CULTIVATION OF THE OYSTER MUSHROOM (PLEUROTUS SP.)ON WOOD SUBSTRATES IN HAWAII

A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THEUNIVERSITY OF HAWAI'IIN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

MASTER OF SCIENCE

IN

TROPICAL PLANT AND SOIL SCIENCE

DECEMBER 2004

By

Tracy E. Tisdale

Thesis Committee:

Susan C. Miyasaka, ChairpersonMitiku HabteDon Hemmes

Acknowledgements

I would first like to acknowledge Susan C. Miyasaka, my major advisor, for

her generosity, thoughtfulness, patience and infinite support throughout this project.

I'd like to thank Don Hemmes and Mitiku Habte for taking time out of their schedules

to serve on my committee and offer valuable insight. Thanks to Jim Hollyer for the

much needed advising he provided on the economic aspect of this project. Thanks

also to J.B. Friday, Bernie Kratky and all the smiling faces at Beaumont, Komohana,

Waiakea and Volcano Research Stations who provided constant encouragement

and delight throughout my mushroom growing days in Hilo.

111

Table of Contents

Acknowledgements , iii

List of Tables , , , , vi

List of Figures vii

Chapter 1: Introduction '" 1

Chapter 2: Literature Review , 3

Industry , ,.. , , , , , , 3

Substrates 6

Oyster Mushroom " '" 19

Production Overview 24

Chapter 3: Research Objectives , '" 32

Chapter 4: Materials and Methods 33

Substrate Wood 33

Cultivation Methods 34

Crop Yield , , 39

Nutrients 43

Taste 44

Fruiting Site Assessment. .46

Economic Analysis .46

Chapter 5: Results and Discussion , , .48

Substrate Wood , , 48

Preliminary Experiment. '" 52

IV

Final Experiment. , , 53

Crop yield , , 53

Nutrients 67

Taste 68

Fruiting Site Assessment. , , , , , 72

Economic Analysis 74

Chapter 6: Summary 84

Appendix A: Taste test I survey questions 86

Appendix S: Taste test" survey questions 87

Appendix C: Wood concentrations of Mg, Fe, ln, S 88

Appendix 0: Yield correlations with substrate lignin and cellulose composition 89

Appendix E: Crop period data 90

Appendix F: Percent moisture data , , , , 91

Appendix G: Nutrient concentrations of Pleurotus fruit bodies 92

References , , '" '" ., 93

v

List of Tables

Table Page

4.1 Analysis of variance table for initial wood composition 34

4.2 Analysis of variance table for economic yield .40

4.3 Analysis of variance table for yield distribution throughout multiple flushes.. .41

4.4 Analysis of variance table for flush period and number of flushes .42

4.5 Analysis of variance table for percent moisture 43

4.6 Analysis of variance table for nutrient concentration .44

4.7 Analysis of variance table for taste test I .45

4.8 Analysis of variance table for taste test 11. .45

5.1 Initial nutrient concentrations of five substrate woods , '" ., 50

5.2 Lignin and cellulose composition of five substrate woods 51

5.3 Preliminary experiment results 52

5.4 Maximum and minimum economic yields , , 55

5.5 Initial bag weight and biological efficiency of Pleurotus sp. on five substrate

woods 62

5.6 Number of flushes produced by Pleurotus sp. on five substrate woods 65

5.7 Temperature and relative humidity of fruiting site 72

5.8 Cost assumptions for small-scale outdoor production , , 77

5.9 Estimated production costs of small-scale outdoor production 78

5.10 Summary of potential revenues, costs, and profits of small-scale, outdoor

oyster mushroom production 83

VI

List of Figures

Figure Page

5.1 Economic yield of Pleurotus sp. cultivated on five wood substrates , 54

5.2 Effect of batch on economic yield , , , 59

5.3 Change in yield over multiple flushes , , , , 64

5.4 Mushroom taste ratings, test I. 69

5.5 Mushroom taste ratings, test 11. 70

5.6 Preferred sample for taste, test 11. , 71

Vll

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

1

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. On the Island of Hawaii, a great deal of former

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.

2

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

practices. By early 17th century, cultivation in France began with Agaricus (lvors

2003). Mushroom production quickly spread to England and other European

countries, reaching the United States by the end of the 19th century (Flegg et al.

1985).

In the last 25 years, worldwide mushroom production has increased over

300%, reaching approximately 2,961,493 tons in 2002 (USDA 2003). China has

become the top-producing nation for all edible mushrooms, turning out over 40% of

the world's supply (USDA 2003). The U.S. is the next largest producer of

mushrooms, contributing about 13%, while the Netherlands and France produce

about 9.5 and 5%, respectively (USDA 2003). Overall U.S. production by volume

has been steadily rising over the last decade. Operations are also diversifying,

adding production of various specialty mushrooms. Industry expansion, in both

output and diversity, is largely due to improvements in cultivation technologies and

the expansion of market demand (Yamanaka 1997).

3

Cultivation methods for edible mushrooms vary considerably around the

world. Methods primarily depend on the type of mushroom. While species of

Agaricus, which include the white button mushroom, portabello and crimini, require

composted substrate, white-rot fungi can be cultivated on uncomposted organic

materials. The majority of the specialty mushrooms are white rot fungi; including

shiitake (Lentinula edodes), wood ears (Auricularia spp.), paddy straw mushrooms

(Volvariella volvacea) , oyster mushrooms (Pleurotus spp.) and many others.

Shiitake and wood ears are known to grow best on hardwoods, while paddy straws,

like their name indicates, grow best on straw. Oyster mushrooms are renowned for

their ability to grow well on a wide array of substrates.

Depending on location of production, many specialty mushrooms can be

grown using either indoor or outdoor methods. In China the majority of cultivation is

done outdoors, while in the US most cultivation is indoors (Shen et al. 2004).

Outdoor cultivation, in general, is a much lower cost operation and relies on nature

to create the environmental conditions necessary. Indoor cultivation allows for far

more control over environmental conditions and hygienic operation. Indoor methods

generally produce higher yields but are far more expensive to establish and operate.

Market Value

Edible mushrooms, especially specialty mushrooms such as the oyster

mushroom and shiitake, have a relatively high market value. The value of the

specialty mushroom crop in the United States reached $37,676,000 in 2002-3

(USDA 2003). On average, growers received $6.40 per kilogram for these

4

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

5

(B. Stenga, personal communication). Koa is a very expensive, prized wood. It is a

relatively slow growing tree and is in high demand in the Hawaiian Islands.

SUBSTRATE

Substrate Availability in Hawaii

Whether introduced or native, an array of tree species can be found growing

at significant densities in Hawaii. If certain species were to be used for cultivation of

mushrooms, two criteria would have to be met. First, the particular wood needs to

be capable of supporting mushroom growth. Second, it needs to be available locally

in substantial, sustainable quantities.

In Oregon, mushroom production has been evaluated in conjunction with

commercial forestry. Thinned trees from timber plantations are used as substrate for

cultivation of the matsutake mushroom (Tricholoma magnivelare) (Weigand 1998).

In Hawaii, Eucalyptus trees (Eucalyptus spp.) are being farmed on the Big Island for

timber and paper pulp. There is interest in finding a functional use for excess wood,

but labor and transportation costs would have to be considered. Also, edibility of

mushrooms cultivated on Eucalyptus would need to be confirmed. Rumors exist that

mushrooms grown on Eucalyptus can cause stomach upset (Stamets 2000). In

short, the plantations must exist locally, be willing to cooperate, and provide a wood

type suitable for mushroom production.

Paul Stamet's successful operation in Olympia, WA relies on the city and

county for a continuous supply of alder trees which have been cleared from

roadsides. In Hawaii, there are several trees that are viewed as weeds, including

6

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

moluccana) and ironwood (Casuarina equisetifolia L. ex J. R. & G. Forst) indicate

that both species grow very fast and can be harvested on short rotations (Salim et

al. 2002). Biomass yields for these trees in a seven year rotation are quite high

(Whitesell et al. 1992, Rockwood et al. 1990). In addition, both are nitrogen fixing

trees, which would reduce negative impacts on the soil and fertilization needs if

farmed over the long term.

