Potato Waste as a Substrate for Single Cell Protein and ...

University of Rhode Island University of Rhode Island

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1981

Potato Waste as a Substrate for Single Cell Protein and Enzyme Potato Waste as a Substrate for Single Cell Protein and Enzyme

Production Production

Adilades A. Arenas Santiago University of Rhode Island

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POTATO WASTE AS A SUBSTRAT~ FOR

SINGLE CELL PROTEIN AND ENZYME

PRODUCTION

BY

ADALIDES A. ARENAS SANTIAGO

A THESIS SUBMITTED IN PARTIAL FULFILLMENT

OF THE REQUIREMENT FOR THE DEGREE OF

MASTER OF SCIENCE

IN

CHEMICAL ENGINEERING

UNIVERSITY OF RHODE ISLAND

1 9 8 41

MASTER OF SCIENCE TI:fESIS

OF

ADALIDES A. ARENAS s.

Approved:

Thesis

Major

Dean of the Graduate

School

UNIVERSITY OF RHODE ISLAND

1981

ABSTRACT

The feasibility of using potato wastes as substrate

f single cell protein (SCP) and extracellular enzyme _or

production by Pleurotus ostreatus was · studied in submerged

culture .

Cell mass y ield and e nzyme production of R· ostreatus

we r e studied as a function of (1) substrate concentration ,

(2) source of nitrogen , (3) tempe rature , (4) pH and (5) the

a ddition of sodium bisulfite on the medium. The fermen-

tat ion process was carried out to batch cultures in 250 ml

flasks . Protein content on a dry cell mass basis , alpha-

amylase activity and reducing sugar content in the broth

were determined in all the growth ste ps of R· ostreatus .

Ammonium sulfate was found to be the better nitrogen

source than either urea or ammonium nitrate for cell mass

yield , protein content and alpha - amylase production.

Two additional batch experiments were carried out in

a 5- liter fermenter as scale up of the optimum growth

conditions determined in 250 ml flask culture . The results

we re close to those obtained in 250 ml flask .

ii

DEDIC ADO

A MIS PADRES, ESPOSA, RIJOS Y HERMANOS

CON AMOR Y GRATITUD

ACKNOWLEDGEMENTS

The author is indebted to his adviser , Dr . Stanley

M. Barnett , for suggesting this topic , for his help and

encoura gement at every stage of this research . Dr . Arthur

G. Rand Jr. , for his valuable advise and for the use of

his lab and equipment . Dr . Chester W. Houston for his

time and technical assistance .

Thanks are due to the Universidad Nacional de San

Cristobal de Huarnanga - Ayacucho - Peru: Iatin American

Scholarship Program of American University and University

of Rhode I sland for allowing the author to study in this

country .

iv

TABLE OF CONTENTS

PAGE

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii

ACKNOWLEDGEMENTS ••••••••••••••••••••••••••••••••• iv

LIST OF TABLES

LIST OF FIGURES

I NTRODUCTION

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THEORY AND LITERATURE SURVEY • • • • • • • • • • • • • • • • • • • • •

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . RESULTS AND DISCUSSIONS . . . . . . . . . . . . . . . . . . . . . . . . . CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RECOMMENDAT IONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BI BLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . APPENDIX

v

v.i

vi i

1

3

25

39

78

80

81

LIST OF TABLES

PAGE

Table 1o- Possible Substrate for SCP Production ••••• 6

Table 2.- Aver age Potato Production af the World •••• 13

Table 3.- Proximate Analysis of White Potatoes . . . . . . Table 4- .- Essential Amino Acid Distribution in

Protein Sources . . . . . . . . . . . . . . . . . . . . . . . . . Table 5.- Results of Substrate Concentration Va

riation Studies of P . Ostreatus grown

18

24-

on Potato Wastes. •••••••••••••••••••••••• 4-9

Table 6.- Results of Different Nitrogen Source

Variation Studies of P. Ostreatus

grown on Potato Wastes . . . . . . . . . . . . . . . . . . . . Table 7.- Resul ts of Temperature Variation Studies

58

of P. Ostreatus grown on Potato Wastes ••• 63

Table 8 .- Results of Sodium Bisulfite Variation

Studies of P. Ostreatus grown on Potato

Wastes ••••••••••••••••••••••••••••••••••• 68

Table 9.-Results of pH Variation Studies of P .

