials such as straw-bedded horse manure, hay, poultry litter,
distillers 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
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/m2) and BE (59.8%) compared to medium (9.22 kg/m2, 44.9%)
and larger (4.95 kg/m2, 24.1%) particle size substrates.
Supplements (cottonseed meal, ground soybean, distillers 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/m2) and BE (92.9%) than distillers 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 Remos 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 Remos All
Season supplement used at casing.
iv
Use of synthetic-type spawn (Sylvan Matrix) resulted in higher yields (15.8

kg/m2) and BE (73.0%) than millet-based spawn (13.0 kg/m2, 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/m2) compared to the lower
spawn rate (12.7 kg/m2), 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.
TABLE OF CONTENTS

List of Figures
ix
List of Tables ..
Acknowledgements
xii
Chapter 1: General introduction
1.1 Introduction
1.2 Economic importance of Agaricus bisporus
1.3 Cultivation history
1.4 Cultivation methods .
1.5 Nutritional requirements .
1.6 Supplementation
1.7 Environmental impact
1.8 Raw materials used for cultivation
1.9 Shortening or eliminating the composting process
1.10 Objectives .
13

substrate ..
15
2.1 Introduction ..
15
2.2 Methods
17
2.2.1 Ingredients ..
17
2.2.2 Substrate preparation .
18
2.2.3 Phase II-only composting ..
21
2.2.4 Experimental design and data analysis
24
2.2.5 Cropping trial
24
and mushroom size .
25
Chapter 2: Phase II-only composting of milled corn stover for use as
2.2.6 Harvesting, determination of yield, biological efficiency
vi
2.3 Results .
26
2.3.1 Substrate particle sizes .
26
2.3.2 Yield, biological efficiency, and mushroom size ....
27
2.4 Discussion ..
28
and spawn type on yield, biological efficiency and mushroom size
31

Chapter 3: Influence of nutrient supplementation at fill and at casing,
temperature zones in minibunkers during phase II composting,

3.1 Introduction
31
3.2 Methods
34
3.2.1 Substrate ingredients ..
34
34
3.2.3 Cropping trials
35
3.2.4 Harvesting and determination of yield, biological
efficiency, and size .
38
3.3 Results
38
3.3.1 Crop 0903
38
3.3.2 Crop 0905
41
3.3.3 Crop 0906
44
3.4 Discussion ..
48
Chapter 4: The influence of spawn rate and SMS addition to milled corn
stover compost on yield, biological efficiency, and mushroom size
51
4.1 Introduction
51
4.2 Methods
53
4.2.1 Substrate ..
53
53
4.2.2 Mushroom cropping trial ..
54
4.2.4 Harvesting and determination of yield, biological

efficiency and mushroom size .
56
4.3 Results ..
56
vii
4.3.1 ANOVA ..
56
4.3.2 Spawn rate .
57
4.3.3 SMS additions ..
58
4.4 Discussion ..
59
Chapter 5: Summary and future work .
61
5.1 Summary .
61
5.2 Future work .
62
64
References

viii
LIST OF FIGURES

Fig. 2.1. WIC bale chopper used for pre-chopping of corn stover used for

preparation of milled corn stover substrate ... 18
Fig. 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) .
19
Fig. 2.3. (A) Wetting and mixing of milled corn stover (MCS) by hand for use as
substrate. (B) Compacting MCS in minibunker. .
20
Fig. 2.4. (A) HOBO Water Temp Pro v2 data logger. (B) Location of HOBO
data loggers within minibunker....
21
Fig. 2.5. Milled corn stover (MCS) substrate following phase II-only composting

in minibunker. Note white-tinged substrate (Actinomycete fire-fang
growth) ..
23
Fig. 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 .
25
Fig. 2.7. Mushroom production on milled corn stover substrate subjected to

phase II-only composting in a minibunker
26
Fig. 3.1. Assignment of temperature zones in substrate during phase II-only

composting in minibunkers. H = high, M = middle, L = low. .
36
Fig. 4.1. Physical appearance of (from left to right) milled corn stover formulas

1, 2, and 3. Formula compositions are listed in Table 5.1............................ 55
ix

