Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
)
ON WOOD SUBSTRATES IN HAWAII
By
Tracy E. Tisdale
Thesis Committee:
Susan C. Miyasaka, Chairperson
Mitiku Habte
Don Hemmes
Acknowledgements
111
Table of Contents
Acknowledgements
iii
List of Tables
, vi
List of Figures
vii
Chapter 1: Introduction
'"
,.. ,
Substrates
Oyster Mushroom
3
6
" '"
19
Production Overview
24
'"
32
33
Substrate Wood
33
Cultivation Methods
34
Crop Yield
39
Nutrients
43
Taste
44
.46
Economic Analysis
.46
Substrate Wood
Preliminary Experiment.
,
,
'"
.48
,
48
52
IV
Final Experiment.
Crop yield
53
53
Nutrients
67
Taste
68
Economic Analysis
72
74
Chapter 6: Summary
84
86
87
88
89
90
91
92
'"
'" .,
93
List of Tables
Table
Page
4.1
34
4.2
.40
4.3
Analysis of variance table for yield distribution throughout multiple flushes .. .41
4.4
4.5
43
4.6
.44
4.7
.45
4.8
.45
5.1
5.2
51
5.3
52
5.4
5.5
Initial bag weight and biological efficiency of Pleurotus sp. on five substrate
.42
'" .,
50
55
woods
62
5.6
65
5.7
72
5.8
5.9
5.10
77
78
83
VI
List of Figures
Figure
Page
5.1
5.2
5.3
5.4
69
5.5
70
5.6
,
,
,
,
Vll
,
,
54
59
64
71
Chapter 1
Introduction
Problem
Agriculture remains one of the top industries in Hawaii. To strengthen both
this industry and Hawaii's overall economic situation, there has been a committed
effort to diversify Hawaii's agriculture. As plantation agriculture (sugarcane and
pineapple) have declined tremendously, there is a great opportunity for small,
diversified agriculture in the state.
The U. S. mushroom industry is of substantial value, producing over $889
million dollars of fresh mushrooms in the 2002 - 2003 season (USDA 2003).
However, there are very few producers of edible mushrooms in Hawaii. Substrate is
a key component in mushroom cultivation. First, the substrate must be suitable for
the growth and fruiting of the fungus. Second, the substrate should be available
locally in sustainable quantities and at low cost.
Climate is another factor in successful mushroom cultivation. The majority of
mushroom operations in the United States are indoor operations, which allow for
precise climate control. Such operations are generally extremely expensive to
establish and operate (Shen et al. 2004). High investment costs can be prohibitive
to many farmers, especially small farmers or those interested in producing
mushrooms as an additional crop. Outdoor cultivation methods, used primarily in
China and many other countries, are far less costly but produce relatively lower
yields (Shen et al. 2004). In the end, production must be economically feasible for
farmers in Hawaii.
Significance
With agriculture as one of Hawaii's major industries, the availability of
substrate for mushroom cultivation is promising. Many of the common edible
mushrooms can be grown on plant derived materials such as wood, straw and
various agricultural wastes.
sugarcane land has been shifted to timber forests. Approximately 11,740 hectares
of land on the Big Island have or will be planted using Eucalyptus grandis for short
rotation forests and Acacia koa for long rotation (Martin et al. 2001). There is a
definite potential for thinned trees to serve as a local and sustainable source of
substrate for mushroom cultivation in Hawaii. There are also a number of fast
growing tree species that have been introduced to the islands for various reasons.
Whether they are growing wild or intentionally farmed, wood from rapidly growing
trees is a potential substrate.
The tropical climate of Hawaii, the east coast of the Big Island in particular,
provides a wet, humid environment with an average precipitation rate of 3,404 mm
annually (NOAA 2004). It also offers a long growing season, uninterrupted by a
harsh winter season. With natural environmental conditions conducive to
mushrooms, outdoor cultivation may be a feasible option in Hawaii.
Chapter 2
Literature Review
MUSHROOM INDUSTRY
World Production
The Chinese were the first to grow mushrooms for human consumption. As
early as 600 AD, varieties of Auricularia were being cultivated. Around 1000 AD
Lenfinula edodes, commonly known as shiitake, entered mushroom farming
th
Market Value
mushrooms, 32 cents more than the previous year (USDA 2003). The oyster
mushroom is considered a choice mushroom for cooking and eating and has the
reputation of being easy to cultivate (Stamets 2000). The current market value of
the oyster mushroom in the US is approximately $4.50 per kg (USDA 2003),
although niche markets can generate higher values. Shiitake mushrooms have a
higher selling value, around $6.60 per kg in 2002-3 (USDA 2003). In Hawaii, the
retail value of such mushroom can reach $17.64 to $22.05 per kg. They too are
considered choice eating mushrooms, highly desired in many Asian cuisines. Based
on historical and more recent trends, it is believed that the specialty mushroom
industry will continue to flourish (Royce 1996). The high value, unique production
requirements, and relatively rapid growth cycle make mushroom production an
attractive addition to Hawaii's agricultural repertoire.
Hawaii Production
According to the Census of Agriculture, there are only three mushroom
growers operating in Hawaii (USDA 2004). Within the last few years, the Hamakua
Mushroom Farm has begun operation in Lapaehoehoe on the Big Island. Using
Japanese bottle cultivation methods (indoor), they produce specialty mushrooms
including hon shemeiji (Hypsizygus spp.), shiitake, and a variety of oyster
mushrooms. They have been using sawdust from the native hardwood tree Acacia
koa as the main substrate material. Communications with the operation owners
revealed that they were obtaining the wood locally, as scrap from a nearby koa mill
SUBSTRATE
strawberry guava (Psidium caftleianum Sabine) and the gunpowder tree (Trema
orientalis (L.) Blume) for which eradication efforts are being considered. The costs
and consistency of supply from local tree clearing would have to be evaluated.
Another possible means of obtaining substrate is to grow trees specifically for
mushroom cultivation. There are several fast-growing species in Hawaii which
would be good candidates. Reported growth rates for both albizia (Falcataria
Eucalyptus grandis
There are roughly 500 species of Eucalyptus, most of which came from
Australia. Over 90 of these species have been introduced to the Hawaiian Islands
(Little & Skolmen 1989). Eucalpytus spp. are among the world's tallest hardwood
trees and are often propagated because of their size and rapid growth. Eucalyptus
trees tend to have good form, with branching only at the upper one-third of the total
height (Little & Skolmen 1989).
The natural ranges of E. grandis are coastal regions of Eastern Australia with
tropical temperatures and frequent rainfall. Usually forming pure or nearly pure
stands, E. grandis grows in open forests, valleys or hillside slopes. It is found from
sea level up to approximately 2,700 m in elevation where mean annual temperatures
range from -1 to 40 C and rainfall is 100 to 1800 mm per year. Deep, well-drained
soils are best for E. grandis growth and survival (Salim et al. 2002). It is able to
grow in marginal to poor soils (Campinhos 1999), however nitrogen (N) and
phosphorus (P) have been identified as limiting factors for Eucalpytus growth
(DeBell et al. 1985).
Coppicing is quite successful with E. grandis, and therefore a common means
of regenerating plantations (Campinhos 1999). Vegetative reproduction by cuttings
is also a popular method. Cuttings can be made from trees as young as 4 to 5 years
(Salim et al. 2002). Regeneration by seedling plantings is also a viable option.
Greenhouse raised seedlings can be out planted within 3 to 4 months (Whitesell et
al. 1992).
Yields of Eucalyptus spp. in Hawaii are quite favorable as compared to some
countries (Khamoui & Baker 1982). Spacing of Eucalyptus does influence growth
and biomass. Whitesell et al. (1992) found that denser spacing generates higher
mean annual production. If biomass yield is the main objective in a short-rotation
plantation, planting trees at densities of 1.5 m2 and 3.0 m2 appear to give optimal
yields. Yields as high as 94 t/ha can be achieved in just 4 years with moderate N
Fa/cataria mo/uccana
The Molucca Albizia, Fa/cataria mo/uccana, previously named A/bizia
fa/cataria (L.) Fosberg, is native to the Molucca Islands of Indonesia and was
introduced to Hawaii in 1917 for reforestation efforts. This deciduous tree grows tall
with a long, slender trunk and large spreading crown. It has compound leaves and
seed born in narrow, flat, splitting pods (Little & Skolmen 1989). As a member of the
Legume family, Fa/cataria has N-fixing nodules containing leghaemoglobin on the
roots and fixes N at a beneficial rate (Salim et al. 2002).
germinate irregularly and require sufficient light for survival. Seedlings are ready for
transplanting to the field after 4 to 5 months in the nursery. Coppicing is natural for
F. mo/uccana, but may be strong or weak depending on environmental conditions
(Salim et al. 2002).
A/bizia is commonly used in agroforestry systems. In Hawaii, Fa/cataria
forests yielded over 5,150 m3 of hardwood (Little & Skolmen 1989). It has a very
fast growth rate and has been known to reach a height of seven meters in its first
year under optimal conditions (Salim et al. 2002). In Hawaii, growth rates up to 4.5
m per year have been recorded (Little & Skolmen 1989). In short-rotation
plantations, Fa/cataria has high biomass yields. Both 5 and 7 year rotations resulted
in annual biomass yields of 24.5 tlha (9.9 tlacre) (Whitesell et aI.1992).