Potential Substrate Trees

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

grandis Hill ex Maid, commonly known as the rose-gum or flooded-gum, is one of

the favored species for wood and fuel production. This species tends to grow

slender (dbh of 0.6-0.9m) and tall (42 to 54 m) (Little & Skolmen 1989). It is known

to produce high yields on short rotation (DeBell et al. 1985). When planted densely,

7

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

8

fertilization. Supplementation with N, and P to a lesser degree, are known to

significantly increase tree biomass (Yost et al. 1987). Planting Euca/yptus in mixed

plots with N-fixing trees, such as Fa/cataria mo/uccana, can decrease the need for N

fertilization. Mixed plantings of Euca/yptus and Fa/cataria have shown substantial

biomass yields (Whitesell et al. 1992).

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).

Fa/cataria establishes naturally in abandoned sugarcane fields in Hawaii. It is

also found lowland rainforests, montane forests, and along roadsides. It reproduces

prolifically at sites with 203 to 381 cm rain per year and elevation of 305 m or less

(Little & Skolmen 1989). This pioneer species does best in fertile, deep, well­

drained soils. It is, however, known to survive on relatively poor soil without

fertilization (Salim et al. 2002). Another advantage of Fa/cataria is its ability to

improve soil fertility by dropped foliage (Whitesell et al. 1992).

Fa/cataria can be planted with seedlings, cuttings, or by direct seeding.

Natural re-seeding by its wind-borne seeds is unpredictable. Untreated seeds

9

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

G.equisetifolia is a fast-growing tree and mature individuals reach an average

size of 24 to 40 m tall and 40 to 50 cm dbh (diameter at breast height) (Dommergues

1990). During the first seven years, growth is rapid (1.5 to 2.5 m/yr) and then it

starts to slow down (Dommergues 1990). Thus, a short rotation (seven years) is

said to be best for wood volume yields. Short rotation plantings, if spaced two

meters apart, can yield roughly 10 to 20 tlha/yr (Duke 1983). A more accurate mean

annual biomass was reported by Rockwell et al. (1990) as 18.3 tlha/yr in the United

States.

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).

P. cattleianum readily establishes in wet to moist lowland forests. It is found in

areas where elevations range from 100 to 1,300 m and annual rainfall from 127 to

699 cm. This species is highly shade tolerant and capable of enduring moderate to

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/m2

(Huenneke & Vitousek 1990). This introduced species also produces fruit at a

plentiful rate. Seeds of the fragrant fruits are dispersed over vast areas by non­

native 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.

T. orienta/is is a pioneer species, colonizing cleared lands, bare soils, and

flood-damaged banks. It is found at sites where annual rainfall is between 1,000

and 2,000 mm and the average temperature range is 20 to 27 degrees C. It can

grow on a wide range of soil types at elevation from 0 to 2500 m and tolerates

moderate soil salinity. The extensive root system of T. orientalis makes it a drought

tolerant species, but it is unable to endure waterlogged soils for extended duration

(Salim et al. 2002).

Fruits are small, round drupes that are pink to black in color. Seeds are said

to germinate in 10 to 30 days with a 70 to 80% success rate. Seedlings do best in

full sunlight and can be ready for out planting in three months (Hines & Eckman

1993). The trees produce numerous seeds and regenerate plentifully in nature.

Both coppicing and cuttings are said to be successful methods for propagation

(Salim et al. 2002).

T. orienta/is has a short life span and lives about 8 to 10 years. It also has an

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).

Cellular Properties of Wood

Lignocellulosic materials, such as wood and wood byproducts (chips,

sawdust) are composed mainly of three polymers: cellulose, hemicellulose, and

lignin. Lignin, the most complex of the polymers, is especially difficult to break down

biologically and blocks the bioavailability of cellulose and hemicellulose. Lignin's

complex polymer structure, with individual units cross-linked to each other by many

different chemical bonds, makes it resistant to most microbial degradation (Richard

1996). The strong lignin matrix surrounds the organized cellulose and hemicellulose

microfibrils, comprising 20 to 30% of the wood tissue (Kirk & Farrell 1987). Among

contiguous cells, high concentrations of lignin act like glue binding adjacent cells

together, forming the middle lamella.

Lignin polymers are formed by precursor alcohols which give rise to three

main types of polymer units. All three related polymer types are referred to as lignin,

but individually they are termed: guaiacyl units, syringyl units, and p-hydroxyphenyl

units. Quantities of these units vary by wood type. In most gymnosperms, lignin is

primarily composed of guaiacyl units, while lignin in angiosperms is approximately

half guaiacyl and half syringyl units. Both wood types contain only small quantities

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).

Degradation of Wood by White Rot Fungi

White-rot fungi possess the ability to break down all major components of

wood. Of the few organisms with this capacity, they are the most efficient and

therefore the primary agents of lignin degradation in natural ecosystems (Buswell &

Odier 1987). The enzymatic breakdown of lignin allows access to the carbon and

energy rich celluloses and hemicelluloses. The lignin destroying properties of

Phanerochaete chrysosporium have been studied extensively for practical

application in the paper industry. It serves as a model organism for the

physiological, biochemical, and genetic factors related to lignin biodegradation.

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

peroxidases, H202 producing enzymes, laccase and other phenol-oxidizing enzymes

(Kirk & Farrell 1987). Ligninase of P. chrysosporium has a molecular mass of 41 to

42 kd and is said to induce a non specific oxidation of lignin allowing subsequent

reactions to occur. A Mn peroxidase enzyme isolated from P.chrysosporium has a

molecular mass of 46 kd and functioned in phenol-oxidization and possibly H20 2

production. Laccase and H202-producing enzymes are involved in the subsequent

reactions and have important roles in the biochemical combustion of lignin

degradation (Kirk & Farrell 1987).

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

P. chrysosporium has confirmed that fruiting is dependant on N limitation. Also

genetic manipulation work is being done in hopes of finding strains of greater lignin­

degradation capacity. Overall, the genetic approach holds much hope for clarifying

the many questions within bioligninolytic systems for P. chrysosporium and other

white-rot fungi.

OYSTER MUSHROOM

Historical Information

Like other types of edible mushrooms, oyster mushrooms have been

collected in the wild for many centuries. Cultivation of these mushrooms only began

in the early 1900's. Early techniques and methods for growing Pleurotus involved

tree stumps and logs as substrate, mimicking their growth in nature (lvors 2003). In

Germany in the 1950's, successful attempts to grow oyster mushrooms on sawdust

became a historic milestone for mushroom cultivation. Mass production of oyster

mushrooms first started in the last 1960's using a straw based substrate (Chang &

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).

Taxonomy and Morphology

Species of Pleurotus are wood-inhabiting ligninolytic white-rot Basidiomycetes

belonging to the order Agaricales. There are over 30 species of Pleurotus

mushrooms. Although Pleurotus are considered saprophytic fungi, they have been

known to grow parasitically on trees as well. Fruit bodies range in color from blue­

gray, to white, to gray-brown and are mostly shell or spatula shaped, with a non-

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.

Nutritional & Medicinal Value

Mushrooms have extremely high moisture content. Fresh mushrooms

contain approximately 90% water; when dried, they contain from 5 to 20%.

However, conditions during growth, harvest, and post-harvest storage affect

moisture content (Crisan & Sands 1978). Oyster mushrooms provide good

nutritional value. Yang et al. (2001) reported crude protein content, on dry weight

basis, as 15.4% and 23.9% in P. cystidiosus and P. ostreatus respectively. They

contain about 60% carbohydrates (dry weight), within the ranges for other edible

mushrooms (Crisan & Sands 1978, Bano & Rajarathnam 1988). In addition, they

were reported to be low in fat (2 to 3% by dry weight), a good source of essential

amino acids, and contain approximately 5 to 9% fiber (Yang et al. 2001).

Literature does provide good references for oyster mushroom nutritional

values. However it has been demonstrated that substrate contributes to variation in

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 cholesterol­

controlling 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.

pulmonarius grown on lignin-rich substrate (sawdust), resulted in laccase activity

23

much greater than activity of cellulases and hemicellulases. When grown on

cellulosic cotton substrate, production of cellulase enzyme complexes was

considerably higher than with the lignin-rich substrate.

Inorganic nutrient effect, particularly Ca, Mg, Mn, Fe, and Zn, on lignin

degradation by P. chrysosporium, was studied by Jeffries et al. (1981). It was found

that Mn levels had a strong influence on lignin degradation, rates being greatest

when Mn was removed. Increased levels of Mn resulted lignin degradation

inhibition, however, inhibition was alleviated by increased concentration of either Ca

or Mg. Iron and zinc did not show a major effect on lignin degradation.

Lignin, cellulose, and mineral contents of substrates have also been shown to

influence growth and fruiting of Pleurotus. Philippoussis et al. (2001) demonstrated

that the cellulose:lignin ratio of the substrates was positively correlated to the

mycelial growth rate and mushroom yield of both P. ostreatus and P. pulmonarius.