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 0903 ....
39
Table 3.2. Probabilities > F from analysis of variance for substrate type,
temperature zone and Remos added at casing tested for yield
and biological efficiency (BE %) ..
39
Table 3.3. Effects of distillers 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 ....
40
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).....
43
of milled corn stover substrate at fill using ground soybean and

soybean meal for yield and biological efficiency (BE %) of A. bisporus

(Crop 0905)
43
Table 3.10. 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 0906 ...
45
Table 3.12. 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 3.13. Effects of adding cottonseed meal and distillers 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
of milled corn stover substrate at fill using distillers grain and
cottonseed meal for yield and biological efficiency (BE %) for A.
bisporus (Crop 0906)
47
Table 3.15. 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,
(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,
(Crop 1001b).

57
xi
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

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
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 1900. 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).

1.2 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 2009-10 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 2009-10 season. California ranks
second (14%) in the U.S. in terms of mushroom production, with a value of $184 million
in 2009-10. Production of A. bisporus in 2009-10 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
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/m2
of bed surface, in 1964, this increased to about 10.8 kg/m2 of bed surface (Snetsinger,
1970). Today, farmers can expect average yields of 29 kg/m2 of bed surface.
Mushroom Research at Penn State continues to provide solutions to industry problems.
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 63C, although, they can range between 25 and 80C, 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
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,
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 Mn2+ 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, distillers grain (24% protein, 0.8% fat) is often added to
stimulate microbial communities to initiate organic material breakdown.
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
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, brewers grains and gypsum (John DAmico,
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
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
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 Murphys (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/m2. 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
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
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/m2. 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/m2 (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
substrate. Then the substrate may be spawned normally as demonstrated by Sanchez

and Royse (2009, Sanchez et al., 2008). The mechanisms of S. thermophylums ability to
impart selectivity are not fully known (Sanchez & Royse, 2009). Yields of 21.6 kg/m2
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).

1.10 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
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
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 distillers
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
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 (1.7-2.5% 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
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 m2)(expressed as kg/m2). 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
Murphys 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/m2), biological efficiency (%) and mushroom size
(g/mushroom) is presented and discussed.

2.2 Methods
2.2.1 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
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

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 (250-400 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 (Mn2+) 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 Mn2+ 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

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
Figure 2.4. (A) HOBO Water Temp Pro v2 data

logger. (B) Location of HOBO data loggers
within minibunker.

2.2.3 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 18-20
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 12-
15 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
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 53-54 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

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

23

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=0.05.

2.2.5 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 Remos All Season mushroom supplement (Remos 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 18-19C 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

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.

2.2.6 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/m2. 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

Figure 2.7. Mushroom production (1st break) on milled corn stover substrate subjected to
phase II-only composting in a minibunker.

2.3 Results
2.3.1 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 3-9.5 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
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
<3
3-9.5
9.5-16
16-25
25-50
>50

------------------------------------%----------------------------------------
Phase II compost
27.3
16.8
14.7
34.4
6.9
0
MCS (small) 0.64 cmA
89.2
11.8
0
0
0
0
MCS (medium) 1.27 cmA
79.9
21.1
MCS (large) 1.91 cmA
59.4
41.6
A Discharge screen on hammermill shredder/chipper.

B Particle sizes were determined according to U.S. Composting Council (2010)

2.3.2 Yield, biological efficiency (BE), and average mushroom size

The highest mushroom yields (12.28 kg/m2) 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/m2) 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
Table 2.2. The effect of substrate particle size on mushroom yield, biological efficiency (BE %),
and mushroom size (Crop 0904).

Substrate particle size
Yield (kg/m2)A
BE (%)A
Avg. Size (g)
(screen size)
Small (0.64)
12.28 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 575-650
28
kg/m3 (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 m3) 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
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
B
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

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 0.8-1.0% 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/m2 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
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 1 -1.5 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
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
3.2 Methods
3.2.1 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.
Distillers grain, whole soybeans, soybean meal and cottonseed meal were obtained
from Martins Feed and Fertilizer, Coburn, PA. Whole soybeans were processed
through a hammer mill at the Mushroom Research Center to provide ground soybean.