Casuarina eguisetifolia
There are about 70 species in the Casuarina genus, native to Australia and
neighboring areas (Salim et al. 2002). Casuarina equisetifo/ia is the most
widespread species in the family and is commonly known by many names:
ironwood, Australian pine, she-oak, horse-tail tree, beefwood, whistling pine, and
many others. C. equisetifolia is a tall tree (10 to 40 m) and commonly grows in
dense stands along coasts and roadsides in Hawaii. Despite its conifer-like
appearance, with drooping green needle-like branchlets and cone-like fruits, C.
equisetifolia is actually an angiosperm. Individual trees may be either dioecious or
monoecious, and are known to exhibit great phenotypic variation in shape and size
of cones, branchlets, and crown. Their N-fixing capacity differs from many of the
10
leguminous trees in that their root nodules support an actinorhiza symbiont, rather
than rhizobium (Dommergues 1990).
C. equisetifolia thrives in humid tropical and subtropical climates. It is
intolerant of frost and, although generally a lowland tree, grows at elevations of 600
m in Hawaii. It tolerates a wide range of water availability. In its native range, annual
rainfall ranges from 700 to 5000 mm with a 6 to 8 month dry season (Parrotta 1993).
In more arid climates, it grows best along the coast where additional moisture from
sea spray is available. Its N-fixing potential is believed to be highly dependant on
adequate soil-moisture availability (Dommergues 1990).
Casuarina grows best on well drained, porous soils with sufficient moisture
and nutrient supply. It can withstand nutrient-poor sands, moderately calcareous
soil, moderately saline soils, and soils with a pH ranging from 5.0 to 9.5. However, it
is said to grow best in slightly acidic, sandy soils with moderate nutrient availability
(Rockwood et al. 1990).
Seedlings will reach a height of 10 to 15 cm approximately 6 to 10 weeks
after germination and achieve a plantable size in the nursery within 4 to 8 months. If
grown from seed, inoculation with the N-fixing symbiont is highly recommended.
Actinomycetes of the genus Frankia are symbiotic with Casuarina, forming woody
nodules on the roots (Dommergues 1990). Vegetative propagation from cuttings or
lateral shoots is quite successful. Trees have a tendency to spread horizontally via
root suckering and rooting of branches when trees are damaged. Unlike other
species in the family, C.equisetifolia does not coppice readily (Duke 1983).
11
Psidium cattleianum
Psidium cattleianum Sabine is commonly known as strawberry guava or
purple guava. It is native to Brazil and belongs to the myrtle family (Myrtaceae). In
the early nineteenth century, strawberry guava was introduced intentionally as a fruit
tree. It escaped cultivation and rapidly spread throughout the main Hawaiian Islands
(Staples & Cowie 2001). It grows as a shrub or small tree up to 4.6 m in height and
bears golf-ball sized fruits yellow or purple in color. P. cattleianum forms highly
crowded, single-species stands which cast heavy shade. The prolific surface roots
form dense mats, preventing other species from establishing within the thicket.
Strawberry guava is so prolific and aggressive in Hawaii that it is considered an
invasive weed, threatening populations of native plant species (Tunison 1991).
12
highly acidic soils. Another quality that makes P. cattleianum a threatening species
in Hawaii is its ability to withstand heavy leaf litter. Huenneke and Vitousek (1990)
found that seedlings crushed by fallen tree fern fronds survived and produced
vigorous shoots, while seedlings of native species were killed.
Strawberry guava has very aggressive reproduction both vegetatively and by
seed. Root suckers are sent out so rapidly and abundantly that clonal growth often
dominates the stands. Such stands have stem densities as high as 9 stems/m 2
(Huenneke & Vitousek 1990). This introduced species also produces fruit at a
plentiful rate. Seeds of the fragrant fruits are dispersed over vast areas by nonnative birds and feral pigs. Germination of seeds is neither dependant on animal
processing (Tunison 1991), nor on soil disturbance (Huenneke & Vitousek 1990).
Growth of the strawberry guava is quite rapid. Since this species poses such
a threat to native forest species, current research efforts are focused on eradication,
rather than cultivation. A relative growth rate of seedlings was calculated by
Pattison et al. (1998) as 0.25 grams per week. This rate was approximately two
and a half times greater than that of several small native tree species of similar
habitat. In shaded conditions, growth rate of strawberry guava seedlings was nearly
three times greater than the native species (Pattison 1998). A survey of Hawaiian
angiosperms showed that P. cattleianum individuals in Hawaii are highly mychorrizal
(Koske et al. 1992).
13
Trema orientalis
Trema orientalis (L.) Blume is now widely distributed throughout the tropics,
from the Himalayas, through the Pacific, Australia, and tropical Africa. Its native
range is from southeastern Asia through Malaysia (Little & Skolmen 1989). This
evergreen member of the Ulmaceae family grows as mid-sized tree or large shrub,
reaching approximately 18 m in height. In many countries, the tree has many
functional uses including medicine, fodder, fuel, fiber, and land improvement. In
Hawaii, it is considered a weed tree that quickly invades wet, lowland areas.
especially rapid growth rate, reaching harvestable size for pulpwood in 3 to 4 years.
14
One study in the subtropical islands of Japan reported a stem diameter growth rate
of 0.261 mm per month (Yamashita et al. 2000). This species, unlike others in the
Elm family, is a N-fixing tree. Roots of trees growing in its native regions are said to
have nodules and be associated with bacteria of the Bradyrhizobium genus (F.
Hughes, personal communication).
15
of p-hydroxyphenyl units (Kirk & Farrell 1987). Guaiacyl units are more resistant to
degradation than are syringyl units (Hatakka 2004).
Only a small group of organisms, including the fungi, have developed the
ability to degrade lignin. It is an aerobic process involving many specific enzymes.
The decay rate of a particular plant debris is said to be proportional to its lignin
content. A mathematical formula, developed in 1980 calculates the bioavailability of
a substrate based on its lignin content. It suggests a linear relationship and has
proven relatively accurate for substrates of low lignin content (Chandler et al. 1980).
An adaptation of this equation accommodating high lignin substrates was presented
several years later and involves computing a biodegradable carbon (C): nitrogen (N)
ratio (Richard 1996). In addition to the composition of the substrate itself, conditions
that favor the decomposer (adequate N, temperature and moisture) are significant
factors in lignin decomposition (Richard 1996).
16
Using P. chrysosporium, optimal culture conditions for lignin degradation have been
described and the highest degradation rates have been reported. When grown
using wood pulp, P. chrysosporium's degradation rate was approximately 200 mg
lignin per gram of mycelium per day (Yang et al. 1980).
Lignin degradation appears to be associated with the vegetative phase of
fungal growth, while cellulose degradation is associated with fruit-body formation.
Tan and Wahab (1997) found activity of lignin degrading enzymes increased in P.
pulmonarius mycelium up until the formation of fruiting bodies, at which point activity
of cellulose-degrading enzymes increased. The same pattern in enzyme production
was observed in P. ostreatus (Velazquez-Cedeno et al. 2002). Some reports
suggest that substrates with high cellulose are favored by the white rot fungi P.
pulmonarius, over those with high lignin contents (Sivaprakasam & Kandaswamy
1981).
White rot fungi can degrade the syringyl units of lignin more effectively than
the guaiacyl units. As mentioned earlier, the lignin of gymnosperms have
considerably higher levels of guaiacyl units than that of angiosperms. The
resistance of guaiacyl to degradation may explain why white rot fungi are found
more commonly on angiosperm wood than on gymnosperms (Hatakka 2004).
Biochemistry
White rot fungi secrete an arsenal of extracellular enzymes capable of
disassembling lignin polymers and other wood components. The extracellular
enzymes involved in the lignin mineralization include ligninases, manganese (Mn)
17
Physiology
Nutritional and cultural parameters do influence the physiology of lignin
degradation by white rot fungi. The following parameters have been noted to affect
lignin decomposition: appropriate substrate, oxygen availability, correct level of
certain minerals and trace elements, and nutrient N level (Kirk & Farrell 1987).
Recent studies have revealed that many white rot fungi metabolize lignin in
conjunction with cellulose and hemicellulose, and will only degrade lignin when the
other energy sources are present. Molecular oxygen is essential for lignin
degradation and increased O2 levels resulted in heightened ligninase reactions.
Several inorganic nutrients are closely associated with lignin degradation processes
and correct levels of Mn, copper (Cu), calcium (Ca), zinc (Zn), and iron (Fe) showed
beneficial effects in lignin degradation (Kirk & Farrell 1987). N-limitation was found
to stimulate lignin degradation of P. chrysosporium (Kirk & Farrell 1987). However,
18
P. ostreatus and L. edodes degrade lignin at higher rates when grown in N-rich
versus N-limited media (Kaal et al. 1995).
Genetics
The enzymes involved in lignin degradation are unlike those of regular
metabolic processes. The genetics of lignin-degrading organisms is being
investigated for answers about this unique enzymatic system. Genetic research on
OYSTER MUSHROOM
Historical Information
19
Hayes 1978). Popularity and production of the oyster mushroom has been
increasing ever since.
Commercial production techniques for this edible basidiomycete are well
developed (Stamets 2000; Oei 1996; Zadrazil1978). Compared to other edible
mushrooms, species of Pleurotus are relatively simple to cultivate (Zadrazil 1978).
