Study of the Growth and Biomass Composition of the Edible Mushroom Pleurotus Ostreatus

G. C h a r a l a m b o u s (Ed.), F o o d S c i e n c e a n d H u m a n N u t r i t i o n © 1992 Elsevier Science Publishers B.V. All rights reserved.

239

STUDY OF THE GROWTH AND BIOMASS COMPOSITION OF THE EDIBLE MUSHROOM PLEUROTUS OSTREATUS.

A. M.

Martin

Food Science Program, Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, Canada, A1B 3X9.

SUMMARY The edible mushroom Pleurotus ostreatus. also known as the o y s t e r m u s h r o o m , w a s c u l t i v a t e d in l i q u i d a n d s o l i d m e d i a u s i n g peat as the main substrate source. Amino acids, fat, fibre, moisture, nitrogen, and protein contents were determined for the mushroom biomass produced. T h e p r o t e i n contents of the m u s h r o o m s cultivated on liquid and solid media were approximately 4 0% and 36%, respectively, calculated on a dry weight b a s i s . These are promising values, when compared to those reported with the use of other substrates. In addition, the amino acid p r o f i l e of the m u s h r o o m b i o m a s s showed that the essential amino acids were present in fairly good concentrations.

1.

INTRODUCTION Edible mushrooms have been increasingly recognized by the

Industry worldwide years,

the

as nutritious

consumption

and p o p u l a r

of m u s h r o o m s

has

foods

attained

(1).

an

In

approximately

1 0 % g r o w t h r a t e p e r y e a r in i n d u s t r i a l i z e d W e s t e r n c o u n t r i e s including Canada becoming

the

food

(4), where this interest has resulted

second

tonnes valued

at

most

valuable

136 m i l l i o n

Mushrooms

are mostly

staples.

Only

vegetable

dollars

used

one-third

as of

flavoring the

fresh, while the remaining two-thirds agents

or canned

Only

a

cultivated.

few

the

with

1985

agents

world's

are processed

51,4 00

(5).

rather

annual

(2,3),

mushrooms

crop

into

than is

as

sold

flavoring

the

edible

mushroom

species

are

commercially

The five most important cultivated mushrooms are:

mushroom/shiitake (Flammulina

crop, in

in

(6). of

white mushroom/button mushroom

and

produced

Food

recent

(Lentinus

velutipes),

oyster mushroom

the

(Agaricus b i s p o r u s ) ,the b l a c k edodes),

straw

(Pleurotus

mushroom

the

winter

mushroom

(Volvariella

ostreatus)

(3,7).

the

forest

volvacea)

Agaricus

is

240 cultivated mainly

in

commonly

in

Europe grown

Volvariella, Pleurotus

seventy-four and

North

in C h i n a

but

it

and

is

is g a i n i n g

countries America.

popularity

is

commonly

Lentinus

Japan.

familiar

but

China to

Flammulina

is t h e m a i n

all

in E u r o p e

and

Southeast and

consumed

Asians,

source as

of the

in

in

world

edible

fungi

accepted

the

delicate and

other

(5),

and

foods

ostreatus

has been flavor

so

far

f

and

There

are

more

only

about

25

It

is

reported

Cultivation

more

commonly

favor

among

valued 2,000

species

are

known

mushroom

as

the

lovers

oyster

due

to

its

sources

such

as

straw,

corn

cobs

and

(11)

sawdust

(12, 1 3 ) .

that

for

both

mycelium

lignin-cellulose of

in

(10).

Pleurotus species have a relatively simple growth

development,

are than

P. o s t r e a t u s m a y b e g r o w n o n w a s t e p a p e r

cullulose in p e a t

in c a l o r i e s ,

consumption

gaining

(8).

low

(3,6).

but

for human

Pleurotus mushroom,

(9), are

agents

widely

(8)

In a d d i t i o n to p r o t e i n s , m u s h r o o m s a r e a good

Β vitamins

flavoring

of and

Asia.

Mushroom protein contains all the essential amino acids high concentrations.

are

producer

Pleurotus

growth

materials

species

does

not

and

are

requirement.

fruiting

sufficient

require

body (14).

composting.

In

s o m e c a s e s , p a s t e u r i z a t i o n or s t e r i l i z a t i o n of t h e s u b s t r a t e is not necessary

either.

Pleurotus

species

Comprehensive has

been

literature

presented

on the

(15).