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
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).

3.2.3 Cropping trials

For Crop 0903, distillers 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
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) Remos 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.

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
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) Remos All Season, Lambert T6, or a 50:50 mixture of
Remos 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 + distillers 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 Remos 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
humidity. The casing layer was watered daily with a rose-face hose attachment to
maintain adequate moisture for mushroom production.

3.2.4 Harvesting and determination of yield, biological efficiency and size

Mushrooms (with closed veil) were harvested, counted and weighed daily. At
the end of 3th flush, yield, biological efficiency (BE), and average mushroom size were
determined. Yield was expressed as kg/m2 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
3.3.1 Crop 0903

MCS-only substrate contained less nitrogen than MCS with distillers grain (10%
d/w) (Table 3.1). However, distillers 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 Remos 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/m2 to 17.1 kg/m2, while BEs ranged from 0.3% to 70.3%
(Table 3.3). The highest yields (17.1 kg/m2) and BE (70.3%) were obtained from MCS
38
supplemented with distillers grain (10% d/w) at fill and selected from the middle
zone in the minibunker and supplemented with 5% (d/w) Remos All Season at
casing (Table 3.3). Lowest yields (0.1 kg/m2) and BE (0.3%) were obtained from MCS
supplemented with distillers grain (10% d/w) at fill, selected from the lower zone in
the minibunker and supplemented with 10% (d/w) Remos All Season at casing.

Table 3.1. Substrate type and moisture and nitrogen content of milled corn stover (MCS)
substrates at fill for Crop 0903.
Substrate
Moisture (%)
Total Nitrogen (% d/w)
MCS
75.7
0.97
MCS + DGA
75.2
1.37
A DG = Distillers grain (10% d/w)

Table 3.2. Probabilities > F from analysis of variance for substrate type, temperature zone and
Remos added at casing tested for yield and biological efficiency (BE %).

Source
df
YieldA
BE (%)A
Substrate (S)
1
0.4658
0.4635
Temperature zone (TZ)
2
< .0001
< .0001
S x TZ
2
< .0001
< .0001
1
< .0001
< .0001
Remos @ casing (RAC)
S x RAC
1
0.0271
0.0499
TZ x RAC
2
0.0010
0.0017
S x TZ x RAC
2
0.0011
0.0038
A Values of less than 0.05 were considered significant according to Fishers LSD.
39
Table 3.3. Effects of distillers 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 ZoneB
Yield (kg/m2)C BE (%)C
Remos @
casing (% d/w)
1
None
H
5
8.1 bc
38.9 b
2
None
H
10
1.6 de
7.2 cd
3
None
M
5
15.0 a
64.3 a
4
None
M
10
8.3 bc
33.9 b
5
None
L
5
7.2 c
29.4 b
6
None
L
10
0.4 e
1.5 d
7
DGA
H
5
12.6 ab
59.7 a
8
DG
H
10
5.9 cd
26.6 bc
9
DG
M
5
17.1 a
70.3 a
10
DG
M
10
1.7 de
6.6 cd
11
DG
L
5
5.6 cd
22.7 bc
12
DG
L
10
0.1 e
0.3 d
A DG = distillers 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 Tukeys honestly significant difference (HSD) (P = 0.05).

Yields from the different temperature zones ranged from a low of 3.3 kg/m2
(lower zone) to a high of 10.5 kg/m2 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/m2 compared to 4.8 kg/m2 from the high zone and 1.2 kg/m2 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) Remos All Season at
40
casing resulted in higher yields (10.9 kg/m2) and BEs (47.5%) than those supplemented
with 10% (d/w) Remos at casing (3.0 kg/m2, 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/m2)A
BE
st
nd
rd
th
zone
Total
1 Break 2 Break 3 Break 4 break (%)A
H
36
7.1 b
4.8 a
1.5 b
0.2 a
0.5 b
33.1 a
M
36
10.5 a
6.7 a
2.4 a
0.5 a
1.2 a
43.8 a
L
36
3.3 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 %).

No. Reps
Yield (kg/m2)A

BE
Remos @
st
nd
rd
th
casing (%)
Total
1 Break 2 Break 3 Break 4 break (%)A
5
54
10.9 a
5.7 a
2.6 a
0.6 a
1.3 a
47.5 a
10
54
3.0 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).