In addition, Pleurotus are considered the most adaptable genera of edible fungi, able
to grow on a wide range of lignocellulotic materials (Stamets 2000). Cultivation of
oyster mushrooms around the world occurs using many different organic materials
as substrates, often depending on substrate availability in a particular region. In
nature, Pleurotus spp. grow on the wood of broad leaf trees, thus wood and wood
products are common substrates for oyster mushroom cultivation (Zandrazil 1978).
Wheat straw is a common substrate for oyster mushroom cultivation in the
continental US, while the abundance of rice straw available in China is utilized as
substrate (Chang & Hayes 1978). Other substrates used successfully include
cotton waste, corn cobs, palm fronds, tea waste, and peanut shells (Cohen et al.
2002, Thomas et al. 1998, Kalita & Mazumder 2001, Philippoussis et al. 2001).
20
central stalk. Gills are thin, broad, dense, continuous to the upper part of the stipe
(stalk), and vary in color from white to light gray. Spores of Pleurotus spp. range in
color from white to buff to gray-lilac, and are often produced in large quantities,
sometimes provoking allergic or irritation reaction in growers (Eger 1978).
Even within a species, fruit body morphology is quite variable. Culture
conditions can affect color of the sporophore and gills, as well as stipe texture. Early
means of species identification required specific mating tests and isolations (Eger
1978). Today, molecular analysis and DNA sequencing allow for a more efficient,
reliable, and timely identification.
21
nutrient value of fruit bodies (Crisan & Sands 1978). Specifically, Patrabansh and
Madan (1997) found that mineral content, specifically P, magnesium (Mg), Fe, Mn
and ln, of P. pulmonarius fruit bodies increased when grown on substrates with
higher mineral content. Substrate composition has also been shown to influence
fruit body flavor. Oyster mushrooms (P. f1abellatus) grown on rice straw
supplemented with cotton seed were reported to have a distinctly different flavor
component concentrations than those grown on unsupplemented straw (Bano &
Rajarathnam 1998).
Oyster mushrooms are also known to have multiple medicinal properties.
Two of the more prominent medical attributes are cardiovascular and cholesterolcontrolling benefits. Oyster mushrooms naturally produce mevinolin (Iovastatin) in
portions of the fruiting bodies (Gunde-Cimerman 1999). Mevinolin inhibits the key
enzyme in cholesterol biosynthesis in the liver and reduces cholesterol absorption
(Bobek et al. 1998). P. ostreatus is a known producer of many biologically active
substances. It has been demonstrated to have antibacterial properties (Wasser &
Weis 1999) in addition to antiviral, anti-inflamatory and immune modulation activities
(Jose et al. 2002). It is also believed to be effective in the treatment of cancer.
Gunde-Cimierman (1999) showed its effectiveness as an anticancer agent, while
Gerasimenya et al. (2002) found it useful in decreasing the toxic effects of common
cancer drugs. Cohen et al. (2002) provides a comprehensive list of medicinal
substances found in six species of Pleurotus.
22
Physiology
Like other white rot fungi, species of Pleurotus secrete an arsenal of enzymes
specific for the digestion of lignocellulose materials. Degradation of substrate,
including types and quantities of enzymes produced, differs among different species
of white-rot fungi and different growth conditions (Freer & Detroy 1982, Boyle et al.
1992). Buswell et al. (1996) has studied and summarized the enzymatic profiles of
three edible mushrooms, L. edodes, P. pulmonarius, and Volvariella volvacea
(paddy straw). L. edodes grows naturally, and is cultivated, on high lignin substrates
such as wood logs and sawdust. This fungus is known to produce both Mn
peroxidase and laccase, two enzymes specific for lignin degradation. The paddy
straw mushroom prefers high cellulose substrates such as straw. It produces many
cellulolytic enzymes but none of the lignin-degrading enzymes. When enzyme
production was quantified, P. pulmonarius produced higher levels of both cellulolytic
and ligninolytic enzymes (Buswell et al. 1996). Specific lignin-degrading enzymes
produced by Pleurotus spp. include lignin peroxidase, Mn peroxidase, and laccase
(Orth et al. 1993, Kaal et al. 1995). Cellulolytic enzymes of Pleurotus spp. include
endoglucanase, exoglucanase, r3-glucosidase (Buswell et al. 1996, Tan & Wahab
1997) and cellobiohydrolase (Tan & Wahab 1997, Velazquez-Cedeno et al. 2002).
Several studies show that substrate composition does influence enzymatic
activity in Pleurotus. Sivaprakasam and Kandaswamy (1981) determined that lignin
content of the substrate did affect cellulase activity of P. pulmonarius and,
consequently, cellulose utilization. Tan and Wahab (1997) demonstrated that P.
23
PRODUCTION OVERVIEW
A simplified life cycle of the oyster mushroom can be separated into two
biological stages: the vegetative phase, consisting of mycelial expansion and
24
Like the cultivation of Pleurotus spp. and all white-rot fungi, several factors
are critical for successful fruiting. Certain environmental conditions are required to
cue the organism into the reproductive phase. Moisture, temperature, gas
exchange, and light are involved in mushroom initiation and development. When
choosing or creating a site for mushroom production, consideration of environmental
conditions is important.
Moisture
Extremely high humidity (90 to 100%) is recommended for optimal primordial
formation. Once primordia have formed, humidity should be lowered to 85 to 90%.
Ideally, humidity levels should be managed so that mushrooms are regularly
25
receiving moisture but excess moisture can evaporate from fruit body surfaces
(Stamets 2000). Excessive moisture can cause lack of oxygen in the substrate, as
well as encourage certain contaminates. Inadequate moisture can prevent primordia
formation and stunt fruit body growth.
Temperature
Oyster mushrooms are able to grow and thrive in a wide range of temperature
environments. Stamets (2000) recommends temperatures between 10 and 21 C
for development of oyster mushrooms.
Gas exchange
Since growth of the fungus produces carbon dioxide as it decomposes the
substrate, introduction of 'outside' air reduces carbon dioxide build up and increases
oxygen levels. Fungal mycelium is extremely tolerant of carbon dioxide, thriving at
20% C02 levels. Oxygen is required for formation of fruit bodies. A significant
decrease in ambient CO 2 level and increase in oxygen is critical for the initiation and
development of primordia. Thus sufficient air circulation within a mushroom fruiting
site is vital. Excessive influx of outside air, however, greatly affects both
temperature and humidity of the environment (Stamets 2000).
26
Light
As a forest-dwelling mushroom, indirect natural light is considered ideal for
the formation of Pleurotus spp. fruit bodies. Although the mycelium of the oyster
mushroom does not require light, proper fruit body formation requires moderate light.
Too little or too much light can lead to discolored, malformed fruit bodies or the
inability to fruit. Kalberer (1974) found that oyster mushroom yield was maximized
using light levels of 60 to 86 IJmol/m2/sec (300 to 430 lux) for twelve hour days.
Stamets (2000) recommends levels around 200 to 300 IJmol/m2/sec (1,000 to 1,500
lux) for commercial production.
Spawn Rate
Grain spawn provides many points of inoculation and a nutritional boost to the
substrate. Spawn rate is the amount of spawn used to inoculate the substrate and is
defined as the weight ratio of spawn to substrate. For example, a spawn rate of 5%
would entail using 50 grams of spawn (wet weight) for every 1000 grams of
substrate. Increasing the amount of spawn used in inoculation greatly increases
yield and accelerates the rate of mycelial growth and colonization, which decreases
27
spawn run time. Royce (2000) showed that increasing spawn rate from 1.25% to
5% resulted in yield increase of approximately 50% for Pleurotus and decreased
spawn run by more than seven days. Faster colonization is also advantageous in
deterring fungal competitors, by decreasing the window of opportunity for
contaminates to establish. Since spawn must be made or purchased, spawn rate
will affect production costs of cultivation. Optimal spawn rates will vary depending
on mushroom species, substrate types, and cultivation conditions (Zhang et al.
2002).
28
addition of only 1.2 g/kg N (dry weight) (Yang et al. 1980). That of hemlock sawdust
was increased 2.2% to 3.9% dry weight (Yang et al. 1980). Boyle (1998) showed
that most N-containing supplements increased the growth of several white-rot fungi.
Royce et al. (2004) showed similar yield increases using supplements with Pleurotus
cornucopiae, commonly known as the golden oyster mushroom. However, addition
of an N supplement makes the substrate more suitable for competitor fungi and
bacteria. Along with adding additional cost to production, use of supplements
creates the need for stricter sanitation (Stamets 2000). As for other nutritional
components, Boyle (1998) showed that addition of simple carbohydrates, vitamins,
and micronutrients (other than N) had limited effects on growth rates.
29
Trichoderma are the major green mold causing pathogens in the United States.
Insects also cause major problems in mushroom operations. Several
varieties of mushroom-infesting flies, including Phorids and Sciarids are persistent
pests. Maturing larvae feeds on mycelium and burrow into mushroom fruit bodies,
resulting in significant crop loss (Chang & Hayes 1978). Not only do mushroom
pests destroy mycelium and fruit bodies, they also act as vectors of contamination.
Beetles, ants, and flies of all kinds can carry bacterial and fungal disease throughout
the fruiting site, making bacterial and fungal contamination even more difficult to
control. Rats can also be a problem, as they eat the mushrooms and substrate,
creating varied levels of damage to the crop (Pettipher 1987).