It

cultivation

has

been

that supplementing the substrate with some organic and/or sources

of

contents

of

Pleurotus

nitrogen

could

Pleurotus.

have

been

increase

yield

The possibilities

comprehensively

and

of

by

33°C)

and

relative

The production use

as

with

a

the

food

or

in

humidity

of mushroom food

traditional

(67 t o

mycelium

products

technique

is of

(or s p o r o p h o r e s )

s i n c e W o r l d W a r II in a n t i b i o t i c

to

idea

fermenters. produced

growing

In

this

throughout

such as food

mushroom way,

the

a

submerged

producing

gained

of

in

or

In

beds.

addition,

industry wastes, can be utilized

and

led

aerated could

be

materials,

as substrate

for m u s h r o o m p r o d u c t i o n and at t h e s a m e t i m e r e d u c e t h e oxygen demand of the w a s t e s .

fruiting Experience

product

low-cost

for

compared

processes

agitated

mushroom-flavored

year.

culture

recent,

fermentation in

by

(16) .

temperature

mushroom

in c o m p o s t

mycelium

fixation

(17).

relatively

bodies

the

in m a n u r e

72%)

nitrogen

Kurtzman

Pleurotus species can be cultivated over w i d e ranges of (21 t o

reported inorganic

total

nitrogen

reviewed

of

sources

biochemical

The flavored mushroom mycelium

should

241 be

suitable

for

sauces, etc. was

use

(6) .

initiated

in

in

formulated

foods,

such

as

instant

Commercial production of morel mushroom 1963

by

a company

in t h e U n i t e d

States

soups,

mycelium using

the

Szuecs process.

The production was discontinued because

apparently

the

not

of

market

was

yet

prepared

for

the

acceptance

such

a

product. The

process

in s u b m e r g e d with (18).

in

A

commercial

review

extracts

the

extracting

of

the

or peat

Soviet

Union

States and Canada.

the

submerged

(18-20)

and

value

literature

on

from

a

been

peat

shows has

lesser

studied that

been

to be

scale

acid hydrolysates of

the

in

employed

microorganisms

for

a

long

time

fermentation

conducted

The bulk of the research

culture

fungi

has

hydrolysates

and

been done utilizing in

nutrients

fermentations for the growth of v a r i o u s

potential

peat

of

Ireland,

the

United

in t h e s e c o u n t r i e s

from peat

microorganisms,

as the basic

mainly

of

extensively

yeast

has

media

species

(21-25).

This work reviews the activities which have been conducted the

Department

Newfoundland, in

liquid

source

of

Biochemistry,

Canada,

and

solid

in t h e media

utilizing

MATERIALS AND METHODS

2.1

Organism Pleurotus

University Peat The

ostreatus

of Western

No.

was

samples,

work

reported

here

and

peat

preparation

The

of

also of

tends

the

approximately

results

2 4

the

nutrient

main

of

Plant

Sciences,

Canada).

H S0

reported

2 A

(121°C)

solution

a

high-moor

The

to

initial moisture there

was

some

lose moisture the

in

Sphagnum

for

in

this

work

content

are mixed

of

this

variation

between

storage.

Before

peat

was

correspond

18 t o 6 0 m e s h p a r t i c l e

two

peat

taken from a bog

dried

in

a

moisture

10%.

solution

dry-peat:

utilized

hydrolysates,

a

H S0

as

(Blue M E l e c t r i c Co.) to o b t a i n a s t a n d a r d

of

psig

mushroom

(Department

London,

80%, but

using ground peat

15

of

ostreatus

low degree of decomposition,

approximately

laboratory oven content

152

Ontario,

near the city of St. J o h n ' s .

the

peat

University

P.

hydrolysates

moss, of a relatively

peat

of

(12, 1 3 ) .

2.

2.2

Memorial

cultivation

at

ratio

hours. together

of

1:6

Before

size,

(w/w),

to

1.5%

and

process

(v/v)

H SO ,

2 A

autoclaving

autoclaving,

thoroughly.

the

the

peat

at and

242 The

hydrolysates

product

in

Inc.),

a

modified

followed

paper.

The

nutrients

were

by

laboratory

vacuum

hydrolysates

of

several

sources,

or

in

alone

as possible

minimizing

the

by

press

on the

have

tested

been

and

with

inorganic

nutrients of

(Model

through

The

C,

F.S.

of

organic

has

been

to

the

ones,

although better growth could be achieved with more formulations. or with

concentrated

2.3

nitrogen Culture

NH OH

solutions.