Ostreatus grown on Potato Wastes •••• 73

Table 10.- 5-Liter Fermenter Studies •••••••••••• 74-

Table 11.-P. Ostreatus grown on Different Substrat es 77

vi

LIST OF FIGURES

PAGE

Fig. 1.- General scheme for SCP production from

a gricultural wastes •••••••••••••••••••••• 10

Fig. 2.- Scheme for yeast-starch SCP process ••••••• 11

Fig. 3.- Scheme for fungus-starch SCP process •••••• 12

Fig. 4.- Longitudinal secti on of a Russet Burbank

p ot ato showing principal structural

features •••..•.•••.•••••.•••••••••....••• 17

Fig. 5.- Molecula r structure of starch •••••••••••• 20

Fig. 6.- P . ostreatus growth on 1 % glucose

concentration • . . . . . . . . . . . . . . . . . . . . . . . . . 43

Fig. 7.- P . ostreatus growth on 1 96 substrate

concentration ••••••••••••••••••••••••••• 44

Fig. 8. - P . ostreatus growth on 2 96 substrate

concentration ••••••••••••••••••••••••••• 46

Fig. 9.- P . ostreatus growth on 3 % substrate

concentration ••••••••••••••••••••••••••• 47

Fig. 10.- P. ostreatus growth on 4 % substrate

concentration.......................... 48

Fig. 11.- P . ostreatus growth using ammonium nitrat e

as nitrog en source ••••••••••••••••••••••• 53

Fig.12.- P . ostreatus growt h using urea as nitrogen

source ••••••••••••••••••••••••••••••••• 54

vi i

Fi g . 13 .- Comp arison of ce l l weight obtained using dif-

ferent nitrogen s ourc es •••••••••••• • ••••• • • 55

Fi g . 14.- P . ostreatus growth using ammonium nitrate

as nitrogen source plus sulfuric acid ••••• 56

Fi g . 15 . - P . ostreatus gr owth using urea as nitrogen

source plus sulfuric ac i d . . . . . . . . . . . . . . . . 57 Fi g . 16.- P . ostreatus growth on 1 % substrate

concentration at 20° C. ••••••••••••••••• 6 1

Fi g . 17.- P . ostreatus growth on 1 % substrate

concentration at 30° C . . . . . . . . . . . . . . . . . . . . 62

Fi g . 18 .- P . ostreatus growth on 1 % substrate

c oncentration and 50 ppm Na HSO, ••••••••• 65 ?

Fig . 1 9 .- P . ostreatus growth on 1 °/o subst r ate

conc entration and 100 ppm NaHso3•••••••• 66

Fi g . 20 .- P . ostreatus gr owth on 1 % substrate

concentration and 150 ppm NaHso3•••••••• 67

Fi g . 21 .- P. ostreatus g r owth on 1 '% substrate

c oncentration at pH 4 . 5 . . . . . . . . . . . . . . 70

Fig . 22 .- P. ostr eatus g r owth on 1 °/o substrate

conc entrat ion at pH 6 . 0 . . . . . . . . . . . . . 71

Fig . 23 .- P. ostreatus g r owth on 1 % substrate

concentrat ion at pH 8 . 0 . . . . . . . . . . . . 72

viii

I. INTRODUCTION

Recently , there has been considerable emphasis on

the world food shortage, energy crisis and the availabi

lity of agricultural waste products (9, 32). The food

shortage due to increasing world population and food pro

duction has remained constant over the years. In many

of the developing countries, where two-thirds of the world's

population reside, the relationship between food avail

ability and rising population is increasingly perilous~

Some countries have already reached crisis proportions,

where a great part of the population suffers poor nutri-

tion or malnutrition: especially protein malnutrition

(e.g., Asia , S . E . Asia, India, Africa, Central America ,

Sou th Arne ri ca).