Fasidi and Olorunmaiye (1994) verified that certain macroelements and trace

elements are essential for Pleurotus growth. Liquid cultures of P. tuber-regium

showed significant decrease in mycelial growth when potassium (K) was removed

from the media, and also when Ca was removed. Additionally, medium

supplemented with Cu, Fe, Mn and Zn produced greater fungal growth than basal

medium.

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

maturation, and the reproductive phase of fruit-body production. An initial mycelial

culture can be obtained from a pre-existing stock culture or through tissue culture.

These methods yield more predictably performing cultures than those grown from

spores (Royce 2003). Cultivation begins with propagation of mycelium on sterilized

cereal grains, creating spawn. The spawn is used to inoculate the mushroom

substrate. Once the sterilized substrate has been inoculated, it is allowed to

incubate. During the incubation, also called spawn run, the mycelium grows

throughout the substrate and matures. If environmental conditions are adequate,

the mature fungus will progress to the reproductive phase. Primordia will form and

develop into harvestable mushrooms.

Fruit-body Initiation & Production

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. Pettipher (1987) achieved successful

fruiting of P. ostreatus with daily temperatures ranging between 8° and 33° C.

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 CO2 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.

Factors Affecting Yield

Species of Pleurotus are very efficient at breaking down lignocellulotic waste.

Almost 50% of the substrate by mass is liberated as C02 gas, 20% is lost as water,

20% remains as spent substrate, and 10% is converted to dry mushrooms (Stamets

2000). Certain techniques are used to achieve high production rates and yields.

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).

Substrate Composition & Supplements

Though variation exists among different species of Pleurotus, composition of

substrate does playa role in vegetative and fruit-body growth. Lignin, cellulose and

hemicellulose availability plays a key role in the growth of wood-decaying fungi.

Philippoussis et al. (2001) determined that the cellulose:lignin ratio of a substrate

was positively correlated to rate of mycelial growth and mushroom yield of both P.

ostreatus and P. pulmonarius. Research on two other species of oyster mushroom

(P. citrinopileatus and P. florida) concluded that wood based substrate rich in

cellulose supported higher yields and resulted in more nutritious mushrooms than

substrates of other agricultural wastes (Kalita & Mazumder 2001).

Enhancing base substrate with a nitrogen rich supplement is common

practice to increase yields in mushroom cultivation. Commonly used supplements

are grain products, such as bran and meal. The lignin degradation rate of alder

sawdust by P. chrysosporium was shown to increase from 5.2% to 29.8% with the

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.

Pests &Contaminants

Warm, humid cultivation conditions ideal for mushrooms are also favorable to

many pests and pathogens. Outdoor mushroom fruiting sites, filled with nutrient rich

substrate, attract many of these contamination-causing organisms, which are difficult

to control once present. Oyster mushroom growers using outdoor fruiting sites can

face serious problems due to bacterial contamination, fungal contamination, insect

damage, and other pests.

The most common bacterial problem in Pleurotus cultivation is infection by

Pseudomonas tolaasii (Royce 2003). The bacteria cause a splotch disease of

Pleurotus similar to that of Agaricus spp. Infected mushrooms are discolored, brittle,

and have a shorter shelf life. Constantly excessive moisture levels, insufficient

ventilation and high temperatures worsen P. tolaasii cases. Infection causes

29

significant reduction in yield and is considered a major concern to growers

throughout the world.

Fungal contamination is also a big problem in cultivation operations. Most are

not parasitic, but will develop on the substrate. They compete with the cultivated

fungus and negatively impact its growth. Green mold is a major fungal problem for

mushroom growers. Infection of green mold has been known to cause severe crop

losses. From 1994 to 1996 the mushroom industry in Pennsylvania experienced

losses from 30 to 100% due to green mold infestation (Beyer et al. 1999). Over 30

species of fungi cause green mold in mushroom cultivation (Cha 2004). Species of

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

of contaminated bags is required in both incubation and fruiting stages. Efforts

should be made to keep insects and pests out of fruiting and incubation sites using

screening, proper windows and doors. Also, floors should be kept clean and sticky

mats should be used to reduce damage by crawling insects. Finally, it is imperative

to clean and disinfect the fruiting site and equipment between crops.

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. Crop yield in terms of:

i. Economic yield

ii. Biological efficiency

iii. Flush

iv. Crop period

v. Percent moisture

b. Nutrient concentration of mushroom fruit bodies

c. Flavor and aroma of mushroom fruit bodies;

3. To assess the of suitability of an outdoor shade house as a low-cost

fruiting site;

4. To estimate the economic feasibility of small scale mushroom

production in Hawaii using similar methods and substrates.

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

mo/uccana, Casuarina equisetifo/ia L. ex J. R. & G. Forst, Euca/yptus grandis W. Hill

ex Maid., Psidium catt/eianum Sabine, and Trema orienta/is (L.) Blume. Major

considerations involved were availability of the wood, cost and ease of obtaining it.

All five wood types were harvested locally on the east side of the Big Island.

Young E. grandis trees were obtained from commercial forests with the permission

and assistance of Forest Solutions, Inc. Young F. mo/uccana and C.equisetifolia,

growing along the edges of the commercial forests were also obtained with the help

of Forest Solutions. P. cattleianum and T. orientalis were cut from the overgrowth

along property edges of the University of Hawaii at Hilo (UHH) Farm. The wood was

chipped at the UHH Farm using a farm grade brush chipper. Larger logs (greater

than 15 cm in diameter) were first split manually to fit through the chipper. All wood

chips were stored outdoor and covered loosely with plastic until needed.

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 85°C 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.

Table 4.1. Analysis of substrate woods' initial composition.

ANOVA dfReplicate 2Substrate 4Error 8

--------------------------------------------------_.Total 14

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 25°C.

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 25°C 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.

Inoculation & Incubation

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

(155°W longitude, 19°N 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

SUBSTRATE EFFECT ON CROP YIELD

Economic Yield

Economic yield was determined by fresh weight of mushrooms harvested.

Mushrooms were checked regularly and harvested when fruit bodies averaged

approximately 6 to 10 em in diameter. According to Stamets (2000) maturity of

mushrooms at harvest does not greatly affect overall yield. Each bag's yield was

harvested in its entirety and weighed the same day using an Ohaus digital scale,

model GT8000. Date of each harvest was also recorded.

Only yields from the first three flushes were used in calculating economic

yield. In commercial production, yield becomes so low in later flushes that it is not

economically viable to continue cropping the bags. All yield-related analyses, with

the exception of the flush, utilize economic yield values.

The SAS computer program (SAS 1982) was used to perform the statistical

analyses of economic yield data. Using the general linear model procedure (GLM),

an analysis of variance (ANOVA) was performed to determine the effects of

substrate treatment on fresh weight yield (Table 4.2). Single degree of freedom

contrasts were utilized to further analyze the data. The GLM procedure was also

used to analyze correlations between mean yield and mean cellulose:lignin ratios of

the substrates, as well as between yield and cellulose concentrations or lignin

concentrations.

39

Table 4.2. Analysis of variance of substrate effects on economic yield.

ANOVA OfBatch 1Block 9

__~rr9-~ A_(~~!~!(~!~_~~l ~__Substrate 4

N-fixers vs. Others {1}Fruit-tree vs. Others {1}High N vs. Others {1}High Mn vs. Others {1}

Batch*Substrate 4Error B 72

---~----------------------------------------------------

Total 99

Biological Efficiency

Economic yield data was used to calculate biological efficiency (% B.E.)

values for each treatment. Biological efficiency, a term used specifically in the

mushroom industry, quantifies how efficiently the fungus utilizes the substrate for

growth and development. The value, expressed as a percent, calculates the rate at

which substrate is converted to fresh mushrooms. The conversion rate (CR.) is

derived using the following equation:

Equation 4.2. C. R. = Total Fresh Weight of Yield (g) X 100%Total Weight of Moist Substrate (g)

By definition, 100% B.E. is achieved by a 25% conversion of moist substrate to fresh

mushrooms (Stamets 2000). This ratio was then used to calculate B.E. values from

the calculated conversion rates. B.E. values were analyzed using the SAS GLM

procedure, with an ANOVA similar to that of economic yield (Table 4.2). In addition,

40

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).

Table 4.3. Analysis of variance of substrate effects on distribution of yield throughout

multiple flushes.