A

supplements

was

being

aseptically

inoculated

(100 m L

with

expensive 15N

NaOH was utilized

when

in t h e p e a t e x t r a c t o r

in 2 5 0 m L s h a k e

blended

pure

incubated

in a G y r o t o r y w a t e r b a t h s h a k e r Inc.).

aerated

Fermentations

and

Co.,

Inc.).

pH,

fermentation

optimize

itself, cheapest

in

tested.

Sci. litre

as

conditions

The sterile growth media

Co.,

rely

The pH of the h y d r o l y s a t e s w a s adjusted by

the effect of the nitrogen concentrations other

various nutrient

hydrolysate

choosing

filter

additional

media NaOH

Carver,

#1

addition

aim

and

autoclaved

Whatman

and

in t h e p e a t

supplements

the

without

the

salts

combinations.

addition

pressing

filtration

(non-supplemented),

concentrations

much

obtained

agitated

have

flasks)

were

mycelia

and

(Model G 7 6 , N e w

also

fermenter

culture

been

(Bioflo,

Brunswick

conducted New

the

growth

and

agitation

conditions

of

speed

the

have

been

microbial

a

Brunswick

V a r i o u s v a l u e s of inoculum ratio, temperature, time

in

1-

Sci.

initial

tested

species

to

(13,

24,

for

the

25) . 2.4

Spawn

preparation

Wheat

grain,

preparation water

and

of

raw peat

the

then

and

1%

CaC0 ,

3

psig

inoculated

on

The

temperature

20

was

minutes

at

for

14

boiled

room

grain

15

weight.

with

temperature the

inoculated days

first

and

growth

psig

used

as

one

was

spawn

to

in

(121°C) .

10% raw

Glass

peat

bottles

for 20 m i n u t e s at

(approximately

of

mixture

immersed

slant

25°C)

culture

15 and

of

P.

incubated

at

room

inoculate

the

solid

substrate.

Substrate Sphagnum

support

for

carbonate were used grain

and the grain was mixed with the

to

aseptically

ostreatus.

2.5

based

wheat

200g of the mixture were sterilized

(121°C) , c o o l e d

culture

The

autoclaved

Excess water was removed

containing

and calcium

spawn.

for peat

for the

treatment

P.

involved

fruiting moss

was

ostreatus only

body

production

used

as

fruiting

addition

of

the body

substrate

source

development.

sufficient

water

to

The the

and preraw

243 peat to obtain an initial substrate m o i s t u r e content of 7 5 - 8 0 % ,

and

addition

The

of

substrate bran,

and

(121°C) . was

to

raise

by

at

the

of

rate

the

were

trays

made

tungsten

near

for

neutral

20

of

values.

dry

peat)

minutes

in

the

twice

a

the

sterilized

substrate χ

15

χ

at

was

aseptically cm)

and

polythene

cover

to

allow

day.

The

air

The

were

2.6

Analytical

considered

from

substrate

was

cultivation of

chamber

mushrooms

method

to have

attained

their maximum

fermented media were determined

(2 6 ) .

nitrogen

The moisture

by

the

AOAC

gravimetrically

For with

the

with

Beckman

121

MB

All

results

when

growth.

analyses,

0.2N

amino are

determined

for

24 h o u r s

Fat was

ash

was

reagent drying,

and

crude

determined

determined

at

samples 110°C

were The

by

(27) .

hydrolysed

samples

were

citrate buffer and analysed with

analyser

average

oven

fibre by the A O A C 7.061 m e t h o d freeze-dried

lithium

acid

by

method,

6.25.

extraction,

hydrolysates

by the anthrone

micro-Kjeldahl

ether

under vacuum

reconstituted

the

after

acid

was

the % nitrogen by

at 600°C, a n d c r u d e

amino

6N H C 1

content

47.003

protein by multiplying

combustion

was

appeared

methods

T h e t o t a l c a r b o h y d r a t e c o n c e n t r a t i o n s in t h e p e a t in t h e

with

to light

the

flush

a

diameter

exchange

colonized

in

into

with

i n c u b a t e d a t 2 7 + 2°C

the

first

packed

1.5 m m

f o r 14 d a y s , a n d t h e n e x p o s e d Thereafter,

spawn

weight.

covered

gas

psig

14 d a y s , a n d t h e f r u i t i n g b o d i e s w e r e h a r v e s t e d

they

and

wheat

15

substrate

2.5

The covered containers were

humidified.