Political instability is going to get worse, not

better, if proper actions are not taken by governments

or world organizations (10, 20, 21) to alleviate these

nutrit ional conditions.

Protein can be obtained from several sources. These

sources are classifie d under the following three main

headings: 1. traditional a gricultural and fisheries s ystems

2. "non conventional" or other biological s ystems

3. chemical and biochemical syntheses

Traditional or conventional a gricultural and f isherie.s

s ystems yield protein from animal (meat and milk ), plants

(grains,. le gume s , cereals, seeds, roots, etc.) and f ish

(fish and others). Plants convert inorganic nitrogen into

protein but most animals cannot, ex cept for ruminants t ha t

are able to convert urea into protein. Technology (e. g.,

fertilizer, equipment, improved genetic changes and good

nanagement) has improved protein yield but competition

for grains and other a gricultural products exists between

people and animals.Animal pr otein s are of good quality

but are too costly to be a major source of protein for a

considerable proportion of the world's population. More-

over, world fish catches are reaching or ex ceeding the

natural l i mits for many species. Recently the escalating

cost of petroleum has increased the price of fertilizer

and other a gricultural inputs to achieve gTeater food pro-

duction (4).

Proteins coming f rom chemical and biochemical s yntheses

are expensive due to the continuous rising cost of petro

leum since most of the raw materials used are derived from

petroleum.

I n additi on to the above considerations, availability

and disposal of a gricultural waste products have created

pollution problems. New protein sources from a gricultural

wastes could be an alternative for helping to solve food,

energy , and pollution probfuems.

II . THEORY AND LITERATURE SURVEY

Single Cell Prote in. - Because the words "microbial" and

"bacterial" have somewhat undesirable connotations with

respect to food, the term "Single Cell Protein" was pro

posed to cover the concept of utilizing microat:'ganism as

food (62). It is a term used to describe the protein

contained in microorganisms capable of independent exis

tence as single cells, in particular yeasts, bacteria,

algae, and fungi .

Yeasts.- Several species have been studied by resear

chers in order to use as food, however two of them have

been the most used for human food and animal feed, Candida

utilis, also known as torula yeast and Saccharomyces carls

bergensis or brewer's yeast. The main disadvantage to

using yeasts as human food is the high level content of

nucleic acids which may cause certain physiological prob

lems because uric acid, the final metabolic product of

the purines contained in nucleic acids, is relatively

insoluble ( 57, 66 , 69 ).

Algae.- Members of Chlorella (green algae) and Spiru

~ (blue-green algae) have been studied extensively as

producers of edible protein. These species ma y con tain

50% and 60% protein respectively , on a dry weight basis.

Algal prote in contains all of the essential amino acids

however, low in sulfur-containing amino acids, but it is,

particularly meth ionine ( 10).

Bacteria.- Due to their ability to use petroleum hydro

carbons as carbon sources, species of Nocardia, Mycobacterium,

Micrococcus, Bacillus, and Pseudomonas are being investi--gated for protein production and pollution control.

Fungi.- Fungi have been used as food and in food process-

. The macroscopic sporophores have been advocated as ing.

a potentially valuable source of protein for man and domes-

tic animals (25). Most fungi have been obtained by solid

state fermentation processes.

Researchers have proposed that a large amount of food

protein can be produced by growing microorganisms on a

wide variety of substrates. These substrates are classi-

fied into three categories in Table 1 (15, 29) : materials

that have a high value as a source of energy or are derived

from such materials~ materials that are essentially waste

and should be recycled back into the· ecosystem by some

non-polluting method; and materials that can be derived

from plants and hence are a renewable re source.

From an economic and energy standpoint, and avail

ability , the last two categories have an advantage over

oil or its derived products due to constantly rising price

of oil.