ANOVA dfBatch 1Block 9Error A 9--------------------------------------------------_.Substrate 4Batch*Substrate 4Error B 72

----------------------------------------------------Flush 4Flush*Substrate 16Flush*Batch 4Flush*Substrate*Batch 16E~rC ~O--------------------------------------------------_.Total 499

41

Table 4.4. Analysis of variance of substrate effects in two batches on flush period

and flush number.

ANOVA dfBatch 1Block 9

__~~~~~ _~ {~~!~_~~_~!9_~~1 ~__Substrate 4Batch*Substrate 4Error 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 85°C 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 dfBatch 1Replicate 5

__~~~~~ _~ {~~R~~~<?_~~1 ~_.

Substrate 4Batch*Substrate 4Error B 10

----------------------------------------------------Total 29

SUBSTRATE EFFECT ON NUTRIENT CONCENTRATIONS

Three sub-samples of fruit bodies grown on each substrate were dried in a

drying oven for 6 to 8 hours to constant dry weight at 85°C. Samples were ground

using a ball mill for ten minute intervals. Samples were sent to the University of

Hawaii's Agricultural Diagnostic Service Center for plant tissue analyses.

Concentrations of N, P, K, Ca, Mg, Fe, Mn, Zn, Cu and B were quantified using

methods described for nutrient concentration of wood. For each nutrient, results

were statistically analyzed using ANOVA (Table 4.6), as well as comparison of the

means using Duncan's multiple range test.

43

Table 4.6. Analysis of substrate effects on fruit body nutrient concentration.

ANOVA dfReplicate 2Substrate 4Error 8

-------------------------------------------------~_.

Total 14

SUBSTRATE EFFECT ON TASTE

Two blind taste tests were performed evaluating both aroma and flavor.

Mushrooms were stir-fried lightly in olive oil for six minutes at medium heat and

served warm. Salt was added while cooking for the first test only.

The first test was performed at the Beaumont Research Station in Hilo, HI on

May 6,2004. Mushrooms grown on four of the five substrates were included.

Those grown on Eucalyptus were excluded from this taste test due to reported

possible toxicities. Volunteers were asked to first smell each cooked mushroom

sample and rate the aroma on a scale of 1 to 5. They were also instructed to taste

the cooked mushrooms and rate the flavor on a scale of 1 to 5 (Appendix A). For

both flavor and aroma, the scale was defined as: 1= unappealing, 2=poor, 3=fair,

4=good,5=excellent. Four additional questions were asked - two for the aroma

section and two for the flavor component. Questions 1 and 3 asked whether or not a

difference among the four samples could be detected (in aroma and flavor,

respectively). Questions 2 and 4 asked to select a best sample in terms of

preference (for aroma and flavor, respectively).

The second test was performed at the Komohana Research Station in Hilo, HI

on July 8,2004. This test also had both aroma and flavor components. Changes

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 dfRater 22Substrate 3Error 66

----------------------------------------------------Total 91

Table 4.8. Analysis of variance of substrate effects on fruit body taste - test II.

ANOVA dfRater 27Substrate 4Error 108

--------------------------------------------------_.Total 139

45

FRUITING SITE ASSESSMENT

Environmental conditions of the fruiting site are critical for optimal production.

In sophisticated indoor operations, mushroom grow rooms are designed and built for

accurate control of these conditions. Attempts were made to evaluate the shade

house's environmental conditions. A HOBO ProSeries RHfTemp Data Logger

(Onset Instruments, Bourne, MA) was used to measure and record both temperature

and relative humidity levels inside the fruiting shade house. Temperature and

relative humidity data was monitored for a portion of the production. Monitoring

began in mid-May and continued through mid-June.

A Quantum Light Meter (Apogee Instruments, Auburn, CA) was used to

measure photon flux density inside the fruiting site. Light readings were taken in

both sunny and cloudy conditions. Readings were used to calculate the percent

shade of the shade house. Contamination and pests were recorded as observations

and notes, but not quantified.

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

significantly greater N concentration.

A highly significant difference (p=.001) in P concentration was found.

Euca/yptus wood contained 0.5 g/kg P, significantly more than the other woods, but

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.

With respect to K, a significant difference (p=0.034) was found among wood

types. Highest K concentration was observed in Trema, followed by Psidium,

Casuarina, Euca/yptus and Fa/cataria, respectively. Mean comparison by Duncan's

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 aresignificantly different at p=0.05 as determined by Duncan's multiple range test.

Lignin & Cellulose

Differences in both cellulose content (p=0.004) and lignin content (p=0.015)

were found among the five woods. Psidium had the greatest cellulose content,

28.67%, while Fa/cataria and Euca/yptus were on the lower end. The lignin content

of Fa/cataria wood was significantly lower, while those of the other four woods were

similar. The cellulose:lignin ratio was greatest in Psidium, followed by Fa/cataria,

Casuarina, Trema, and Eucalyptus respectively, however differences were not

significant at p=0.05 (Table 5.2).

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 aresignificantly 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

other substrate treatment (Falcataria, Eucalyptus, and Casuarina). Results of

preliminary Pleurotus sp. cultivation trials are displayed in Table 5.3. No significant

differences (p=0.919) in overall yield were found among substrates. Overall mean

bag weight was 3.4 kg and biological efficiency values of the three substrates were

not statistically different (p=0.926). Crop period was relatively short, with no

differences among substrates (p=0.933). However, the short crop period is relative

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 2

Casuarina 710.5 82.4 2.5 53.0 2

Eucalyptus 425.7 50.5 2.0 56.5 2

Psidium NF NF NF NF 0

Trema NF NF NF NF 0

* NF denotes no fruiting of Pleurotus sp. occurred.

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 -enEcoL-

a>200--~a>

~ 150~en(I)L-

100U-

50

0Falcataria 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

* Yields are measurements of fresh weight (grams) per bag.

Statistical analysis of economic yield revealed differences among substrates

to be nearly significant (p=0.06). Using single degree of freedom contrasts,

relationships among substrate treatments were evaluated. Of the five substrates

tested, three are N-fixing trees: Fa/cataria, Casuarina and Trema. Single degree of

55

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.

Similar results were seen with N. The effect of N on substrate degradation

and growth is a common component of white-rot fungi research. Literature indicates

that N availability of wood is beneficial for P/eurotus growth and that the addition of

N to substrate increases growth (Boyle 1998). Although Fa/cataria, Casuarina and

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

significant difference was seen (p=0.966). Likewise, no significant correlation

between mean N concentrations and mean yield was found (p=0.575). Perhaps, the

variations in N levels between wood types were not great enough to affect yield. Or,

it is possible that this particular species of P/eurotus is not influenced by N levels.

Though mushroom yield did not correspond directly to substrate N concentrations,

the wood of N-fixing trees did support higher yields than the wood of non-fixers.

Results did not show definite patterns between substrate lignin and cellulose

levels and economic yield (Appendix D). Psidum wood, although richer in cellulose,

did not support greater yields. Additionally, Fa/cataria wood had the lowest lignin

content, but was not the lowest producer of mushrooms. Using SAS's GLM

procedure, the correlation between mean yield and substrates' cellulose:lignin ratios

was not significant (p=0.353). Similarly those between mean yield and cellulose

composition (p=0.187) and between mean yield and lignin composition (p=0.671)

were not significant.

From results observed, several conclusions can be drawn about substrate's

effect on economic yield of P/eurotus sp. All woods tested do support growth and

fructification of this P/eurotus species. All treatments have the potential to achieve

high yield, as seen by maximum values. Findings agree with previous studies that

wood of fruit trees is not the best selection for mushroom production. Nitrogen-fixing

trees appear to be better substrate choices for P/eurotus production, giving higher

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 shade­

house. 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>-"0Q) c:::=:=J Falcataria>= ~ Casuarinau 200 ~"E r= ~ Eucalyptus

~0 ~ ~ Psidiumc::

~ ~ Trema0~u

UJ ~~

100 ~~~

2

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

cattleianum is also a heavy wood, similar to related species P. guajava and P.

friedrichsthalianum, which have specific gravities of 0.8 and 0.7, respectively (Little &

Skolmen 1989, Morton 1987). Wood density could be a noteworthy factor in the

60

economic analysis of the production, depending on how substrate materials are

obtained and handled.