Management of pests and pathogens is critical for successful mushroom
cultivation. According to Cha (2004), some basic steps can be taken for proper
management. Overall sanitation and stringent hygiene throughout the cultivation
operation are key preventative measures. Also, regular inspection for and removal
30
31
Chapter 3
Research Objectives
This study intends to evaluate the suitability of wood from five locally
occurring, fast-growing tree species as fruiting substrate for cultivation of the edible
mushroom Pleurotus sp., in Hawaii. Several components are involved in the
evaluation. Objectives of this study are:
1. To obtain substrate wood locally and evaluate its initial composition, in
terms of nutrient concentration, lignin content and cellulose content;
2. To determine substrate effect on:
a.
Economic yield
ii.
Biological efficiency
iii.
Flush
iv.
Crop period
v.
Percent moisture
32
Chapter 4
Materials and Methods
SUBSTRATE WOOD
Consultation with advisors and local foresters resulted in a general consensus
that the following five wood types are highly appropriate for this study: Fa/cataria
Initial Composition
Nutrient analysis and forage analysis were performed on samples of all five
wood species. Three samples of each wood chip type were dried for approximately
48 hours at 85C to constant weight and ground using a ball mill (Spex Inc.,
33
Metuchen, NJ). Ground samples were sent to the University of Hawaii's Agricultural
Diagnostic Service Center for analysis. Total N concentrations were quantified
using the Dumas combustion method (Bellomonte et al. 1987). P, K, Ca, Mg, Fe,
Mn, Cu, Zn, and B concentrations were quantified using an inductively coupled
plasma emission spectroscope (Issac & Jones 1972). All woods were also analyzed
for cellulose and lignin content. Classical methods (acid detergent fiber) were used
to quantify cellulose and lignin (Van Soest 1963). From these data, cellulose:lignin
ratios were calculated for each sample. Initial compositions (lignin, cellulose, and
each individual nutrient) were statistically analyzed by analysis of variance (ANOVA)
(Table 4.1) at p=0.05. SAS's general linear model procedure (GLM) was used for
the analysis (SAS 1982). Mean comparison was performed using Duncan's multiple
range test.
CULTIVATION METHODS
The method of oyster mushroom production will follow those described by
Paul Stamets in his book Growing Gourmet and Medicinal Mushroom (2000). The
Pleurotus culture was obtained from Dr. Don Hemmes of UH Hilo. A preliminary
34
cultivation experiment was conducted during the summer of 2003 using the five
wood species.
Plate Cultures
Antibiotic malt agar, purchased from Fungi Perfecti (www.fungiperfectLcom)
was used to grow pure plate cultures. Cultures were allowed 7 to 10 days to grow
across the entire petri plate. Transfer of cultures was performed in a laboratory
facility. The cultures were stored in an incubator set at 25C.
Spawn
Hard winter wheat berries were ordered through a local grocer. Grain spawn
jars were prepared by mixing 200 ml of wheat berries and 175 ml of tap water in
glass mason jars. The jars were capped with special filter lids purchased from Fungi
Perfecti (www.fungiperfectLcom). Jars were autoclaved for 60 minutes and allowed
to cool for at least 3.5 hours before inoculation. Colonized agar from plate cultures
was cut into small sections and used to create grain spawn. Sections from one plate
were used to inoculate three jars of grain. The inoculated grain was incubated in the
laboratory at approximately 25C and shaken regularly to prevent aggregation.
Spawn was ready for use after 10 to 14 days.
Substrate Preparation
The substrate recipe followed that outlined by Stamets (2000), calling for a
4:1:1 ratio of wood chips, wheat bran and water. Wheat bran provides a protein rich
35
supplement proven to improve yield and quality of Pleurotus spp. (JinTorng et al.
2000). Commercial grade flaky red wheat bran, ordered through a local grocer, was
used. Wood chips, bran and tap water were measured by volume and thoroughly
mixed by hand in large plastic bins. Mushroom cultivation bags purchased from
Fungi Perfecti (www.fungiperfecti.com) were used as fruiting containers. A three
liter bucket was used for filling bags, each bag receiving one bucket of substrate
mixture. Bags were folded loosely, autoclaved for three hours, and allowed to cool
overnight.
Sterilized substrate bags were inoculated with grain spawn in a laminar flow
hood. One jar of spawn was used to inoculate three bags of substrate. Using mean
initial bag weight, the inoculation rate (wet weight) was calculated to be 6.5%, an
acceptable rate for outdoor bag cultivation using grain spawn. Inoculated bags were
sealed using an impulse sealer manufactured by Clamco Corporation (Cleveland,
OH) and transferred to the incubation room. An existing room at the University of
Hawaii's Beaumont Research Station was modified and used as the incubation
room. The 14.8 m2 , uninsulated room was surfaced sterilized and all windows were
covered using black plastic sheeting and duct tape. Two HEPA air cleaners
manufactured by Honeywell (www.honeywell.com) were set up in the room in
attempt to reduce air-borne contaminates. The existing air-conditioning unit was
fitted with an additional filter used to maintain a 25 to 30 C temperature. All bags
were incubated for 15 to 16 days.
36
Fruiting
At the end of the 15 to 16 day incubation period, the mycelium had grown
throughout the substrate and the bags were transported to the fruiting site. An
existing shade house at the University of Hawaii's Waiakea Experiment Station
(155W longitude, 19N latitude) was selected as the fruiting site. The 30.5 m2
shade house had been constructed using fine screen for insect control experiments.
Several adaptations were made to the shade house in order to use it as a mushroom
fruiting site. Major tears in the screen were repaired or covered. A seven centimeter
layer of rock gravel was spread on the floor of the house to control weeds and
potential contaminates. A specially designed, timer-operated misting system was
installed to achieve necessary humidity levels in the house. Poly tubing was
positioned centrally over each bench-top, approximately one meter above the bench
surface. One-GPH barbed fogging nozzles (DIG Irrigation Products, Vista, CA) were
installed in the poly tubing, one every 1.2 meters. The misting system was
controlled by a one-station battery operated controller manufactured by DIG
Irrigation Products.
For each bag, the bags were cut open and holes cut in the sides to drastically
decrease ambient CO2 levels and initiate formation of primordia. To create high
humidity, the misting system was set to operate for two minutes every hour
throughout the day and night. All bags were kept in the fruiting site for
approximately 8 to 10 weeks, allowing for multiple flushes of fruit bodies.
37
Experimental Design
The experiment followed a split-plot design, where the main plot was batch
and the split-plot was substrate. The experiment was blocked on location among the
greenhouse benches. The five woods served as substrate treatments and all
treatments replicated into ten blocks. With each batch consisting of ten replicates,
treatments were tested in two batches 15 days apart. In total, 100 bags
(experimental units) were created, 20 per substrate treatment. The following
equation was used to estimate that 20 was a sufficient number of replicates to
achieve high statistical power:
Equation 4.1.
where 0=15% and p=0.05. The coefficient of variation (CV) of 12.5% was obtained
from previous studies on mushroom cultivation (Zhang et al. 2000). Using a 0 value
of 15 allows for the detection of true differences between treatments as 15% of the
mean. The t values, at p=0.05, were found in the standard table of t-values; t1 = 1.96
and t2 = 2.1816. All statistical analyses performed were analyzed at p=0.05.
38
39
Of
1
9
__ ~rr9-~ A_(~~!~!(~!~_~~l
Substrate
N-fixers vs. Others
Fruit-tree vs. Others
High N vs. Others
High Mn vs. Others
Batch*Substrate
Error B
Total
~__
4
{1}
{1}
{1}
{1}
4
72
99
---~----------------------------------------------------
Biological Efficiency
Equation 4.2.
C. R. =
X 100%
mean B.E. values were correlated with lignin: cellulose, lignin, and cellulose
concentrations.
Flush
In addition to date, flush number was also recorded at each harvest. Three
components of flush were studied: flush number, distribution of yield per flush, and
flush period. Total number of flushes produced was noted for each bag and is
referred to as flush number. The distribution of yield per flush was tabulated in order
to look at changes in yield over the course of multiple flushes. Flush periods were
determined for each bag by quantifying the intervals between subsequent flushes.
Using SAS, yield distribution throughout flushes was analyzed with a repeated
measure design using the Mixed procedure (Table 4.3) (Ray et al. 1982). The GLM
procedure was used to analyze flush period and flush number (Table 4.4).
~O
Total
499
--------------------------------------------------_.
41
Table 4.4. Analysis of variance of substrate effects in two batches on flush period
and flush number.
AN OVA
Batch
Block
__~~~~~ _~ {~~!~_~~_~!9_~~1
df
1
9
~ __
Substrate
4
Batch*Substrate
4
Error B
72
--------------------------------------------------_.
Total
99
Crop Period
In the mushroom industry, crop period is considered the duration of time from
inoculation through final harvest. The crop period entails two main phases:
incubation and fruiting. Duration of time (days) was calculated for incubation
periods and fruiting periods for each bag. Crop period was analyzed using SAS's
GLM procedure using ANOVA similar to that of flush period (Table 4.4).
Percent Moisture
Six dry weight sub-samples were taken for each substrate treatment. For all
treatments except Casuarina, three of the six samples were from batch 1 and three
from batch 2. In the case of the Casuarina treatment, two samples were from batch
1 and four from batch 2. Sub-samples were weighed, dried for 6 to 8 hours to
constant dry weight at 85C in a drying oven and re-weighed once dry. Fresh
weights and dry weights were used to calculate water content (% moisture) of fruit
42
bodies. Percent moisture values were analyzed using the GLM procedure, including
effects of substrate, batch and replicate (Table 4.5).