3.3.2 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 72.9-78.6%. 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
Table 3.6. Substrate type and moisture and nitrogen contents at fill, spawning and casing for
milled corn stover (MCS) (Crop 0905).

SubstrateA
Moisture (%)B
Total Nitrogen (%)
@ Fill
@ Spawning
@ Casing
MCS
78.6
0.99
0.9
1.2
MCS + GS
74.9
1.55
1.5
1.7
MCS + SM
72.9
1.34
1.5
1.5
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
YieldB (kg/m2)
BE (%)B
Substrate (S)
2
< 0.0001
< 0.0001
Supplement @ casing (SAC)A
2
< 0.0001
< 0.0001
S x SAC
4
< 0.0001
< 0.0001
ARemos All Season, Lambert T6 with Mertect, Lambert T7 with Mertect (4% d/w).
B Values less than 0.05 were considered significant according to Fishers LSD.

The highest yields (14.0 kg/m2) 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/m2) and BE (60.9%) when used alone at casing (Table 3.10). Remos 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 Remos. Yields from substrates
supplemented with a 50:50 mixture of Remos and T6 at casing were not significantly
different from either of the supplements when used alone (Table 3.10). Lambert T6,
42
when used alone, resulted in higher yields than Remos 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).

Trt.
Supplement
Yield
BE (%)C Size (g)C
Remos at
T6 at casingB
A
2
C
No.
at filling
(kg/m )
casingB
1
None
4
0
11.2 bc
57.1 bc
13.3 a
2
None
0
4
9.5 c
48.7 c
12.4 ab
3
None
2
2
10.5 bc
53.9 bc
12.7 ab
4
GS
4
0
9.4 c
47.8 c
12.8 ab
5
GS
0
4
14.0 a
71.6 a
12.4 ab
6
GS
2
2
11.1 bc
56.9 bc
13.7 a
7
SM
4
0
6.5 d
33.3 d
11.7 b
8
SM
0
4
12.2 ab
62.3 ab
12.6 ab
9
SM
2
2
10.2 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 Remos All Season with Topsin (% d/w), Lambert T6 with Mertect (% d/w).

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).

SupplementA
No. Reps
Yield (kg/m2)B
BE (%)
st
nd
rd
Total
1 Break
2 Break 3 Break
None
12
10.4 a
7.7 a
2.3 a
0.6 a
53.2 a
GS
12
11.5 a
6.6 b
4.2 b
0.7 a
58.8 a
SM
12
9.6 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

43
Table 3.10. Means and grouping from analysis of variance for supplementation type at casing
for yield and biological efficiency (BE %) for A. bisporus (Crop 0905).

SupplementA
No.
Yield (kg/m2)B
BE (%)B
Reps
Total
1st Break 2nd Break 3rd Break
12
11.9 a
7.5 a
3.8 a
0.6 a
60.9 a
Lambert T6
12
9.0 b
5.4 b
3.0 b
0.6 a
46.1 b
Remos
12
10.6 ab
6.6 ab
3.2 b
0.8 a
54.3 ab
T6 + Remos
A Supplement added at 4 % d/w. T6 + Remos = 50:50 mixture.

3.3.3 Crop 0906

Supplements (distillers 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
Table 3.11 Substrate type, moisture content and total nitrogen at fill and at spawning for Crop
0906.
SubstrateA
MoistureB
Total Nitrogen Content (%)

@ Fill
@ Spawning
MCS
77.0
0.92
0.8
MCS + DG
78.3
1.95
1.1
MCS + CM
76.8
1.25
1.4
A MCS = milled corn stover, DG = distillers grain (5% d/w), CM = cottonseed meal (5% d/w)
B At spawning

Table 3.12. 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/m2) A
BE (%)A
SizeA
Supplement @ fill (SAF)
2
< 0.0001
< 0.0001
0.0006
Spawn type (ST)
1
< 0.0001
< 0.0001
0.9544
SAF x ST
2
0.0015
0.0015
0.5777
Supplement @ casing (SAC)
2
0.6232
0.6232
0.0473
SAF x SAC
4
0.0457
0.0457
0.2903
ST x SAC
2
0.2694
0.2694
0.0244
SAF x ST x SAC
4
0.9937
0.9937
0.1625