In addition to the oil crisis, protein from microorgan

isms which were grown using hydrocarbons as substrate has

some formi dable obstacles to overcome before this protein

will be accepted for human consumption. The main problem

with these substrates is the possible contamination of t he

feedstoc k with carcinogenic polycyclic aromatics, such as

benzopyrene, which are known to be present in crude oil

( 46).

wastes and by-products have limited uses in single

cell protein production. Domestic wastes have inherent

dangers, various chemical residues could cause acute or

chronic poisoning if the y were taken up by microorganisms.

waste paper, wh ich can easily be handled separately from

other domestic wastes, has been used successfully , but

ma y also be t he source of some toxic materials. ( 44, 64).

Photosynthetically-produced materials are available

in very large quantities and have the virtue of being

renewa ble. The y are mainly composed of cellulose and

starches. (46 , 60, 64).

Microbial protein production ma y become a potential

protein source for the world's growing human population

and animals for t he following reasons (33, 43, 46) :

1.- Substrate for single cell protein production

can be cheap, e. g . agricultural waste products, in com

parison with conventional methods of production from

animals, fish a nd plants.

2.- Quick growth because the life cycles of micro

organisms are relatively short; some yeasts can double

Table 1.- Possible Substrates for Single Cell

Prate in.

subst:ra te

Na tu:ral gas

n-Alkanes

Gas oil

Methanol

Ethanol

Acetic Acid

Baggase

Citrus Waste

Whe y

Sulfite Water Liquor

Molasses

Anim:tl Manure

Sewage

Carbon Dioxide

Starch

Sugar

Cellulose

Re f • ( 1 5 , 2 g ) •

Classification

Energy source

" " II " " II

" " Energy source or waste

Waste Materials

II " " " " "

" " " " " II

Mat .

Waste or Renewable Resource

Renewable Resource Material

" " II

" II "

~age f

their mass in about 30 minutes.

3.- The conditions of growth of microorganism

for SCP production are easy to control.

4.- Pro tein content of microorganisms is high,

some conta in up t o 50% protein on a dry basis.

5.- Production of protein directly from inorganic

ammonium salt is possible.

6.- It can be produced in continuous processes

independently of climate changes and in plants that require

small amount of land.

The principal and desirable factors in selecting

microorganisms as food or feed are (48)

I.- Technical Factors

A.- Rapid growth

B.- Simple media

C.- Suspension culture

TI.- Simple separation

E.- Resistance to infection

F.- Efficient utilization of energy source.

G.- Tii sposable efluent.

II .- Physiological and Organoleptic Factors.

H.- Capable of genetic modification

I.- Nontox ic

J .- Good taste

K.- High digestibility

L.- High nutrient content

M.- Protein, fat and carbohydrate content of

h igh quality .

N.- Economically suitable.

According to the materials used as substrates and

microorganism .used in the f ermentation process for SCP

production, several processes are available and are shown

on Figures 1-3.

POTATO WASTE AS A SUBSTRATE FOR SINGLE CELL

PROTEIN PRODUCTION.

Historically , the potato, Solanum tuberosum, has

been a reliable food source for man and animal. Origi

nally from Peru it is now spread around the world and

presently it is the fourth largest world food crop , fol

lowing wheat, corn and rice. It is one of the most econo

mical food sources per unit area. Its yield per hectare

ranges from 25000 Kg to 40000 depending upon soil quality ,

fertilizer, climate and. available technology .

After harvesting , the potato is highly perishable.

Proper storage and transport are needed to bring this

crop from the farm to the market . During this period a

great deal of potatoes are wasted due to spoilage by micro

organisms, insect damage, sprouting, mechanical damage

or poor handling. In addition to this wastage, potatoes

which are too small or mechanically damaged during the

harvesting process and are not used in any potato process-

( · g chip potato , mash potato , starch , etc . ) are ing dryin ,

also wasted. These wastes reach from 5% to 7% of the total

Plus 3% to 5% due to improper size and mechanical damcrop age on the farm . In the third world countries (developing

countri es or under developed countries) where there is

neither technology for processing nor preserving this crop ,

the wa ste is at the 15 or 20% level (21). (see Table 2) .

s tructure and Chemical Composition of the Potato Tuber .