Table 4.5. Analysis of variance of substrate effects on fruit body percent moisture.
ANOVA
Batch
Replicate
__ ~~~~~ _~ {~~R~~~<?_~~1
df
1
5
~_.
Substrate
4
Batch*Substrate
4
Error B
10
---------------------------------------------------Total
29
43
df
2
4
8
14
-------------------------------------------------~_.
44
were made to the taste test questionnaire to make the test less subjective (Appendix
B). The 1 to 5 rating scale represented varying levels of flavor or aroma intensity.
The scale for the second test was defined as: 1=none, 2=weak, 3=mild, 4=moderate,
5=strong. The two questions regarding selection of a best sample were included in
this test (Questions 2 and 4). However, the two questions regarding detection of
differences (Questions 1 and 3) were omitted. The second test included mushroom
samples from all five substrate treatments. Edible mushrooms grown on Eucalyptus
were confirmed safe to eat by commercial growers in Tasmania, Australia (K. Stott,
personal communication).
Twenty-three individuals participated in the first test. Twenty-eight
volunteered for the second test. Data for each test was statistically analyzed
separately using ANOVA (Table 4.7 and 4.8) and Duncan's multiple range test for a
comparison of means.
Table 4.7. Analysis of variance of substrate effects on fruit body taste - test I.
ANOVA
df
Rater
22
Substrate
3
Error
66
---------------------------------------------------Total
91
Table 4.8. Analysis of variance of substrate effects on fruit body taste - test II.
ANOVA
df
Rater
27
Substrate
4
Error
108
--------------------------------------------------_.
Total
139
45
ECONOMIC ANALYSIS
With the help of Jim Hollyer, Director for CTAHR's Agricultural Development
in the American Pacific (ADAP) program, an existing agricultural economics
spreadsheet was adapted for small-scale outdoor mushroom cultivation in Hawaii.
The spreadsheet designed by the University of Hawaii's College of Tropical
Agriculture and Human Resources for taro production in Hawaii was used as a
template (Fleming & Sato 2001).
46
Time required and labor involved in each step of cultivation was recorded
throughout the experiment. All materials and supplies involved in the cultivation
were tracked also. These values, along with others found in recent literature, were
used in the analysis to compute costs. Economic yield quantities generated in the
cultivation experiment were used to predict potential revenue. Using approximate
costs and potential revenue, the economic feasibility of small-scale, outdoor
mushroom cultivation in Hawaii will be discussed.
47
Chapter 5
Results and Discussion
SUBSTRATE WOOD
Nutrient
Overall, nutrient composition varied considerably among the five woods.
Concentrations of N, P, K, Ca, Mn, and Cu showed the greatest variation. A
significant difference (p=0.014) in N concentration was found among the five
substrate woods. The mean N levels in Fa/cataria was 6.3 g/kg; which is
comparable to that of rice straw, 6.4 g/kg, (Muthukrishnam et al. 2000) and wheat
straw, 6.2 g/kg (Stamets 2000). Casuarina, Psidium, Trema, and Euca/yptus were
all considerably lower in N than Fa/cataria (Table 5.1). Although Fa/cataria,
Casuarina and Trema are all N-fixing species, Fa/cataria was the only wood to have
less than that reported for rice straw (1.7 g/kg) (Muthukrishnam et al. 2000) and
wheat straw (0.7 g/kg) (Stamets 2000). Using Duncan's multiple range test for mean
comparison, Fa/cataria wood showed significantly higher P levels than Casuarina,
Trema, and Psidium, but lower levels than Euca/yptus.
48
multiple range test, revealed that K concentrations of both Trema and Psidium (1.5
g/kg and 1.4 g/kg, respectively) were greater than the other three woods, although
no significant difference existed between Psidium and Casuarina. Straw (wheat) is
reported to contain 7.9 g/kg K (Stamets 2000).
There was a statistically significant difference (p=0.026) in Ca concentration
of the five woods. Calcium level of Psidium cattleianum (14.8 g/kg) was two to three
times the levels in all other woods. Comparatively, straw (rice) is approximately 2.8
g/kg Ca (Muthukrishnam et al. 2000).
The most drastic difference in mineral content between woods was seen in
Mn. Manganese concentration of Eucalyptus grandis was significantly higher
(p<.0001) than that of the other four woods. Mean Mn concentration of Eucalyptus
(235.7 IJg/g) was more than 5 times that of Casuarina and more than 47 times that of
Trema.
Finally, results of Cu concentrations showed a highly significant difference
(p=.002) among the wood substrates. The mean Cu concentration of Falcataria
moluccana (9.7 1J9/g) was statistically greater than the other four woods (5.7 to
6.7IJg/g), as exhibited by Duncan's test for mean comparison (p=0.05).
No significant difference in Mg content existed among the five woods. Nor
was there significant difference in levels of Fe, ln, or B (Appendix C). The use of
wheat bran as a substrate supplement contributed an additional 24.8 g/kg N
(Stamets 2000). The bran supplement also provided additional K (11.8 g/kg), P
(10.1 g/kg), Mg (6.1 g/kg), Ca (0.73 g/kg), Mn (0.12 g/kg), Fe (0.11 g/kg), ln (0.07
49
g/kg), and Cu (0.001 g/kg). Nutrient concentrations were derived from nutritional
label information.
Table 5.1. Initial mineral concentration* of five woods used as substrates for
cultivation of P/eurotus sp.
Substrates
N (g/kg)
P (g/kg)
K (g/kg)
Ca (g/kg)
Mn (l-Ig/g)
Cu (l-Ig/g)
Fa/cataria
6.3a
0.3b
0.8c
5.3b
23.0b
9.7a
Euca/yptus
1.0b
0.5a
0.8c
4.7b
235.7a
6.7b
Casuarina
3.5b
0.2c
1.0bc
7.4b
40.7b
5.7b
Psidium
2.0b
0.1c
1.4ab
14.9a
6.3b
5.7b
Trema
1.6b
0.2c
1.5a
5.8b
5.0b
6.7b
*Values are means of 3 replicates. Values not sharing the same common letters are
significantly different at p=0.05 as determined by Duncan's multiple range test.
50
Table 5.2. Percent lignin, cellulose and cellulose:lignin ratio of five substrate woods*.
Substrate
Lignin (%)
Cellulose (%)
Cellulose: Lignin
Falcataria
30.79b
17.19bc
0.57
Eucalyptus
47.69a
16.53c
0.35
Casuarina
43.74a
22.05b
0.50
Psidium
44.35a
28.67a
0.66
Trema
44.66a
21.98b
0.49
*Values are means of 3 replicates. Values not sharing the same common letters are
significantly different at p=0.05 as determined by Duncan's multiple range test.
It should be noted that wood analyses were done several weeks after the
cultivation experiment had started. The chips used as substrate were considerably
'fresher' than those used for the chemical composition analysis. All wood was
chipped less than a week prior to substrate mixing. Chips used in the chemical
analysis were taken from the pile after the cultivation experiment had started,
approximately ten weeks after they had been chipped. Other microbes active in the
wood chip piles during storage may have affected chemical composition results
observed.
PRELIMINARY EXPERIMENT
Of the 15 bags inoculated in the preliminary experiment, only six successfully
fruited with oyster mushrooms. Trema and Psidium were unsuccessful in producing
Pleurotus sp. fruit bodies. The six producing bags included two replicates of each
51
to the very low number of flushes produced. The quantity of substrate in the
preliminary experiment bags was greater than that of the final experiment. The
increased substrate quantity explains the considerably greater mean yields (fresh
weight, g/bag) obtained in the preliminary experiments.
Table 5.3. Mean fresh weight yield, biological efficiency, flush number, and crop
period of Pleurotus sp. cultivated on five wood substrates in the preliminary
experiment*.
Fresh weight
Biological
Flush
Crop period
Num.
Substrate
(g/bag)
efficiency (%)
num.
(days)
reps
Falcataria
657.1
75.5
1.5
45.0
Casuarina
710.5
82.4
2.5
53.0
Eucalyptus
425.7
50.5
2.0
56.5
Psidium
NF
NF
NF
NF
Trema
NF
NF
NF
NF
52
Results of the preliminary trials suggested that substrate wood did not greatly
affect yield, biological efficiency, or crop period of Pleurotus sp. cultivation.
However, only three of the five wood types were included in the analysis and low
replicate numbers resulted in a weak statistical analysis.
FINAL EXPERIMENT
Substrate Effect on Crop Yield
Economic Yield
Economic yield values reported are the total fresh weight of mushrooms
produced per bag of substrate over the course of three flushes. The highest mean
yield observed was that of the Casuarina substrate; 275.5 grams of fresh
mushrooms per bag. Trema gave the second highest yield (272.4), followed by
Falcataria (268.8), Eucalyptus (250.7) and Psidium (190.5). Standard errors of
these means were substantial (Figure 5.1).
53
350
300
........ 250
en
E
co
L-
-a>
200
a>
150
en
(I)
L-
U-
100
50
0
Falcataria
Casuarina
Eucalyptus
Psidium
Trema
Substrate
Figure 5.1. Mean economic yield per bag of Pleurotus sp. cultivated on five wood
substrates and standard errors of the mean, as indicated by error bars.