Highest mushroom yields (23.6 kg/m2) were obtained from MCS + cottonseed
meal (CM) (5% d/w) spawned with synthetic-type Matrix spawn and Remos 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 Remos All Season was added at casing (Table 3.13).
The poorest treatments with regard to yield and BE were MCS alone combined with
45
millet spawn (Table 3.13). Average size did not differ significantly for most of the
treatments (Table 3.13).

Table 3.13. Effects of adding cottonseed meal and distillers 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).

Trt. No. Supplement
Spawn
Supplement Yield (kg/m2)D
BE (%)D
Size
at FillA
typeB
at casingC
(g)D

1
None
Millet
5.5 gh
25.2 gh
11.9 ab
T6
2
None
Millet

4
.6
h

21.3
h

9.5 b
T7
3
None
Millet
5.3 gh
24.6 gh
12.6 ab
Remos
4
None
Matrix

8
.7
f
gh

4
0.2
f
gh
11.5 ab
T6
5
None
Matrix
9.7 efgh
44.8 efgh 10.9 ab
T7
6
None
Matrix

1
0.5
e
fg
48.6 efg
11.0 ab
Remos
7
DG
Millet
17.1 bcd
78.8 bcd 10.9 ab
T6
8
DG
Millet
16.9
b
cd
77.9 bcd 10.7 ab
T7
9
DG
Millet
63.4 def
11.7 ab
Remos 13.7 def
10
DG
Matrix
15.5
c
de

7
1.4
c
de
12.8 ab
T6
11
DG
Matrix
17.4 bcd
80.4 bcd 12.0 ab
T7
12
DG
Matrix
67.0 def
10.0 ab
Remos 14.5 def
13
CM
Millet
17.6 bcd
81.1 bcd 13.4 a
T6
14
CM
Millet
18.2 abcd
83.9 abcd 12.0 ab
T7
15
CM
Millet
18.6 abcd
86.1 abcd 12.9 a
Remos
16
CM
Matrix
20.5 abc
94.7 abc
12.5 ab
T6
17
CM
Matrix
21.9 ab
101.2 ab
12.2 ab
T7
18
CM
Matrix
109.1 a
12.8 ab
Remos 23.6 a
A DG = Distillers 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. Remos All Season Supplement
D Means followed by the same letter within the same column are not significantly different

Cottonseed meal (5% d/w) addition at fill resulted in the highest yields (20.1
kg/m2) across all treatments, followed by distillers grain (5% d/w) addition (15.8
kg/m2) and MCS alone (7.4 kg/m2) (Table 3.14). BE followed a similar pattern (Table
3.14).

46
Synthetic-type Matrix spawn resulted in higher yields (15.8 kg/m2) across all
treatments compared to millet-type spawn (13.0 kg/m2) (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 3.14. Means and groupings from analysis of variance for supplementation of milled corn
stover substrate at fill using distillers grain and cottonseed meal for yield and biological
efficiency (BE %) for A. bisporus (Crop 0906).

SupplementA No. Reps
Yield (kg/m2)B
BE (%)B
st
nd
rd
th
Total 1 Break 2 Break 3 Break 4 Break
None
54
7.4 a
4.3 a
1.7 a
1.0 a
0.4 a
31.1 a
DG
54
15.8 b 10.5 b
4.1 b
0.8 a
0.4 a
73.1 b
CM
54
20.1 c
14.5 c
4.1 b
0.7 a
0.7 a
92.9 c
A DG = Distillers grain (5% d/w) or CM = Cottonseed meal (5% d/w), added at fill.

Table 3.15. 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/m2)A
BE (%)A
st
nd
rd
th
Millet
81
13.0 a
7.9 a
3.9 a
0.8 a
0.4 a
60.0 a

Matrix
81
15.8 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
47
4.4 Discussion

The addition of distillers 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 Remos 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
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
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
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 2.5-2.7% 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 m3 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
Murphys (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
4.2 Methods
4.2.1 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. Distillers grain, added to adjust
nitrogen content of the different substrates to the same level, was obtained from
Martins 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.