Biological efficiency (B.E.) was calculated using initial bag weight and

economic yield (Equation 4.2). A significant difference (p = 0.002) in B.E. was found

among the five substrate treatments. The average B.E. of the Psidium was 44.2%,

which was statistically lower than the other four substrates. When analyzed by

mean comparison, there was no statistical difference among the other four woods,

which ranged from 78.5% to 69.4% (Table 5.5). Single degree of freedom contrasts

for B.E. revealed results similar to those of economic yield. The following two

contrasts were significant in terms of B.E.: N-fixing trees versus non-fixers (p=0.004)

and fruit tree versus others (p<0.0001). Nitrogen-fixing trees gave greater B.E.

values than non-fixers and the fruit tree gave lower B.E. than non-fruit trees. As

seen in economic yields, when high-N versus low-N woods were contrasted, no

difference in B.E. were found (p=0.228). Similarly, no differences existed between

low-Mn and high-Mn woods (p=0.507).

Biological efficiency did not appear to be related to cellulose:lignin ratios of

substrate. Of all five woods Psidium had the greatest cellulose:lignin ratio, but the

lowest B.E. Correlation studies did not show a significant relationship between

mean cellulose:lignin ratios and mean B.E. (p=0.243). This finding does not agree

with previous studies, which report a positive correlation between cellulose:lignin

and B.E. (Philippoussis et al. 2001). Likewise, no significant correlation between

B.E. and cellulose (p=0.113) or between B.E. and lignin (p=0.733) was found.

Although significance was detected for both lignin concentrations and cellulose

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.

Biological Efficiency (%)**

Bag Weight (g)* 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 (8.5)

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 (8.4)

* 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

Biological efficiency was not significantly influenced by block (p=0.60).

Therefore, it can be concluded that placement of bags within the shade house was

not a major source of variation in B.E.

Biological efficiency values in this study can be compared to those of

successful mushroom operations. Although it is possible to cultivate species of

Pleurotus at even greater B.E., it is common that commercial indoor mushroom

farms operate between 75% and 125% (Stamets 2000). In comparison to other

outdoor cultivation experiments, B.E. results achieved in this study, using all five

woods, were high. Pettipher (1987) reported B.E. yields of 46 to 58% growing P.

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

- 150C>'--" -.- Falcataria-..c. -0-- EucalyptusC>

~ 100-y- Casuarina---v-- Psidium

..c. ----.- Tremaen~u- 50

0

F1 F2 F3

Flush

F4 F5

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

Pleuratus (Philippoussis et al. 2001).

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

Percent moisture of fruit bodies cultivated on all five substrates were

statistically similar (p=0.942). Neither block nor batch significant affected mushroom

moisture content (p= 0.446 and 0.064 for block and batch, respectively). Therefore,

it was concluded that substrate did not affect the moisture content of mushrooms.

Substrate Effect on Nutrient Concentration

Very little variation was found when the mineral concentrations of fruiting

bodies grown on different substrates were analyzed. Of the ten minerals analyzed,

none exhibited significant differences among the fruit bodies grown on various

substrates. Nutrient levels of Pleurotus cultivated on all five woods are comparable

to those reported levels of oyster mushrooms grown on straw (Bano & Rajarathnan

1988). Nitrogen levels, generally reported as protein content, are high. Mean N for

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 1-1gIg , 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

were found when cultivated on different substrates. More specifically, mineral

concentration of the mushrooms increased when grown on substrate with higher

mineral concentration.

All substrates tested in this experiment were angiosperm woods. However,

significant differences were found in wood nutrient levels, specifically Ca, Cu, Mg, N,

P and K. Differences in these nutrient concentrations, although statistically

significant, were not large enough to affect nutrient levels of the mushrooms.

Among the five woods tested, differences in mineral concentrations among wood

species did not alter mushroom nutrient concentration.

Substrate Effect on Taste

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

Analysis of sample ratings from test I showed a significant difference (0.004)

in preference of mushroom flavor. Of the four substrates tested, mushrooms grown

on Trema, Fa/cataria, and Casuarina were preferred over those grown on Psidium

(Figure 5.6). Also, it was evident that a difference in flavor among samples could be

detected; of the 74% of participants that did record an answer for question 3, all

were 'yes' responses.

5

4

I/)

~

8 3Cf)

Q)oc~~ 2Q)a..

o T

c=J Fa/cataria~ Casuarina~ Psidium18888881 Trema

Aroma Flavor

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).

5

4

~8 3(f)

~'wc~ 2c

o

t-

1=f=1=f=1=f=1=1=f=1=f=1=

1=~~f=1=f=1=~

c=J Falcataria~ Casuarina~ Eucalyptus~ Psidium~ Trema

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

Casuarina as the most preferred for flavor (Figure 5.6).

Figure 5.6. Test II's overall preferred sample for flavor.

71

Overall, cultivation substrate did not appear to have an impact on the

mushrooms' aroma. However, results suggested that substrate did influence the

flavor of mushrooms. Flavor preferences and rankings did vary considerably

between the two tests. Further taste tests are suggested in order to draw more

definite conclusions about wood substrate effect on mushroom taste. In addition, it

maybe of interest to analyze for soluble carbohydrates in the future, as several of the

participants' comments implied differences in sweetness.

FRUITING SITE ASSESSMENT

Temperatures within the shade house got as high as 44°C, far above the

recommended range for oyster mushrooms. Relative humidity reached lows far

below recommended for oyster mushroom cultivation. Dramatic diurnal fluctuations

in both temperature and relative humidity occurred during the monitoring period

(Table 5.7).

Table 5.7. Mean, maximum and minimum values for temperature and relative

humidity within the mushroom fruiting shade house.

Week1234

InMayMayJuneJune

Temperature CC)Mean Max Min25.9 44.4 18.325.7 42.2 18.326.0 42.2 17.226.1 43.3 17.8

72

Relative Humidity (%)Mean Max Min71.7 101.5 9.874.3 100.9 12.770.3 100.9 11.265.8 100.6 8.0

Fluctuation in environmental climate is inevitable in outdoor production

systems. Actually, some variation in humidity and temperature is recommended

throughout the fruiting cycle to mitigate contamination (Stamets 2000). Although

the extremes observed are not within the recommended ranges for Pleurotus

production, yield was considerably high for outdoor production (Table 5.4).

Mid-day light readings taken inside the shade house ranged from 78

tJmol/m2/s (4,300 lux), on a clear sunny day, to 17 tJmol/m2/s (940 lux), on an

overcast day. Light reaching the mushroom bags did attain levels exceeding those

recommended for commercial Pleurotus production (Stamets 2000). 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

As in any agricultural endeavor, the economic component is a major factor in

the realistic feasibility of mushroom production in Hawaii. This analysis provides

examples of costs and revenues typically associated with starting and operating a

small-scale outdoor mushroom farm in Hawaii. The projected revenue provided is

based solely on the results obtained from this cultivation experiment. Many factors

influence yield, thus output and revenue may vary considerably. Some of these

factors would include, but are not limited to, mushroom type, substrate, location,

methods and facilities used.

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

year. Since the current wholesale value of oyster mushrooms in Hawaii is

$10.00/kg, annual revenue could amount to $11,500.

Costs and Labor

Major assumptions underlying the costs of a small-scale, outdoor mushroom

operation are outlined in Table 5.8. Some of the costs are merely estimates

provided by local businesses and specialists, others were actual costs incurred

during the experiment. Estimated production costs are outlined in Table 5.9.

Although production and fruiting sites used in the experiment were pre­

existing buildings converted for mushroom production, the site costs used in the

analysis reflect building new structures.

The production rate (P2) utilized in the analysis was calculated to provide

enough work for one full-time farmer throughout the year, but not require additional

labor. Additional labor requirements would vary depending on the means by which

substrate was obtained. Also, if the production rate were to be increased

significantly, maintaining the same methods of production, additional labor would be

necessary.

This analysis uses a moderate cost for substrate. At $4.20/m3, overall annual

substrate costs are relatively low at both P1 and P2 (Table 5.9). Even assuming

zero cost for substrate, overall profits would be negative at both production rates.

Three means of obtaining wood for substrate were suggested in the experiment.

These means will be discussed briefly.

76

Table 5.8. Assumptions underlying cost estimates for small-scale, outdoor oyster

mushroom production in Hilo, HI.

Item

Building/Facilities construction

Production facility

Fruiting facility

Spawn production

Wheat berries (retail)

Filter discs (retail)

Culture agar (retail)

Substrate preparation

Substrate

Wheat bran supplement (retail)

Cultivation bags (retail)

Harvesting

Packaging materials

General operations

Water (liters)

Water charges

Electricity

Labor

77

Cost (in 2004, $)

845 /square m.

430 /square m.

1.3 /kg

49 /set of 100

75 /kg

4.2 /cubic m.