The tuber itself is essentially an abruptly thickened

underground stem closely resembling the a erial stem of the

plant (3) . Figure 4 shows the organization of the princi

pal i nternal tissue s of the mature tuber. The outer skin

consists of a layer of corky periderm, which appears to

serve the purpose of retarding loss of moisture and resa s -

ting atta ck by fungi.

Underly ing the periderm is the cortex , a narrow layer

of pa.re nchyma tissue . Vascular storage parenchyma which

is high in starch content , lies within the shell of the

cortex . Forming a small central core but radiating nar

row branches to each of the eyes, is the pith , sometimes

calle d the water "core".

Proximate Analysi s and Mineral Content.

It is difficult to obtain a clear picture of the

composition of the potato . It varies with variety , area

of gr owth , cultural practices , maturity at harvest , sub

sequent storage history and the methods of analysis used

( 42).

SEPARATION

"NON FERMENTABLE

SUBSTANCES

r+-1 CONVERSION ~

WASTES OR CLASSI FI CATI ON

FERMENTABLE CONCENTRATION I ._I STERILIZATI ON

SUBSTANCES

t I NOCUJ.JATION

--""'

NUTRIENTS I FERMENTATION AIR (02 ) .....

l-

WAREHOUSE ~ I PACKING ,.. PASTEURIZATION ,. I HARVEST

FIG. 1

'--------'

.- GENERAL SCHEME F0l1._SI NGLE CELL PROTEI N PRODUCTION FROM AGRICULTURAL WASTE MATERIALS (15)

HEAT

C02 .....

1-cJ Sl' aq <D

~

0

STARCH (POTATO, CASSAVA )

WATER

~

STARCH

HYDROLYSI S

MI NERAL SALTS

I WATER TREATMENT

MEDIUM ~

STERILIZATION

PROCESSING FOR HUMAN ..._

CONSUMPTION

ANIMAL FEED """ SUPPLEMENT

WATER RECYCLE DUMP ~

~

SEPARATOR FERMENTER

CENTRIFUGE

CfJ. H .. H r-il 0

PROTEI N HUMAN -EXTRACTION GRADE

SPRAY ANIMAL ~

~

DRYING GRADE

FIG . 2 • - SCHEME FOR YEAST- STARCH SINGLE CELL PR01rEIN PROCESS ( 46 )

f-d s:u crg (I)

~ ~

µ:] H 0 ?--I 0 µ:] p::<

P::l µ:]

~ :s:

~' ~ :s

~ ~lw ~-LI 8 ~ H H <I:! ~ UJ.

f:Ill w 0 i:,q ~ 8 <I:! w. E-f <I:! UJ. :s

M I X E R

CULTURE TANK

FERMENTER

A I R

SEPARATOR SCREEN

...___ __ ____. ---l ;-0 0 L E R \'~ STERILIZER

CULTURE

TANK

S T R E A M

DRYER

CULTURE CULTURE

FEED OR FOOD STOCK

, _ ___.....: ~1 ROTARY I .. I

'--~~~~~~--' ~~~~~~~~~

. FIG . 3.- SCHEME FOR FUNGUS~STARCH SI NGLE CELL PROCESS ( 25 ) • 1-LJ P' rrq <D

~ I\)

Table 2.- Average Potato Production in the World 1972 - 1974.

Underdeve loped Re gions Production

( 1000 M. T.)

I. - South America 7921

II .-Central America and Carribean 657

III .- Tropical Africa 1121

IV.-Middle East, North Africa 1734

V.- Non-Arab Muslim Countries 2933

VI.- India, Eangladesh, Nepal 5899

VII .- Southeast Asia 71 2

TOTAL 20977

Source ( 21 ) .

Harvested Area

( 1000 Ha)

939

70

209

156

259

639

96

2281

Yield

M. T. / Ha

8 . 4

9 .4

5.4

1 1 • 1

1 1 • 3

9 . 2

7 .4

9 . 2

~ ()q CD

--" \J.l

Table 2.- Cont. Average Potato Production in the World 1972 - 1974.