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
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).

4.2.3 Mushroom cropping trial

Three substrate formulas were developed using various mixtures of CS, SMS,
distillers grain, and lime (Table 5.1) (Fig 5.1). Distillers 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
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

-----------------------------------------% -----------------------------------------
1
87.5
0
11.1
1.3
2
70.0
22.4
6.2
1.0
3
50.5
48.1
0.7
0.8
A CS = corn stover, SMS = spent mushroom substrate, DG = distillers 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 5.1.
55
4.2.4 Harvesting and determination of yield, biological efficiency and mushroom

size

Mushrooms (with closed veil) were harvested, counted and weighed daily. At
the end of 4th flush, yield, biological efficiency (BE), and average mushroom size were
determined. Yield was expressed as kg/m2 and BE was determined by dividing the
weight of fresh mushrooms harvested (g) by the dry weight of the substrate (g) and
expressing the result as a percentage. Average mushroom size (g/mushroom) was
calculated as fresh mushroom weight divided by the number of fresh mushrooms
harvested.

4.3 Results
4.3.1 ANOVA

Significant sources of variation from the analysis included spawn rate and SMS
for yield and SMS for size. BE was not significantly affect by the treatments. (Table 4.2)
There were no significant interactions between spawn rate and SMS addition at fill for
any of the parameters examined (Table 4.2).
Yields ranged from a low of 11.4 kg/m2 for 22% SMS (0.75% spawn) to a high of
14.5 kg/m2 for no SMS with 1.5% spawn (Table 4.3). Mushroom size ranged from a low
of 11.8 g/mushroom (48% SMS with 1.5% spawn) to a high of 15.1 g/mushroom (22%
SMS with 1.5% spawn) (Table 4.3).
56
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).

Source
df
Yield (kg/m2) A
BE (%)A
SizeA
Spawn rate (SR)
1
0.0353
0.0984
0.7095
SMS
2
0.0004
0.1046
0.0095
SR x SMS
2
0.8319
0.7810
0.0972

Table 4.3. Effects of various levels of spent mushroom substrate (SMS) added to milled corn
stover substrate at fill and spawn rate on mushroom yield, biological efficiency (BE %), and
mushroom size for A. bisporus (Crop 1001b).

Trt. No.
SMS (% d/w)A Spawn
Yield (kg/m2)C
BE (%)C
Size (g)C
B
(% w/w)
1
0
0.75
13.9 a
69.0 a
13.9 ab
2
0
1.5
14.5 a
71.1 a
14.4 ab
3
22
0.75
11.4 b
66.2 a
13.6 ab
4
22
1.5
12.6 ab
72.1 a
15.1 a
5
48
0.75
12.8 ab
62.9 a
13.1 ab
6
48
1.5
13.7 a
66.2 a
11.8 b
A SMS = spent mushroom substrate, from MTDF Crop 3708.
B Sylvan A15 Matrix Spawn.

4.3.2 Spawn rate

A higher spawn rate (1.5%) resulted in increased yields (Table 4.4). Most of the
yield increase, attributable to the higher spawn rate, occurred on the first break. There
was no significant differences in BE and average mushroom size with regard to spawn
rate (Table 4.4).

57
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).

Spawn
No.
Yield (kg/m2)B
BE
Size
Rate
Reps
(g)B
Total
1st Break 2nd Break 3rd Break 4th Break (%)B
(% w/w)A
0.75
18
12.7 a 7.8 a
3.4 a
1.0 a
0.4 a
66.0 a 13.5 a
1.5
18
13.6 b 9.0 b
3.3 a
0.8 a
0.4 a
69.8 a 13.7 a
A Sylvan A15 Matrix Spawn.

4.3.3 SMS additions

Means and groupings from analysis of variance for SMS added at fill for yield, BE
and mushroom size are presented in Table 4.5. Yields were highest from MCS
containing 0% and 44% SMS. Mushrooms were larger from MCS containing 0% and
22% SMS compared to MCS with 48% SMS.

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).