0.72 /kg

399 /set of 1000

4.8 /100

0.19 /1000 liters

13.41 /month

0.06 /MJ

10 /hour

Table 5.9. Estimated production costs associated with small-scale, outdoor oyster

mushroom production in Hilo, HI at two production levels.

Production Level*

Item P1 P2

Cost (in 2004, $/year)

Spawn production

Wheat berries

Filter discs

Culture agar

Bag production

Substrate

Wheat bran supplement

Purchased bags

Harvesting

Packaging Materials

General operations

Water

Electricity

Facilities

Equipment

Labor

Total Annual Cost

61

3

10

18

251

559

169

165

873

2,393

305

6,547

$11,354

184

10

29

53

752

1,676

508

175

2,619

3,726

334

22,835

$32,899

*P1 represents a production rate of 1,400 bags per year, while P2 represents a rate

of 4,200 bags per year.

78

Wood Chip Quantity Needs

Using the methods described and a production rate of 4,200 bags per year,

annual production of oyster mushrooms requires approximately 12.74 m3 (450 ft3) of

wood chips. Wood chip quantities used per bag (3 L volume) were weighed for each

wood type. From these weights, it was approximated that between 1500 kg (1.5 t)

(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.

Obtaining waste chips from Eucalvptus plantations

The commercial Eucalyptus plantations along the Hamakua coast of the Big

Island do provide a potential source of wood suitable for mushroom substrate.

Depending on management practices, excess wood may be available from thinned

trees or slash. Costs of obtaining such excess wood could be substantial and may

include hauling wood from the forest, transporting, and chipping. Such costs would

need to be investigated and incorporated into the economic analysis.

Obtaining waste wood from Clearing Operations

Since many woods are suitable substrate for the production of oyster

mushrooms, the option for using cleared wood is viable. There is potential to work in

conjunction with the city and county to obtain wood from local land clearings. Since

wood from clearings is often a mix of several species, yield and flavor quality may be

affected. If substrate wood were to be obtained from clearings, the sustainability

79

and consistency of such sources would be a major factor to consider. Transporting

the material may be an additional factor involved.

Growing Trees for Chips

Growing trees specifically for substrate purposes ensures the availability and

consistency of substrate. It would also decrease transportation costs if the trees are

grown close to the mushroom operation. It is reported that farmed Fa/cataria

mo/uccana can produces 24,463 kg/ha/yr (9.9 t/acre/yr) in a 7 year rotation in Hawaii

(Whitesell et al. 1992). Considering the relatively low density of the wood, one

hectare of land could produce 74.1 m3 of wood annually. Reports of Casuarina

show an 18,285 kg/ha/yr (7.4 t/acre/yr) biomass production in a 7 year rotation

(Rockwell et al. 1990). As a higher density wood, biomass is equivalent to

approximately 18.3 m3 of wood annually per hectare. Euca/yptus biomass per

hectare per year falls in between that of Fa/cataria and Casuarina, with 61 m3

(27,922.6 kg/ha/yr, or 11.3 t/acre/yr, in a 7 year rotation) (Whitesell et al. 1992).

A potential farmer would need to estimate the amount of land needed to grow

enough wood for mushroom production. A certain amount of land would be allotted

to the mushroom fruiting site and building. The remaining land could be planted with

trees intended for harvest after 7 years of growth. Starting at year seven, one

seventh of the land could be harvested and replanted. The following year, the

second seventh could be harvested and replanted, allowing a continual supply of

trees from year seven onward.

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.

Conclusions and Suggestions

Small-scale, outdoor oyster mushroom production in Hawaii, does not appear

to be economically feasible under the given circumstances and assumptions.

Considering all noted assumptions, annual profits calculated for such a production

are negative at both production rates (Table 5.10). The major component of cost is

labor, being approximately 60-70% of total costs. Cost of constructing buildings and

facilities is also substantial. Converting existing structures (shed, barn, garage,

greenhouse, etc) into mushroom buildings or fruiting sites would likely be more

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

Table 5.10. Summary of potential revenues, costs, and profits of small-scale,

outdoor oyster mushroom production in Hilo, HI at two production rates*.

Economic Summary P1 P2

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).

Under the assumed costs, a significant increase in production would be

required to reach an economic break-even point. A biological efficiency of 220%

would be necessary to create positive profits. If costs were cut by 25%, a B.E. of

only 156% would result in positive profits. Using Pleurofus, such B.E. values are

attainable, however are generally achieved using indoor cultivation.

Overall, several inexpensive, suitable substrates have been identified.

However small-scale outdoor cultivation of oyster mushrooms does not appear to be

economically feasible under given circumstances and assumptions. 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

In summary, wood of Fa/cataria mo/uccana, Casuarina equisetifo/ia,

Euca/yptus grandis, Psidium catt/eianum, and Trema orienta/is all proved to be

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. Please circle your choice.

(unappealing) (poor) (fair) (good) (excellent)Sample A: 1 2 3 4 5 Comment

Sample B: 1 2 3 4 5 Comment

Sample C: 1 2 3 4 5 Comment

Sample D: 1 2 3 4 5 Comment

1. Could you detect a difference among the samples' aroma? (please circle) yes no

000'\

2. Overall Best Sample for aroma: (please circle) A B C D n/a*

Please TASTE the following oyster mushroom samples and rate them for FLAVOR on a scale of 1 - 5. Please circle your choice.

Sample A:

Sample B:

Sample C:

Sample D:

(unappealing)1

1

1

(poor)2

2

2

2

(fair)3

3

3

3

(good)4

4

4

4

(excellent)5

5

5

5

Comment _

Comment _

Comment _

Comment _

3. Could you detect a difference among the samples' flavor? (please circle) yes no

4. Overall Best Sample for flavor: (please circle)

* indicates no detectable difference among samples

A B C D n/a*

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 _

Sample B: 1 2 3 4 5 Comment

Sample C: 1 2 3 4 5 Comment

Sample 0: 1 2 3 4 5 Comment

Sample E: 1 2 3 4 5 Comment

00 Overall Best Sample for aroma: (please circle) A B C 0 E n/a*'I

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)Sample A: 1 2 3 4 5 Comment _

Sample B: 1 2 3 4 5 Comment

Sample C: 1 2 3 4 5 Comment

Sample 0: 1 2 3 4 5 Comment

Sample E: 1 2 3 4 5 Comment

Overall Best Sample for flavor: (please circle) A B C 0 E n/a*

* indicates no detectable difference among samples Date Taken:

Appendix

c. Additional nutrient concentrations* of substrate woods

SubstratesFalcatariaEucalyptusCasuarinaPsidiumTrema

Mg (g/kg)20.008.005.336.339.67

Fe (~g/g)

104.0044.6776.6774.0071.00

Zn (~g/g)

23.6711.0036.337.6729.33

B (~g/g)

10.677.337.009.676.67

*Values are means of 3 replicates.

88

Appendix

D. Correlations between Pleurotus sp. yield and substrate lignin andcellulose composition

Substrate Wood* P/eurotus sp.**Lignin Cellulose Yield B. E.

Substrates (%) (%) Cellulose:Lignin (g/bag) (%)Fa/cataria 30.8 17.2 0.57 268.8 74.0Eucalyptus 47.7 16.5 0.35 250.7 71.0Casuarina 43.7 22.1 0.50 275.5 69.4Psidium 44.3 28.7 0.66 190.5 44.2Trema 44.7 22.0 0.49 272.4 78.5

*Values are means of 3 replicates.**Values are means of 20 replicates.

89

Appendix

E. Crop period* of Pleurotus sp. cultivated on five wood substrates

Fruiting Crop Period

Incubation(days) (days)

Batch Substrate(days) Flush 1 Flush 2 Flush 3

1 Falcataria 15 10.2 6.7 15.3 47.2

1 Eucalyptus 15 12.7 10.4 14.7 44.8

1 Casuarina 15 13.0 12.0 14.8 50.6

1 Psidium 15 12.8 17.1 23.0 58.7

1 Trema 15 11.8 7.4 19.3 53.5

Batch 1 Average 15 12.1 10.7 17.4 51.0

2 Falcataria 16 7.9 21.9 24.0 55.4

2 Eucalyptus 16 10.0 24.9 19.5 52.3

2 Casuarina 16 8.2 22.1 16.0 51.1

2 Psidium 16 8.4 33.4 **47.8

2 Trema 16 8.2 23.3 22.0 49.6

Batch 2 Average 16 8.5 25.1 20.4 51.2

* Values are means of 10 replicates

** Crop period calculated for 2 flushes only

90

Appendix

F. Percent moisture of fruit bodi.. cultivated on fiye substrate woods

100

80

-'#.-! 60:::J-;;·0::E-c: 40~Q)a.