The range of individual yields observed was quite wide, from 535.3 to 55.7
glbag. This variation is likely due to contaminants and/or climatic variations within
the greenhouse. Close control of climate and cleanliness, as seen in sophisticated
mushroom operations, are major factors in ensuring high and consistent production.
Much of this control was forfeited with the choice of a low-cost, outdoor growing
facility. Green mold, Trichoderma spp., was noted as the most prevalent
contaminant. Sporulation of the competitor fungi occurred in some of the bags, as
54
evident by green patches on the substrate surface. As an outdoor fruiting site, the
bags were subject to air flow by wind, which could have exacerbated the incidence
of green mold. Finding ways to control climate and sterility in low-tech operations
will likely reduce the variation and increase overall yield.
Regardless of contaminants, exceptionally high economic yields were
achieved in at least one bag of all substrates. Maximum yield values (Table 5.4)
confirm that higher yields are possible with all five substrate types using an outdoor
growing facility.
Table 5.4. Range of economic yield values* of P/eurotus sp. fruit bodies.
Substrate
Mean
Maximum
Minimum
Fa/cataria
268.8
445.9
73.6
Casuarina
275.5
535.4
112.2
Euca/yptus
250.7
398.7
60.9
Psidium
190.5
447.4
55.7
Trema
272.4
515.3
60.0
freedom contrasts between N-fixing trees and non-fixing trees, revealed a significant
difference in yield (p=0.018). Wood of N-fixing trees supported greater yields of
P/eurotus sp. than did wood of the non-fixing trees. The rates at which these trees
can fix N do vary, as did the N concentration found in the wood tissue analysis
(Table 5.1).
In general, fruit trees are reputably poor for mushroom production (Stamets
2000). When Psidium, a prolific producer of sweet, fleshy fruit, was contrasted
against the others, a highly significant difference (p=0.005) was found. Differences
between the wood of fruit trees and wood of non-fruit trees would have to be further
investigated to support this finding.
When low-Mn woods were contrasted against high-Mn woods, no significant
difference (p=0.702) in yield was found. Also, no significant correlation was found
between mean Mn concentration and mean yield (p=0.919). Manganese availability
is known to influence the activity of certain lignin-degrading enzymes. Boyle et al.
(1992) showed that media low in Mn supported faster lignin degradation for P
chrysosporium; however, P. pu/monarius was not affected by Mn levels. It is
possible that variations in Mn levels among woods did not vary sufficiently to affect
fungal growth. Another possible explanation is that this species of P/eurotus, like P.
pu/monarius, is not influenced by Mn levels in substrate.
56
Trema are all N-fixers, only Fa/cataria wood had a significantly higher (p= 0.014)
level of N than other woods (Table 5.1). However, when P/eurotus yield using
Fa/cataria substrate was compared to that of other (Iow-N) wood substrates, no
57
yields than trees that do not fix N. However, N concentration of the wood does not
show a correlation with Pleurotus yield. Manganese is an element of physiological
importance in the lignin degradation process of white-rot fungi. Although a large
portion of the degradation enzymes are Mn dependant, variations in Mn among
wood does not appear to have a serious effect on yield of this Pleurotus species.
Lastly, yield did not seem to be directly related to lignin or cellulose levels of various
woods. Again, such levels may not be great enough among wood tested to produce
a notable effect on yield.
Blocks were set up to reduce yield variation due to position within the shadehouse. However, no statistical significance of block effect on economic yield was
found. Anticipated sources of variation were wind direction and proximity to the
shade house entrance, both which could be factors in contamination.
Although there was no significant difference among blocks, there was a
significant difference (p=O.017) observed between the two batches. Economic yields
from batch 1 were notably higher than those of batch 2 (Figure 5.2).
58
400.-------------------------,
300
,....,
0>
"0
Q)
>=u
"E
c:::=:=J Falcataria
r=~~
200
0
c::
0
~
~
~
~
~
~
~
UJ
100
Casuarina
Eucalyptus
~ Psidium
~ Trema
~
Batch
Figure 5.2. Economic yield of Pleurotus sp. on five substrates in two batches.
Standard errors of the means are indicated by error bars.
The two batches were separated temporally by 15 days. Fruiting was initiated
on March 1, 2004 for bags of batch 1. Batch 2 bags entered the fruiting stage on
March 15, 2004.
A major difference between the two batches was the state of the fruiting site
when the bags were moved into the fruiting stage. For batch 1, the shade house
was empty and clean when bags were set out. When batch 2 bags were set out, the
site had been in production for 15 days. A higher incidence of contaminants may
have been present in the shade house when batch 2 was set out. Although
contamination was not quantified throughout the experiment, it was noted in several
59
incidences. Species of P/eurotus are thought to be more tolerant of green mold than
other cultivated fungi, but some effect on yield is inevitable (Stamets 2000).
An interaction between substrate and batch effects was not found to be
statistically significant (p=0.714). It can be assumed that substrates do not act
differently when exposed to the effects of batch. Similarly, the interaction between
batch and block effects did not exhibit significance (p=0.152). Thus, all blocks were
influenced by the batch effect similarly.
Biological Efficiency
When initial weights of the inoculated bags were examined, a significant
difference (p<0.0001) among substrates was found. Average bag weight of Psidium
was far greater than that of other substrates. Mean bag weight of Casuarina was
less than that of Psidium, but greater than the lightest three: Fa/cataria, Euca/yptus,
and Trema (Table 5.5).
Cultivation bags were filled using volumetric measurement. Thus the
differences in initial bag weight resulted from density variation among the different
woods. Wood of both Psidium and Casuarina are relatively dense and heavy
woods. Specific gravity of Casuarina equisetifo/ia is 0.81 (Little & Skolmen 1989),
over twice that of either Trema orientalis or Fa/cataria mo/uccana. Psidium
60
61
concentrations of the substrates (Table 5.1), it is possible that the variations among
woods were not great enough to affect Pleurotus growth and fruiting.
Similarly to economic yield, a significant difference in B.E. was found
between batch 1 and 2 (p=0.002). Mean B.E. for batch 1 was 78.6%, while that of
batch 2 was only 54.2%. Again, the B.E. differences between the batches are likely
due to greater contaminants present at the site when fruiting of batch 2 was initiated.
When mean B.E. was analyzed by individual batch, effect of substrate
remained similar. Batch 1 averages were between 75% and 100% for all woods
except Psidium (Table 5.5). The batch 2 average B.E. for Psidium was quite low,
30.6%, while that of other substrates were between 59% and 63% (Table 5.5).
Table 5.5. Initial bag weight and biological efficiency yield of Pleurotus sp. cultivated
on five substrates in two batches.
Batch 1
Batch 2
Fa/cataria
1448.9c
85.6 (6.2)
62.4 (9.6)
Casuarina
1616.8b
77.6 (9.8)
61.1
Eucalyptus
1414.0c
81.5 (7.0)
60.5 (10.5)
Psidium
1753.8a
57.7 (9.7)
30.6 (3.4)
Trema
1414.4c
97.9 (10.8)
59.1
(8.5)
(8.4)
* Values not sharing the same letters are significantly different at p=0.05 as
determined by Duncan's multiple range test.
**Values are means of 20 replicates, followed by standard errors.
62
ostreatus on coco shell waste. Similarly, Thomas et al. (1998) obtained B.E. yields
ranging from 38% to 59% using a low-cost mushroom shed for outdoor production
and coconut palm waste as substrate. In India, P. pulmonarius yields on rice straw
peaked at 71 % using outdoor cultivation sheds (Muthukrishnan et al. 2000). Using
wood chips of Acacia sp. supplemented with wheat bran, B.E. values of 60.7% and
54.3% were achieved for P. ostreatus and P. pulmonarius grown outdoors in South
Africa (Da Serra & Kirby 1999). Although other production yields provide a decent
point of reference, direct comparisons are different due to differences in methods,
substrates, and locations.
63
As expected, there was a definite decline in yield over the course of five
flushes (Figure 5.3). Substrate, however, did not have a significant effect on the
pattern of decline observed between flushes (p=O.058).
250
200
--
150
~
..c.
100
C>
'--"
- . - Falcataria
-0-- Eucalyptus
-y- Casuarina
---v-- Psidium
----.- Trema
..c.
C>
en
~
u-
50
F1
F2
F3
F4
F5
Flush
Figure 5.3. Change in mean yield throughout five flushes. Standard errors of mean
are shown by error bars.
64
Table 5.6 illustrates the average number of flushes produced for each
substrate. There was a difference in flush number observed between the five
substrates (p=0.04). The flush numbers produced by Fa/cataria and Trema were
higher, where those for Euca/yptus and Psidium were lower. On average, bags in
the first batch produced 3.7 flushes, while those in the second batch produced only
2.2. The difference in flush number between the two batches (p=0.0001) is likely
due to contaminants, as discussed earlier. Differences between batches may also
be due to natural fluctuations in environmental conditions. In general, Fa/cataria,
Casuarina and Trema produced greater yield and more flushes than Euca/yptus and
Psidium.
Table 5.6. Mean number of flushes produced by P/eurotus sp. cultivated on five
substrate woods.
Substrate
Flush Num.*
Fa/cataria
3.45a
Casuarina
2.95ab
Euca/yptus
2.65b
Psidium
2.55b
Trema
3.20a
* Values are means of 20 replicates. Values not sharing the same common letters
are significantly different at p=0.05 as determined by Duncan's multiple range test.