SMS (%)A No. Reps
Yield (kg/m2)B
BE(%)B Size(g)B
st
nd
rd
th
0
12
14.2 a 10.6 a
2.3 a
0.8 a
0.5 a
70.1 a
14.1 a
22
12
12.0 b 7.6 b
3.0 a
0.9 ab
0.4 a
69.1 a
14.4 a
48
12
13.2 a 7.2 b
4.6 b
1.1 b
0.3 a
64.5 a
12.4 b
A SMS = Spent mushroom substrate (d/w), from MTDF Crop 3708.
58
4.4 Discussion

Synthetic (Matrix) spawn colonized the substrate well, regardless of spawning
rate (visual observation). No noticeable contaminants were observed in the substrates
in any of the treatments. Higher spawn rates (1.5%) used lead to yield increases on the
first break alone. This may be due to better more extensive colonization throughout the
substrate and more efficient use of the nutrients early in production. Nutrient
depletion in the substrate over time may have accounted for no additional yield
increases. Further work is needed to determine optimum spawning rates for synthetic
spawn on MCS.
SMS addition increased bulk density of the substrates (personal observation).
SMS addition of 48% (d/w) at fill did not affect yields compared to MCS alone, however,
22% SMS addition resulted in decreased yields. 48% SMS addition gave better second
yield breaks than the other two treatments. Perhaps nutrients in SMS are less available
compared to those in MCS.
Recycling SMS for incorporation into new compost is a way to reduce the solid
waste disposal problem associated with mushroom production. After mushroom
harvest is completed, SMS must be moved off the farm unless it is used for another
purpose. In recent years, research has lead to development of SMS as a nutrient-rich
soil amendment and even more recently as an ingredient in mulch in artillery fungus
suppression (Davis et al., 2006). In the past, mushroom farmers piled SMS in their
fields for disposal or further composting, creating the potential for contamination of
surface and groundwater from runoff. Currently, environmental regulations are
59
becoming increasingly strict with regard to waste production and disposal. Current
research is being conducted (disease suppression on Fraser Fir and artillery fungus
suppression, Don Davis, personal communication) on the use of fresh SMS directly from
mushroom houses that does not require aging. Preliminary results indicate that fresh
mushroom compost is able to suppress artillery fungus similar to aged mushroom
compost (D.D. Davis, personal communication).
SMS can also be used as an ingredient in non-composted substrate. Mamiro and
Royse (2007) found that addition up to 50% SMS to a non-composted substrate mixture
had no effect on mushroom yield, BE and mushroom size, compared to a phase II
compost control. By re-using SMS, mushroom farmers may expect increased profits
while minimizing environmental impact, thereby, improving popular perception of the
mushroom industry. In addition, growers may also benefit from shortened spawn runs
using synthetic spawn.

60
Chapter 5: Summary and Future Work

5.1 Summary

Results presented here are consistent with Murphy (1972) in that phase I
composting is not needed in order to produce high yields and good quality mushrooms.
This may be of use to smaller scale or specialty mushroom growers that may be
interested in producing white button mushrooms, but do not have the space or
equipment for large composting operations. In addition, we noticed no foul odors
during composting except from ammonia.
Minimizing the dry weight loss during composting is of upmost importance to a
mushroom grower, since organic matter is ultimately the nutrient source for the
mushroom and affects yields. Eliminating phase I composting greatly reduces the time
needed to produce a suitable mushroom substrate. Also, ash content in MCS is much
lower, about 5.6% (Huang et al., 2009) compared to conventional phase II compost
(approximately 30%). Therefore, MCS has more available organic matter for mycelium
growth and mushroom production.
Mushrooms produced from MCS were significantly smaller (11-13g/mushroom)
compared to mushrooms produced on conventional mushroom substrate
(17g/mushroom, Daniel Royse, personal communication). This remains a disadvantage
of using MCS since many consumers prefer large white mushrooms. Also, several
treatments in some crops had poor quality mushrooms (small or deformed). This may
61
be due the particular treatment and/or to improper management practices (humidity,

watering, CO2) throughout production.