20

o

T T T T T

Fa/cataria Casuarina Eucalyptus Psidium Trema

91

Appendix

G. Nutrient content of Pleurotus fruit bodies grown on five substrate woods*

N P K Ca Mg Fe Mn Zn Cu B

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 1.67'0N

* Values are means of 3 replicates. Units of N, P, K, Ca and Mg are g/kg, while all other are J,Jg/g.

References

Bano, Z. and Rajarathnam, S. 1988. Pleurotus Mushrooms. Part II. Chemicalcomposition, nutritional vlue, pot-harvest physiology, preservation and roleas human food. CRC Critical Reviews in Food Science and Nutrition 27,87-102.

Bellomonte, G. Constantine, A, and Giammariolo, S. 1987. Comparison ofmodified automatic Dumas method and the traditional Kjeldahl method fornitrogen determination in infant food. Journal ofAssociation of OfficialAnalytical Chemists 70, 227-229.

Bobek, P., Ozdin, L., and Galbavy, S. 1998. Dose- and time-dependenthypocholesterolemic effect of oyster mushroom (Pleurotus ostreatus) inrats. Nutrition 14, 282-286.

Boyle, C. D., Kropp, 8. R., and Reid, I. D. 1992. Solubilization and mineralizationof lignin by white rot fungi. Applied and Environmental Microbiology 58,3217-3224.

Boyle, C. D. 1998. Nutritional factors limiting the growth of Lentinula edodes andother white rot fungi in wood. Soil Biology and Biochemistry 30,817-823.

Buswell, J. A, Cai, Y. J., Chang, S. T., Peberdy, J. F., Fu, S. Y., and Yu, H. S.1996. Lignocellulolytic enzyme profiles of edible mushroom fungi. WorldJournal of Microbiology and Biotechnology 12,537-542.

Buswell, J. A and Odier, O. 1987. Lignin biodegradation. Critical Review ofBiotechnology 6, 1-60.

Campinhos, E. Jr. 1999. Sustainable plantations of high-yield Eucalyptus treesfor production of fiber: the Aracurz case. New Forests 17,129-143.

Cha, J.-S. Pest Disease and Management. Mushroom Growers HandbookVolume 1. 2004. MushWorld. July 2004.

Chandler, J. A, Jewell, W. J., Gossett, J. M., Van Soest, P. J., and Robertson, J.B. 1980. Predicting methane fermentation biodegradability. Biotechnologyand Bioengineering Symposium 93-107.

Chang, S. T. and Hayes, W. A 1978. The Biology and Cultivation ofEdibleMushrooms, Academic Press, Inc., New York.

Cohen, R., Persky, L., and Hadar, Y. 2002. Biotechnological applications andpotential of wood-degrading mushrooms of the genus Pleurotus. AppliedMicrobiology and Biotechnology.

93

Crisan, E. V. and Sands, A 1978. Nutritional value. In The biology and cultivationof edible mushrooms. (S. Chang and W. A Hayes, Eds.), pp. 137-165.Academic Press, INC., New York, NY.

Da Serra, M. and Kirby, R. 1999. Development of Pleurotus (oyster) mushroomproduction in South Africa using alien wood species as lignocellulosesubstrate. The International Journal of Mushroom Sciences 2, 49-55.

DeBell, D. S., Whitesell, C. D., and Schubert, 1. H. Mixed Plantations ofEucalyptus and Leguminous Trees Enhance Biomass Production. USDA­Forest Service PSW-175. 1985. Berkeley, CA, Pacific Southwest Forestand Range Experiment Station.

Dommergues, Y. 1990. Casuarina equisetifolia: An old-timer with a new future.NFTA (Nitrogen Fixing Tree Association) 90.

Duke, J. A Handbook of Energy Crops- Casuarina equisetifolia J.R. & G. Forst.1983. May 2004.

Eger, G. 1978. Biology and breeding of Pleurotus. In The biology and cultivationofedible mushrooms (S. 1. Chang and W. A Hayes, Eds.), pp. 497-517.Academic Press, New York.

Fasidi, I. O. and Olorunmaiye, K. S. 1994. Studies on the requirements forvegetative growth of Pleurotus tuber-regium (Fr.) Singer, a Nigerianmushroom. Food Chemistry 50, 397-401.

Flegg, P. B., Spencer, D. M., and Wood, D. A 1985. The biology and technologyof the cultivated mushroom, Wiley, Chichester West Sussex, New York.

Fleming, K. and Sato, D. CTAHR. Cost of Production Spreadsheet: WetlandTaro. 2. March 31,2001. June 2004.

Freer, S. N. and Detroy, R. W. 1982. Biological delignification of 14C-labelledlignocelluloses by basidiomycetes: degradation and solubilization of thelignin and the cellulose components. Mycologia 74,943-951.

Gerasimenya, V. P., Efremenkova, O. V., Kamzolkina, O. V., Bogush, 1. A,Tolstych, I. V., and Zenkova, V. A 2002. Antimicrobial and antitoxicalaction of edible and medicinal mushroom Pleurotus ostreatus (Jacq.: Fr.)Kumm. extracts. International Journal of Medicinal Mushrooms 4, 127­132.

Gunde-Cimerman, N. 1999. Medicinal value of the genus Pleurotus (Fr.) P.Darst. (Agaricales s.l., Basidiomycetes). International Journal of MedicinalMushrooms 1, 69-80.

94

Hatakka, A Biodegradation of Lignin. Hofrichter, Martin and Steinbuchel,Alexander. Lignin, Humic Substances and Coal. Volume 1, 130-167.2004.Wiley-VCH. Biopolymers. Steinbuchel, Alexander.

Hines, D. A and Eckman, K. 1993. Indigenous multipurpose trees of Tanzania:Uses and economic benefits for people, FAa, Ottawa, Ontario, Canada.

Huenneke, L. F. and Vitousek, P. M. 1990. Seedling and clonal recruitment of theinvasive tree Psidium caffleianum: Implications for management of nativeHawaiian forests. Biological Conservation 53, 199-211.

Issac, R. A and Jones Jr., B. J. 1972. Effects of various dry ashing temperatureson the determination of 13 nutrient elements in five plant tissues.Communications in Soil Science and Plant Analysis 3,261-269.

Ivors, K. Tracing the roots of mushroom cultivation. Mycena News 54 [4]. 2003.San Fransicsco, Mycological Society of San Francisco.

Jeffries, T. W., Choi, S., and Kirk, T. K. 1981. Nutritional regulation of lignindegradation by Phanerochaete chrysosporium. Applied and EnvironmentalMicrobiology 42, 290-296.

JinTorng, P., ChienMing, L., and YingFung, T. 2000. Effect of different organicsupplements on the production of King oyster mushroom by using bottlecultivation technology. Journal ofAgricultural Research of China 49,56­64.

Jose, N., Ajith, T. A, and Jananrdhanan, K. K. 2002. Antioxidant, anti­inflammatory, and antitumor activities of culinary-medicinal mushroomPleurotus pulmonarius (Fr.) Quel. (Agaricomycetideae). InternationalJournal of Medicinal Mushrooms 4, 329-335.

Kaal, E. E. J., Field, J. A, and Joyce, T. W. 1995. Increasing ligninolytic enzymeactivities in several white-rot Basidiomycetes by nitrogen-sufficient media.Bioresource Technology 53, 133-139.

Kalberer, P. P. 1974. The cultivation of Pleurotus ostreatus: Experiments toelucidate the influence of different culture conditions on the crop yield.Mushroom Science 9,653-662.

Kalita, P. and Mazumder, N. 2001. Performance of oyster mushroom (Pleurotusspp.) on certain plant wastes. Journal ofAgricultural Science Society ofNorth-East India 14, 221-224.

Khamoui, T. and Baker, H. L. An economic analysis of Hawaii eucalypt woodchipproduction for export. May 1981. University of Hawaii, Hawaii Institute ofTropical Agriculture and Human Resources.

95

Kirk, 1. K. and Farrell, R L. 1987. Enzymatic "Combustion": The Microbialdegradation of lignin. Annual Review of Microbiology. 41, 465-505.

Koske, R E., Gemma, J. N., and Flynn, 1. 1992. Mycorrhizae in Hawaiianangiosperms: A survey with implications for the origin of the native flora.American Journal of Botany 79, 853-862.