10 and

of

Six h o l e s of a p p r o x i m a t e l y

source.

constantly between

(by w e i g h t

(22

in t h e a b s e n c e o f l i g h t

watered

to

5%

7%

spawned

aluminum

the environment.

a

pH

autoclaving

of

sterile p o l y t h e n e film. each

the with

After the substrate was cooled to room temperature,

3 00g

sterile

3

supplemented

sterilized

added

About

CaC0

was

values

using from

a at

single least

column three

a

method.

different

determinations.

3.

RESULTS AND DISCUSSION

3.1

Composition

of

substrates

T a b l e 1 shows the composition of the peat extracts utilized the submerged c u l t i v a t i o n of Pleurotus ostreatus m u s h r o o m and

of

the

solid

peat

utilized

for the

growth

of

in

mycelium,

fruiting

bodies.

244 TABLE

1

Basic c o m pa osition substrates.

of

the

Component

peat

Peat

Total carbohydrate Moisture Nitrogen

hydrolysates

hydrolysates

32.75

+

1.23 g / L

±

0.01 g / L

0.60

Not

of three determinations determined.

3.2

Growth

parameters

The production

+

(29)

Peat

of mushroom

deviations.

cultivation

mycelium

was

optimized

the growth parameters.

The conditions resulting

are

2.

in

concentration

Table

produced,

In

addition

the value

(12)

b 77.5 ± 3 . 5 % 1.3 ± 0 . 1 %

standard

in s u b m e r g e d

peat

solid

b

a bM e a n s

presented

and

to

the

by

varying

in t h e b e s t total

for the b i o m a s s

dry

yield

growth

mycelium (grams

of

dry mycelium

produced p e r gram of total carbohydrate

consumed)

calculated.

As

approximately

60%,

was

processes TABLE

it m a y

relatively

be

seen,

high

in

for the production

the yield

obtained,

comparison

of microbial

to

other

fermentation

biomass.

2

Growth parameters for the mushroom Pleurotus ostreatus produced s u b m e r g e d c u l t i v a t i o n in p e a t h y d r o l y s a t e s ( 1 3 ) . Parameter

150 5 5 28 5 60

(g/L)

Composition of the mycelium and fruiting bodies o s t r e a t u s m u s h r o o m c u l t i v a t e d in p e a t s u b s t r a t e s The concentration of the main components

biomass Table

and

The

fruiting

of

for both the

ostreatus

in

for the same

mushroom

case

it

has

concentration cellulosic

in

been the

substrates

substrates. reported peat

(14)

In

that

substrates,

generally

the

employed

as

the

is

mycelial

reported

species

other

characteristic

is

content, which was higher than those reported in

significant

P.

P.

protein

grown

most

bodies

of

the

bodies,

3.

the

in

Value

A g i t a t i o n (rpm) I n o c u l u m r a t i o % (v/v) pH T e m p e r a t u r e (°C) Final biomass concentration Biomass yield (%)

3.3

was

of

higher

compared

in t h e

the

solid

fruiting nitrogen

with

other

cultivation

245 of

P.

ostreatus,

fungus

contributed

to the higher

protein

content

of

the

grown.

Finally, Table 4 shows the essential amino acid composition P.

ostreatus

mycelium

grown

in

submerged

f r u i t i n g b o d i e s g r o w n in s o l i d m e d i u m . amino acids were present mycelium

presented

than

and

It s h o w s t h a t a l l

in s a t i s f a c t o r y

higher values

culture

amounts.

the

of

essential

In g e n e r a l ,

fruiting

of the

bodies

for

the the

crude protein content and for the individual essential amino acids. These

findings

mycelium TABLE

as

could

highlight

a protein

supplement

the for

potential

of

the

mushroom

foods.

3

C o m p o s i t i o n o f t h e m y c e l i u m a n d f r u i t i n8g m u s h r o o m c u l t i v a t e d in p e a t s u b s t r a t e s .

Component

(%)

Mycelium

Ash Crude Protein Fat Fibre Moisture

(3 0)

bodies

Fruiting

6.5 + 0.5 4 0 . 1 + 1.8 2.6 + 0.2 5.9 + 0.4 7 8 . 2 + 2.5

of

P.

bodies

8.6 36.0 1.9 6.5 89.6

ostreatus

(12)

+ 0.5 + 4.9

± ± ±

0.1 0.3 0.6

"Means of t h r e e d e t e r m i n a t i o n s ± s t a n d a r d d e v i a t i o n s . With the exception of the % m o i s t u r e content, all v a l u e s are reported on a dry weight basis.

TABLE

4

3

Essential amino acid composition h y d r o l y s a t e s (g/100g p r o t e i n ) .