5.2 Future work

Additional research involving supplementation prior to phase II-only
composting should be conducted to improve the basal substrate formula described
here. Perhaps mixtures of several agricultural byproducts could be incorporated to
provide adequate nutrients to the mushroom. Murphys T1 formula (1972) might be
used as a basal formula, with substitution of MCS for corncobs. Corn stover is already
being used as a compost ingredient in conventional mushroom compost, but not as a
minimally-composted substrate.
In order to achieve higher yields, nitrogen content in the substrate must be
increased further. This may be possible by adding poultry manure or other high
nitrogen containing ingredients to the basal mixture. Future work should focus on
increasing nitrogen content at spawning to at least 1.5-1.7%. Other nutrients may be
limiting in MCS. A more in-depth analysis of the nutrients content of MCS may address
this issue.
The research reported herein was done using minibunkers. Further research,
needed to develop MCS as a viable commercial mushroom substrate, should center on
scaling-up and performing phase II-only composting in computer-controlled phase II
tunnels. This is a critical part of determining whether MCS, subjected to phase II-only
composting, has commercial potential within the mushroom industry.
62
In order for a new substrate to become adopted by the industry, there needs to
be a definite advantage for its use. An economic analysis should be done to determine
the cost savings, if any, that MCS confers over conventional substrate preparation.
Conventional phase I composting involves the use of very expensive pre-wetting and
compost turning machines, bunkers, and/or large concrete areas for ricks, thereby
contributing to the high costs of conventional substrate preparation. MCS does not
require any of these, however, mechanical processing and the fuel associated with it
may drive up the price of MCS substrate preparation.

63
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The

Pennsylvania State University

MINIMALLY COMPOSTED SUBSTRATE FOR THE PRODUCTION

Submitted in Partial Fulfillment

Agaricus bisporus is cultivated commercially on a composted substrate that

Use of synthetic-type spawn (Sylvan Matrix) resulted in higher yields (15.8

Chapter 1: General introduction

1.2 Economic importance of Agaricus bisporus

1.3 Cultivation history

1.4 Cultivation methods .

1.5 Nutritional requirements .

1.7 Environmental impact

1.8 Raw materials used for cultivation

1.9 Shortening or eliminating the composting process

2.2.2 Substrate preparation .

2.2.3 Phase II-only composting ..

2.2.4 Experimental design and data analysis

2.2.5 Cropping trial

and mushroom size .

Chapter 2: Phase II-only composting of milled corn stover for use as

2.2.6 Harvesting, determination of yield, biological efficiency

2.3.1 Substrate particle sizes .

2.3.2 Yield, biological efficiency, and mushroom size ....

and spawn type on yield, biological efficiency and mushroom size

3.2.1 Substrate ingredients ..

3.2.2 Experimental design and data analysis

3.2.3 Cropping trials

3.2.4 Harvesting and determination of yield, biological

efficiency, and size .

3.3.1 Crop 0903

3.3.2 Crop 0905

3.3.3 Crop 0906

4.2.2 Experimental design and data analysis

4.2.2 Mushroom cropping trial ..

4.2.4 Harvesting and determination of yield, biological

4.3.2 Spawn rate .

4.3.3 SMS additions ..

Chapter 5: Summary and future work .

5.2 Future work .

preparation of milled corn stover substrate ... 18

screens used to prepare mushroom substrate of various particle

sizes (from left: 0.64, 1.27, 1.91 cm sieve size) .

in minibunker. Note white-tinged substrate (Actinomycete fire-fang

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 .

Fig. 2.7. Mushroom production on milled corn stover substrate subjected to

phase II-only composting in a minibunker

Fig. 3.1. Assignment of temperature zones in substrate during phase II-only

composting in minibunkers. H = high, M = middle, L = low. .

1, 2, and 3. Formula compositions are listed in Table 5.1............................ 55

soybean meal for yield and biological efficiency (BE %) of A. bisporus

Table 3.13. Effects of adding cottonseed meal and distillers grain

Table 4.1. Percentages of various ingredients in three formulas of phase II-only

Chapter 1: General introduction

1.2 Economic importance of Agaricus bisporus

1.4 Cultivation methods

microorganisms that may affect growth of A. bisporus and 2) conditioning of the