Little, E. L. Jr. and Skolmen, R G. Common Forest Tress of Hawaii (Native andIntroduced). Agriculture Handbook 679. 1989.

Martin, D. A, Nakamura, R, and Taka, E. 2001. A special report on Hawaii'sprivately-owned forest lands. NASS- Hawaii Forestry.

Morton, J. F. 1987. Costa Rican Guava Psidium friedrichsthalianum Ndz. InFruits of warm climates (J. F. Morton, Ed.), pp. 365. Miami, FL.

Muthukrishnan, N., Venugopal, M. S., and Janarthanan, R 2000. Recyclingspent larval food of Corcyra cephalonica Stainton for preparing spawn andsporophore of Pleurotus sajor-caju (Fr.) Singer. World Journal ofMicrobiology & Biotechnology 16,265-270.

NOAA. National Climactic Data Center. www.prn.noaa.gov. September 2004.

Oei, P. Mushroom cultivation: with special emphasis on appropriate techniquesfor developing countries. 274. 1996. Leiden, The Netherlands, TOOLPublications.

Orth, A 8., Royse, D. J., and Tien, M. 1993. Ubiquity of lignin-degradingperoxidases among various wood-degrading fungi. Applied andEnvironmental Microbiology 59,4017-4023.

Parrotta, J. A Casuarina equisetifolia L. ex J.R & G. Forst. 1-11. 1993. UnitedStates Department of Agriculture, Forest Service.

Patrabansh, S. and Madan, M. 1999. Mineral content of the fruiting bodies ofPleurotus sajor-caju (FR) Singer cultivated on different kinds of biomass.Acta Biotechnologica 19,101-109.

Patrabansh, S. and Madan, M. 1997. Studies on cultivation, biological efficiencyand chemical analysis of Pleurotus sajor-caju (FR) Singer on different bio­wastes. Acta Biotechnologica 2.

Pattison, R R, Goldstein, G., and Ares, A 1998. Growth, biomass allocation andphotosynthesis of invasive and native Hawaiian rainforest species.Oecologia 177, 449-459.

Pettipher, G. L. 1987. Cultivation of the oyster mushroom (Pleurotus ostreatus)

96

on lignocellulosic waste. Journal of the Science of Food and Agriculture.41, 259-265.

Philippoussis, A, Zervakis, G., and Diamantopoulou, P. 2001. Bioconversion ofagricultural lignocellulosic wastes through the cultivation of ediblemushrooms Agrocybe aegerita, Volvariella volvacea and Pleurotus spp.World Journal of Microbiology & Biotechnology 17, 191-200.

Ray, A A Sail, J. P., Saffer, M., Joyner, S. P., and Whatley, J. K. 1982. SASUser's Guide: Statistics, SAS Institute, Inc., North Carolina.

Richard, 1. The Effect of Lignin on Biodegradability. 1996. April 28, 2004.

Rockwood, D. L., Fisher, R. F., Conde, L. F., and Huffman, J. B. 1990.Casuarina. In Agricultural Handbook 654, Silvics of North America (R. M.Burns and B. H. Honkala, Eds.), USDA, Washington DC.

Royce, D. J., Rhodes, 1. W., Ohga, S., and Sanchez, J. E. 2004. Yield,mushroom size and time to production of Pleurotus cornucopiae (oystermushroom) grown on switch grass substrate spawned and supplementedat various rates. Bioresource Technology 19,85-91.

Royse, D. J. 2003. Cultivation of Oyster Mushrooms. Penn State College ofAgricultural Sciences, Agricultural Research and Cooperative Extension 1­11.

Royse, D. J. 2002. Influence of spawn rate and commercial delayed releasenutrient levels on Pleurotus cornucopiae (oyster mushroom) yield, size,and time to production. Applied Microbiology & Biotechnology. 58, 527­531.

Royse, D. J. Specialty Mushrooms. In Janick, J. (Ed.) Progress in New Crops.Specialty Mushrooms, 464-475. 1996. Arlington, VA, ASHS Press.

Salim, A S., Simons, A., Orwa, C., Chege, J., Owuor, B., and Mutua, A. 2002.Agroforestree Database. World Agroforestry Center. 2002.

Statistical Analysis System (SAS). SAS Institute. 1982. Cary, NC, SAS Institute.

Shen, Q., Dan, H., Chen, Y., and Royce, D. J. Comparison of oyster mushroomproduction practices in China and the United States. The FourthInternational Conference of Mushroom Biology and Mushroom Products.2004. World Society of Mushroom Biology and Mushroom Products.

Sivaprakasam, K. and Kandaswamy, 1. K. 1981. Influence of the growth ofPleurotus sajor-caju (Fr.) Singer on cellulose content of the substrates.Madras Agricultural Journal. 68,628-630.

97

Stamets, P. 2000. Growing gourmet and medicinal mushrooms, Ten SpeedPress, Berkeley, CA

Staples, G. W. and Cowie, R H. 2001. Hawaii's Invasive Species: A Guide toInvasive Plants and Animals in the Hawaiian Islands, Mutual Publishingand Bishop Museum Press, Honolulu, HI.

Tan, Y. H. and Wahab, M. N. 1997. Extracellular enzyme production duringanamorphic growth in the edible mushroom, Pleurotus sajor-caju. WorldJournal of Microbiology & Biotechnology 13,613-617.

Thomas, G. V., Prabhu, S. R, Reeny, M. Z., and Bopaiah, B. M. 1998.Evaluation of lignocellulosic biomass from coconut palm as substrate forcultivation of Pleurotus sajor-caju (Fr.) Singer. World Journal ofMicrobiology and Biotechnology 14,879-882.

Tunison, T. 1991. Element Stewardship Abstract for Psidium cattleianum. TheNature Conservancy, Arlington, VA

USDA 2004. 2002 Census of Agriculture. National Agricultural Statistics ServiceAC-02-A-PR.

USDA 2003. Mushrooms. NASS, Agricultural Statistics Board.

Van Soest, P. J. 1963. Use of detergents in the analysis of fibrous feeds. II. Arapid methods for the determination of fiber and lignin. Journal ofAssociation of Official Analytical Chemists 46,829-835.

Velazquez-Cedeno, M. A, Mata, G., and Savoie, J.-M. 2002. Waste-reducingcultivation of Pleurotus ostreatus and Pleurotus pulmonarius on coffeepulp: changes in the production of some lignocellulolytic enzymes. WorldJournal of Microbiology and Biotechnology 18,201-207.

Wasser, S. P. and Weis, A L. 1999. General description of the most importantmedicinal higher basidiomycetes mushrooms. International Journal ofMedicinal Mushrooms 1,351-370.

Weigand, J. F. Management experiments for high-elevation agroforestrysystems jointly producing matsutake mushrooms and high-quality timberin the Cascade Range of Southern Oregon. July 1998. USDA ForestService.

Whitesell, C. D., DeBell, D. S., Schubert, T. H., Strand, R F., and Crabb, T. B.Short-Rotation Management of Eucalyptus: Guidelines for Plantations inHawaii. 1992. Albany, CA, Pacific Southwest Research Station. USDAForest Service General Technical Report.

98

Yamanaka, K. 1997. Production of cultivated edible mushrooms. Food ReviewsIntemationa/13, 327-333.

Yamashita, N., Ishida, A., Kushima, H., and Tanaka, N. 2000. Acclimation tosudden increase in light favoring an invasive over native trees insubtropical islands, Japan. Oecologica 125, 421-419.

Yang, H. H., Effland, M., and Kirk, T. K. 1980. Factors influencing fungaldecomposition of lignin in a representative lignocellulosic,thermomechanical pulp. Biotechnology and Bioengineering 22,65-77.

Yang, J.-H., Lin, H.-C., and Mau, J.-L. 2001. Non-volatile taste components ofseveral commercial mushrooms. Food Chemistry 72, 465-471.

Yost, R. S., DeBell, D. S., Whitesell, C. D., and Miyasaka, S. C. 1987. Earlygrowth and nutrient status of Eucalyptus saligna as affected by nitrogenand phosphorus fertilization. Australian Forestry Resources. 17,203-214.

Zadrazil, F. 1978. Cultivation of Pleurotus. In The biology and cultivation of ediblemushrooms (S. T. Chang and W. A. Hayes, Eds.), pp. 521-553. AcademicPress, New York.

Zhang, R., Li, X., and Fadel, J. G. 2000. Oyster mushroom cultivation with riceand wheat straw. Bioresource Technology 82,277-284.

99