65
Crop Period
The duration of the incubation phase, the fruiting phase, and the overall crop
period (sum of incubation and fruiting) for each substrate treatment are presented in
Appendix E. The average crop period for batch 1 was 50.9 days, while that of batch
2 was only slightly longer at 51.2 days. The difference among batches was not
found to be statistically significant (p=0.915). With regard to substrate, Eucalyptus
exhibited the shortest crop period (48.5 days), while Psidium was the longest (53.3
days). However, differences observed were not statistically significant (p=0.868).
Since crop period indicates time invested per crop, it is important to keep it as low as
possible without negatively affecting yield. Relative to findings of other studies, all
five woods supported a relatively rapid cropping period for this particular species of
Percent Moisture
Sub-sample dry weights were used to calculate the percent moisture of fruit
bodies cultivated on the five substrate woods. Overall, mean moisture content of the
Pleuratus fruit bodies was calculated to be 79% (Appendix F). Most literature states
that fresh mushroom fruit bodies contain approximately 90% water on average
(Crisan & Sands 1978, Stamets 2000, Sano & Rajarathnam 1988). It is also agreed
that environmental factors (temperature and relative humidity) during growth and
storage have effects on mushroom moisture content (Crisan & Sands 1978, Stamets
2000, Sano & Rajarathnam 1988). Fluctuations in environmental conditions are
likely the reason for the lower than average percent moisture content observed.
66
Pleurotus sp. fruit bodies grown on five woods was 56.9 g/kg. Potassium and Pare
the other main mineral constituents of Pleurotus fruit bodies with mean
concentrations of 14.0 and 26.0 glkg, respectively. Both Ca and Mg are present in
comparatively low levels (1.1 and 1.9 glkg, respectively). Mean Fe concentration of
the fruit bodies was 107.27 1-1 gIg , while that of Zn was 105.20 I-Ig/g. Manganese, Cu,
and B were also found only in small concentrations (13.47,18.13, and 3.20 1-19/g,
respectively). Mean levels of all fruit body nutrient concentrations when grown on
various woods are listed in Appendix G.
Patrabansh and Madan (1999) studied the effect of four organic plant waste
substrates on the mineral concentration of Pleurotus pulmonarius fruit bodies.
Variations in mineral concentration (Ca, P, K, Mg, Fe, Mn and Zn) of fruit bodies
67
Taste Test I
Results of the first taste test were fairly subjective since the ranking scale was
based on preference. Also, only four of the five treatments were evaluated, giving
an incomplete analysis. Thus, the first test served as preliminary data for mushroom
aroma and flavor. Using results and comments received from the first test,
improvements were made for the second test.
Ratings for aroma in test I showed no significant difference (p=O.765) among
mushrooms of various substrates (Figure 5.4). When asked if a difference in aroma
could be detected (question 1),52 % of the participants answered 'yes', while 22%
answered 'no', and 26% opted not to answer. Question 2 asked raters to select a
best sample for aroma, the majority of the participants chose N/A, or opted not to
answer.
68
4
I/)
c=J Fa/cataria
Cf)
Q)
Casuarina
Psidium
18888881 Trema
~
o
c
~
~
Q)
a..
Flavor
Aroma
Figure 5.4. Test I's average preference scores for both aroma and flavor of
mushrooms grown on five woods with standard errors of the means indicated by
error bars.
69
Taste Test II
Test II was made more objective by changing the ranking scale from
preference levels to aroma/flavor intensity levels. To streamline the test sheet, the
two questions referring to detection were removed. Ability or inability to detect
aroma or flavor differences was incorporated into questions 1 and 2. Lastly, the test
II was a more complete, as it included samples of mushroom grown on Eucalyptus.
Aroma ratings for test II were similar to those of test I. No significant
difference (p=O.596) was determined among samples. On average, intensity scores
for flavor were higher than scores for aroma (Figure 5.5).
t-
'~
wc
~
1=
f=
1=
f=
1=
f=
1=
1=
f=
1=
f=
1=
~
(f)
c=J Falcataria
~
~
Casuarina
Eucalyptus
Psidium
Trema
1=
~
f=
1=
f=
1=
~
o
Aroma
Flavor
Figure 5.5. Test II's average intensity scores for aroma and flavor of mushrooms
grown on five woods with standard errors of the means indicated by error bars.
70
When asked to select the best sample for aroma (question 1), 50% of the
participants chose N/A or opted not to answer. Other choices were deemed best for
aroma by only a few participants.
There was a highly significant difference (p=O.004) in flavor ratings of the
second test. The rating scale for test II was based on flavor intensity, and results
revealed that mushrooms grown on Casuarina were deemed most flavorful (Figure
5.5).
Results from question 2 showed trends in preference. It was quite evident
that mushrooms grown on Casuarina were highly favored over others. Seventy-five
percent of people who took the test chose the mushroom sample grown on
71
Table 5.7. Mean, maximum and minimum values for temperature and relative
humidity within the mushroom fruiting shade house.
Week
1
2
3
4
In
May
May
June
June
Temperature CC)
Mean
Max
Min
25.9
44.4
18.3
25.7
42.2
18.3
26.0
42.2
17.2
26.1
43.3
17.8
72
Relative
Mean
71.7
74.3
70.3
65.8
Humidity (%)
Max
Min
101.5
9.8
100.9
12.7
100.9
11.2
100.6
8.0
Lack of light is
known to prevent proper formation of Pleurotus caps, while intense, direct sunlight is
said to be harmful to mushroom growth (Stamets 2000). In this experiment,
mushroom formation did not appear to be negatively affected by light levels in the
shade house, as evident by the relatively high yields achieved (Pettipher 1987,
Thomas et al. 1998). The shade house provided light levels suitable, though not
ideal, for Pleurotus production.
Although the shade house was constructed using insect screen, it was not
completely effective in keeping all insects out. Of the 30 days during which
observations were taken (harvest dates), only five included notes of insect(s)
presence in the house. Flies were found inside the shade house at the end of the
production cycle. The presence of earwigs and cockroaches crawling on the
substrate or fruit bodies was also noted. In addition, ants were found crawling inside
shade house, but never on or near the mushroom bags.
73
Presence of, or damage by, other pests was also noted. Rats are known to
cause losses in mushroom production (Pettipher 1987). A rat was assumed to be
the cause of small damages caused to several substrate blocks and fruit bodies.
Several days after rat bait was used, damage to the blocks and mushrooms ceased.
Lizards were also found inside the house on multiple occasions, however rarely on
or near the mushroom bags.
Overall, the shade house was a suitable outdoor fruiting site. Although
environmental conditions fluctuated considerably, mushroom yield was relatively
high for outdoor production. Efforts to make temperature, humidity, and light more
stable may result in higher yields. Providing more shade, by an additional overhead
structure, shade cloth or trees, would reduce maximum light levels and may help to
ameliorate extremely high temperature and low humidity.
ECONOMIC ANALYSIS
Assumptions
74
Similarly, all costs provided are general estimates and assumptions, but are
specific to the production done in this experiment. The cultivation methods used in
this experiment were chosen with intentions of keeping costs as low as possible. In
addition, some of the costs provided are specific to mushroom species, climate, and
substrate. Adjustments to the provided costs can be made by potential mushroom
growers to suit their specific situation and needs. The cost used for substrate in this
analysis was $4.20 per cubic meter and represents the cost of purchasing mixed
species wood chips from a local tree service company, delivery included. Costs
associated with obtaining substrate will vary and depend on source and methods
involved in obtaining it.
One assumption is that land is owned and the cost of land used is zero. All
conclusions drawn are based on the assumption that the land needed for mushroom
production is owned and costs associated are zero. Also, a 5% commercial lending
rate is used throughout the analysis and figures do not incorporate length of loan
period or duration of interest payments. A $10 per hour labor cost is used
consistently throughout the analysis. Return to management costs have been
omitted from the analysis for simplification. In addition, the analysis assumes that
mushrooms were sold wholesale in 100 gram packages.
Potential Revenue
Using yields obtained in the cultivation experiment, potential annual yield was
estimated to be 1,150 kg of fresh mushrooms, at a production rate of 4,200 bags per
75
76
Table 5.8. Assumptions underlying cost estimates for small-scale, outdoor oyster
mushroom production in Hilo, HI.
Item
Building/Facilities construction
Production facility
845 /square m.
Fruiting facility
430 /square m.
Spawn production
Wheat berries (retail)
1.3 /kg
49 /set of 100
75 /kg
Substrate preparation
Substrate
4.2 /cubic m.
0.72 /kg
Harvesting
4.8 /100
Packaging materials
General operations
Water (liters)
Water charges
13.41
Electricity
/month
0.06 /MJ
10 /hour
Labor
77
Table 5.9. Estimated production costs associated with small-scale, outdoor oyster
mushroom production in Hilo, HI at two production levels.
Production Level*
P1
Item
P2
184
Filter discs
10
Culture agar
10
29
18
53
251
752
Purchased bags
559
1,676
169
508
Water
165
175
Electricity
873
2,619
Facilities
2,393
3,726
305
334
6,547
22,835
$11,354
$32,899
Wheat berries
Bag production
Substrate
Harvesting
Packaging Materials
General operations
Equipment
Labor
Total Annual Cost
*P1 represents a production rate of 1,400 bags per year, while P2 represents a rate
of 4,200 bags per year.