Amino

Acid

Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine

b "Means Not

of three reported.

Mycelium 3..5 6..1 5..7 1,.0 3..4 4..9 1..2 3..9

+ + + + + + + +

(31)

of

P.

Fruiting

bodies

2..1 + 3,.5 + 3,.4 + 1..0 + 2..2 + 2,,7 + b 1..8 +

0,.2 0..3 0,.4 0..1 0..3 0..3 0.,1 0.,2

determinations

ostreatus

+

standard

grown

(12)

0..3 0..2 0..4 0..1 0..1 0.,2 0.,1

deviations.

in

peat

246 3.4

Production

of mycelial

Generally,

the

mycelium product.

In

fermentation, important the

center

transfer medium.

production

of

the pellets viscosity the

of

The

(28). of

separation

study

campestris

in

the

production

a safe, of

mushrooms

mycelial

of

flavoured,

pellets

mushroom

marketable

in

submerged

is

reportedly

of

the of

the

In addition, medium, the

biomass

production

(bisporus)

has been

a pellet

enhancing from

of

suspension

mixing,

the

rest

mycelial

reported

in

mass

of

the

pellets

of

(22).

CONCLUSIONS The

edible

mushroom, for the main

cultivation

the and

Agaricus

the

the

objective

consistently,

for flavour development, probably because of autolysis

decreases

4.

main

is t o o b t a i n ,

pellets

mushroom

P.

ostreatus,

was adapted and cultivated

first time using peat

substrate

employed mushroom

a

the

source

mycelial

controlled industry.

is o f t e n u s e d

bisporus. as

known

as

the

oyster

in b o t h s o l i d a n d l i q u i d

and peat hydrolysates

media

as the only

or

in t h e p r o d u c t i o n

of

source.

Peat, which Agaricus

also

of

nutrients

biomass,

conditions

as casing

well-known

a

with

the

soil

button in

mushroom,

the

submerged

objective

mushroom-flavoured

can

be

growth

of producing,

product

for

also

the

of

under food

247

REFERENCES 1.

2.

3. 4. 5. 6. 7.

8. 9.

10. 11. 12. 13. 14. 15.

16.

17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

27.