78
(Falcataria) and 2500 kg (2.5 t) (Casuarina) of wood chips would be needed every
year for mushroom production. This range is only an estimate since chip weight
would vary with water content, wood density, and chip size.
79
80
If Falcataria were planted, less than 2.7 hectares (1.1 acres) of land would be
needed to produce sufficient wood for mushroom cultivation. Harvesting of
approximately 613.3 m2 (6,600 fe) each year would produce the 1,500 kg of wood
chips needed for mushroom production. If Casuarina were planted, approximately 6
hectares (2.5 acres) of land would be required. Thus, roughly 1,390 m2 (15,000 fe)
could be harvested annually to produce necessary substrate quantities.
Growing trees on a short-rotation for substrate would also entail an additional
cost to the mushroom production. Assumptions underlying estimated costs for
short-rotation tree farming are listed and explained in the USDA General Technical
Report PSW-GTR-137 written by Whitesell et al. (1992). These costs and
assumptions were tabulated for Eucalyptus; therefore consideration should be given
to variation due to species, as well as inflation, location, etc. Additionally, the issue
of obtaining substrate in the first seven years of production is of great importance.
81
economically viable than building new ones. Adapting pre-existing structures may
significantly reduce costs involved with facilities.
Annual cost for the cultivation bags is also rather expensive. Reusable
containers, such as plastic crates or metal boxes maybe used to reduce this annual
cost. This would, of course, add to initial investment costs. Lastly, the electricity
cost is high. Electricity requirements were estimated very roughly for the analysis
and a reevaluation of electricity requirements and costs would be suggested.
An increase in the rate of production may result in a positive profit. In order to
significantly increase production, additional labor or equipment would be necessary.
Motorized mixers are often used in larger mushroom operations. Steel mixers
available through Fungi Perfecti can process up to 2000 bags of substrate per day,
but are a costly investment. The price for such a mixer was quoted at approximately
$19,500, not including shipping. Purchasing spawn, rather than making it, would
make more time available for mushroom production. One kilogram of spawn costs
approximately $3.25, not including shipping (Royce 2002). Costs of purchased
spawn should be weighted against costs and benefits of making spawn (time,
supplies, and materials).
82
Economic Summary
P2
P1
Per Year
Per Bag
Per Year
Per Bag
Total REVENUE
$3,920
$2.80
$11,760
$2.80
Total COSTS:
$11,630
$8.31
$34,206
$8.14
Total PROFIT:
-$7,710
-$5.51
-$22,446
-$5.34
*Production rates presented are 1,400 bags/yr (P1) and 4,200 bags/yr (P2).
Further
reductions in costs are necessary to make it a financially viable option for farmers in
Hawaii. Additionally, increased production, through the use of machines versus
manual labor, may be possible to increase profits.
83
Chapter 6
Summary
suitable substrates for oyster mushroom cultivation in Hawaii. Economic yield and
biological efficiencies achieved by the three N-fixing trees (Fa/cataria, Casuarina and
Trema) were greater than those of Psidium and Euca/yptus, which do not fix N.
Also, results do agree with the literature that fruit trees (such as Psidium) are poor
substrates for edible mushroom production. Economic yield and B.E. did not appear
to be affected by variations in nutrient, cellulose, or lignin contents of the five woods.
Also, flush, crop period, fruit body percent moisture were not affected by wood
substrate.
Interestingly, substrate did not affect nutrient content of mushrooms.
Likewise, it appears that substrate did not influence the aroma of the mushrooms.
However, definite differences in mushroom flavor were detected among fruit bodies
grown on the various woods. Mushrooms grown on Psidium received a low
preference score (test I), while those grown on Casuarina were most often chosen
as the best sample (test II). Mushroom flavor, as well as aroma and nutrient
content, are factors involved in the marketing of edible mushrooms. Preferred taste
due to substrate may be beneficial in the marketing aspect of small scale mushroom
industries. Further taste tests are recommended to more accurately evaluate effects
of wood substrate on mushroom taste.
84
All five woods were successful in supporting Pleurotus sp. growth and fruiting.
However, Psidium did produce lower yields in terms of B.E. and low scores for taste.
In addition to growth characteristics which make the trees more difficult to harvest,
these factors make Psidium a less desirable choice for mushroom production. Of
the other four woods, the optimal choice for a potential grower would depend mostly
on costs involved with obtaining chips. As each farmer's situation is unique, so will
be the factors involved in securing a sustainable, cost-effective source of substrate.
Using methods described, small-scale, outdoor mushroom cultivation in
Hawaii does not appear to be financially feasible. Suggested measures to
increasing production or decrease initial costs might help the economic outlook for
this agricultural endeavor.
85
Appendix
A. Taste test I survey questions
Please SMELL the following oyster mushroom samples and rate them for AROMA on a scale of 1 - 5.
Sample A:
00
0'\
(unappealing)
1
(poor)
(good)
(fair)
(excellent)
5
Comment
Sample B:
Comment
Sample C:
Comment
Sample D:
Comment
1. Could you detect a difference among the samples' aroma? (please circle)
yes
no
Please TASTE the following oyster mushroom samples and rate them for FLAVOR on a scale of 1 - 5.
Sample A:
(unappealing)
1
Sample B:
(poor)
(fair)
(good)
n/a*
Please circle your choice.
(excellent)
Comment
Comment
Sample C:
Comment
Sample D:
Comment
yes
no
n/a*
3. Could you detect a difference among the samples' flavor? (please circle)
4. Overall Best Sample for flavor: (please circle)
* indicates no detectable difference among samples
_
_
_
_
Date Taken:
Appendix
B. Taste test" survey questions
Please SMELL the following oyster mushroom samples and rate the MUSHROOM- AROMA on a scale of 1 - 5. Please circle your choice.
INTENSITY
(none)
(weak)
(mild)
(moderate)
(strong)
Sample A:
1
2
3
4
5
Comment
_
00
'I
Sample B:
Comment
Sample C:
Comment
Sample 0:
Comment
Sample E:
Comment
n/a*
Please TASTE the following oyster mushroom samples and rate the MUSHROOM-FLAVOR on a scale of 1 - 5. Please circle your choice.
INTENSITY
(none)
(weak)
(mild)
(moderate)
(strong)
1
2
3
4
5
Comment
_
Sample A:
Sample B:
Comment
Sample C:
Comment
Sample 0:
Comment
Sample E:
Comment
n/a*
Date Taken:
Appendix
c.
Substrates
Falcataria
Eucalyptus
Casuarina
Psidium
Trema
Mg (g/kg)
20.00
8.00
5.33
6.33
9.67
Fe (~g/g)
104.00
44.67
76.67
74.00
71.00
88
Zn (~g/g)
23.67
11.00
36.33
7.67
29.33
B (~g/g)
10.67
7.33
7.00
9.67
6.67
Appendix
D. Correlations between Pleurotus sp. yield and substrate lignin and
cellulose composition
Lignin
Substrates
Fa/cataria
Eucalyptus
Casuarina
Psidium
Trema
(%)
30.8
47.7
43.7
44.3
44.7
Substrate Wood*
Cellulose
Cellulose: Lignin
(%)
17.2
0.57
16.5
0.35
22.1
0.50
28.7
0.66
22.0
0.49
89
P/eurotus sp. **
Yield
B. E.
(g/bag)
(%)
268.8
74.0
250.7
71.0
275.5
69.4
190.5
44.2
272.4
78.5
Appendix
E. Crop period* of Pleurotus sp. cultivated on five wood substrates
Batch
1
Incubation
(days)
Falcataria
15
Substrate
Flush 1
Fruiting
(days)
Flush 2
Flush 3
10.2
6.7
15.3
47.2
Crop Period
(days)
Eucalyptus
15
12.7
10.4
14.7
44.8
Casuarina
15
13.0
12.0
14.8
50.6
Psidium
15
12.8
17.1
23.0
58.7
Trema
15
11.8
7.4
19.3
53.5
15
12.1
10.7
17.4
51.0
Batch 1 Average
2
Falcataria
16
7.9
21.9
24.0
55.4
Eucalyptus
16
10.0
24.9
19.5
52.3
Casuarina
16
8.2
22.1
16.0
51.1
Psidium
16
8.4
33.4
Trema
16
8.2
23.3
22.0
49.6
16
8.5
25.1
20.4
51.2
Batch 2 Average
90
**47.8
Appendix
F. Percent moisture of fruit bodi.. cultivated on fiye substrate woods
100
--
80
Fa/cataria
Casuarina
Eucalyptus
Psidium
Trema
'#.
!
:::J
-;;
60
0
::E
c:
~
Q)
40
a.
20
91
Appendix
G. Nutrient content of Pleurotus fruit bodies grown on five substrate woods*
Ca
Mg
Fe
Mn
Zn
Cu
Fa/cataria
61.67
15.03
28.67
1.09
2.10
102.00
14.00
109.00
17.33
3.33
Euca/yptus
47.17
12.27
22.57
1.68
1.83
99.33
11.33
83.67
12.67
4.67
Casuarina
56.60
13.80
25.53
1.43
2.03
101.00
16.00
114.00
19.67
3.33
Psidium
68.03
16.27
29.00
0.91
2.10
142.67
14.33
143.67
25.00
3.00
Trema
51.17
12.50
24.23
0.58
1.77
91.33
11.67
75.67
16.00
1.67
'0
* Values are means of 3 replicates. Units of N, P, K, Ca and Mg are g/kg, while all other are J,Jg/g.
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93
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94
95
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99