F. Zadrazil and K. Grabbe, in: H. Dellweg (Ed) , Biotechnology. Volume 3, Verlag Chemie, Weinheim, F.R. Germany, 1983, pp. 147-187. W . A . H a y e s a n d N . G. N a i r , in: J. E . S m i t h a n d D . R . B a r r y ( E d s ) , T h e F i l a m e n t o u s F u n g i . V o l u m e 1: Industrial Mycology, Edward Arnold Ltd., London, England, 1975, pp. 212-248. M . C . T s e n g a n d J. H . L o u n g , A n n u a l R e p o r t s o f F e r m e n t a t i o n P r o c e s s e s , 7 (1984) 4 5 - 7 9 . A n o n y m o u s . C a n . I n s t . F o o d S c i . T e c h n o l . J., 14 ( 1 9 8 1 ) 3 5 8 . J. K . S c o t t , R e a d e r ' s D i g e s t , 1 3 0 N o . 7 8 0 ( 1 9 8 7 ) 1 3 - 1 8 . J. H . L i t c h f i e l d , F o o d T e c h n o l . , 2 1 N o . 1 5 9 ( 1 9 6 7 ) 5 5 . S . T . C h a n g a n d P. G . M i l e s , i n : S . T . C h a n g a n d T . H . Q u i m i o (Eds), Tropical Mushrooms. Chinese University Press, Hong Kong, 1982, pp. 3-10. S . T . C h a n g , O . W . L a u , Κ. Y . C h o , E u r o p . J. A p p l . M i c r o b i o l . B i o t e c h n o l . , 12 ( 1 9 8 1 ) 5 8 - 6 2 . Ε. V. Crisan and A. S a n d s , in: S. T. Chang and W. A. H a y e s (Eds), The Biology and Cultivation of Edible Mushrooms. A c a d e m i c P r e s s , N e w York, N . Y . (1978) p p . 1 3 7 - 1 6 8 . E. A. Bessey, Morphology and Taxonomy of Fungi, Hafner Publishing Co., New York, N.Y. (1961). Κ. H . S t e i n k r a u s a n d R. E . C u l l e n , N e w Y o r k ' s F o o d a n d L i f e S c i e n c e s , 11 N o . 4 (1978) 5 - 7 . W . M a n u - T a w i a h a n d A . M . M a r t i n , J. S c i . F o o d A g r i c , 37 (1986) 8 3 3 - 8 3 8 . W. Manu-Tawiah and A. M. Martin, Appl. Biochem. Biotechnol., 14 ( 1 9 8 7 ) 2 2 1 - 2 2 9 . S . S . B l o c k , A p p l . M i c r o b i o l . , 13 ( 1 9 6 5 ) 5 - 9 . F. Z a d r a z i l a n d R . H . K u r t z m a n , i n : S. T. C h a n g and T. H. Quimio (Eds), Tropical Mushrooms. Chinese University Press, Hong Kong, 1982, pp. 277-298. R . H . K u r t z m a n a n d F. Z a d r a z i l , i n : S. T. C h a n g and T. H. Quimio (Eds), Tropical Mushrooms. Chinese University Press, Hong Kong, 1982, pp. 299-348. Z. Bano, H. C. Srinivasan and H. C. Srivastava, Appl. M i c r o b i o l . , 11 ( 1 9 6 3 ) 1 8 4 - 1 8 7 . A . L e D u y , P r o c e s s B i o c h e m . , 15 ( 1 9 7 9 ) 5 - 7 . C. H. Fuchsman, Peat, Industrial Chemistry and Technology, Academic Press, New York, N.Y., 1980, pp. 119-134. P. Q u i e r z y , N . T h e r i e n a n d A . L e D u y , B i o t e c h n o l . B i o e n g . , 2 1 (1979) 1 1 7 5 - 1 1 9 0 . J. M . B o a a n d A . L e D u y , C a n . J. C h e m . E n g . , 6 0 ( 1 9 8 2 ) 5 3 2 - 5 3 7 . A . M . M a r t i n a n d V . I. B a i l e y , A p p l . E n v i r o n . M i c r o b i o l . , 49 (1985) 1 5 0 2 - 1 5 0 6 . A. M. Martin and M. D. White, Appl. M i c r o b i o l . Biotechnol., 2 4 (1986) 8 4 - 8 8 . A . M . M a r t i n , J. F o o d S c i . , 4 8 ( 1 9 8 3 ) 2 0 6 - 2 0 7 . A . M . M a r t i n , C a n . I n s t . F o o d S c i . T e c h n o l . J., 16 ( 1 9 8 3 ) 2 1 5 217. A . L e D u y , N . K o s a r i c a n d J. E . Z a j i c , i n : T. C. H u t c h i n s o n ( E d ) , C o r r e c t i o n F a c t o r for A n t h r o n e C a r b o h y d r a t e in C o l o u r e d Wastewater Samples. Proceedings of the 10th Canadian Symposium on Water Pollution Research, Toronto, Canada, U n i v e r s i t y of T o r o n t o , 1975, pp. 1 2 6 - 1 3 1 . A.O.A.C., Official Methods of Analysis, 13th Edition, A s s o c i a t i o n of Official A n a l y t i c a l C h e m i s t s , W a s h i n g t o n , D.C., 1980.

248 28. 29.

30.

31.

J. C . S u i j d a m , Ν . W . F . K a s s a n a n d P. G . P a u l , E u r o p . J. A p p l . M i c r o b i o l . B i o t e c h n o l . , 10 (1980) 2 1 1 - 2 2 1 . W. M a n u - T a w i a h and A. M. Martin, in: K. G r a b b e a n d O . H i l b e r (Eds), U s e s of N i t r o g e n - Supplemented Peat E x t r a c t s for the Cultivation of Pleurotus ostreatus Mushroom Mycelium. M u s h r o o m S c i e n c e X I I (Part I I ) , P r o c e e d i n g s of t h e T w e l f t h International Congress on the S c i e n c e and Cultivation of E d i b l e F u n g i , B r a u n s c h w e i g , F. R. G e r m a n y , S e p t e m b e r 1 9 8 7 , International Society for Mushroom Science, Berlin, 1989. pp. 157-167. A. M. Martin and W. Manu-Tawiah, in: E. D. Primo Yufera and P. F i t o M a u p o e y (Eds), Biomass Composition of the Edible Mushroom Pleurotus ostreatus. A d v a n c e s in F o o d T e c h n o l o g y , V o l . 1. P r o c e e d i n g s o f t h e S e c o n d W o r l d C o n g r e s s o f Food Technology, Barcelona, Spain, 3-6 March, 1987, PROSEMA, Valencia, 1989, pp. 667-676. W. Manu-Tawiah and A. M. Martin, Food M i c r o b i o l . , 4 (1987) 303-310.