~veloped Coun tries Production Harvested Area Yi eld

( 1 000 M. T . ) ( 1000 Ha) M. T. / Ha

Canada 2202 107 20 . 6

Denmark 785 31 25 . 3

Germany 14420 485 29 . 7

Netherlands 5649 155 36 . 4

Sweden 1083 45 24 . 1

Switzerland 940 26 36 . 2

United Kingd om 6747 227 29 .7

United Sta tes of America 14149 532 26 . 6

Total 45975 1608 28 . 6

Other Countries

u.s. s.R. 89077 7992 1 1 • 2

Poland 50888 2668 19. 1

China 35360 3765 9. 4

Others 54454 3601 1 5. 1 ~ (J"q

TOTAL 22 9779 18026 Cl>

12 . 7 --"

~

Workers have analyzed the whole potato , while others some

Use d peeled tubers . Table 3 gives proximate analy}18.ve

. of a whole potato . sis s tar_£h. - The constituents of potato about which most

is known are the carbohydrates comprised largely of starch.

starch , comprising from 65% to 80% of the dry weight of

potato tuber , is calorically the most important nutritional'

component. In the raw tuber starch is present as micro

scopi c granules in the leucoplasts lining the interior of

the walls of the cells of the parenchyma tissue . The gran

ules a re ellipsoidal in shape , about 100 microme t e rs by

60 .Ltm on the average. The y are thus much large r than the

average starch granules of the cereal grains . The starch

granule r e se mbles an oyster she ll in appearance due to

appare nt striations on the surface .

There is a highly significant correlation betwee n

the starch content of the raw tuber and the textural quali

ties su ch as mealiness , consistency , sloughing , and soggi-

ness. During the cooking process water is taken up by

the s t a rch granule which then starts to swell . I n the

:range of 147 to 160° F , the starch be gins to gelatinize .

In po ta toes of h i gh starch content the cells tend to round

off a nd separate as a result of swe lling of the gela ti

nized s tarch , resulting in a mea l y texture .

The chief constituent of starch grain yields glucose

when hydrolize d and is calle d starch. The material is

actually a mixture of substances of different structure

d roperties. When an P starch is treated with boiling

a substance in the center of the grain passes inwa ter, to the solution , but the greater part of the grain is not

soluble. This insoluble portion absorbs water and swells

to form an elastic sphere, and whole the mass be comes

starch paste. While both the soluble and insoluble frac

tion are mixe d , it is customary to refer to the soluble

component as "amylase " and the insoluble part as "amylo

pectin". The se two main components of starch are present

in a ratio of 1 :3 in potato .

Amylase.- Generally , amylose is present in starch

at from 20 to 28% of total weight . An amylase polymer

consists of 250 to 300 TI- glucose molecules linked by alpha-

1-4-glucosidic bonds (Figure 5). These polymers tend

to twist the chain into a he lix . In amylase, the majori-

ty of the units are similarly connected by alpha-1-4-

glucosidic bonds , but there are occasional alpha- 1- 6-

glucos idic bonds ( 53).

Bo t h granules and the col l oidal solutions starch

react with iodine to give a blue color. This is chiefly

due to amylose, which forms a deep blue complex (71).

Amylopectin.- Amylopectin is a branched-chain glu

cose polymer in which the alpha-1-4- linkages are branched

by an alpha-1-6- linkage (Figure 5) on the average of

every 20 glucos yl residues. Each of these small branches

LATERAL BUD PERI DERM

RING

VASCULAR STORAGE PAREN

STEM END

,,..

-------\

..... ......... __ ,,,...,.,,,.---- ._

------- - - - -' - - -- --·---------

PITH

LATERAL BUD

FIG . 4 .- LONGITUDINAL SECTION OF A RUSSET BURBANK POTATO SHOWI NG PRINCIPAL STRUCTURAL FEATURES

BUD

1-d Pl

(Jtl (J)

~

~

Table 3.- Proximate Analysis of White Potatoes

(Wet Basis)

_9>mponent

water

protein

Fat

carbohydrate

Ash

Total

Fiber

Ref. (70).

Percentage

79. 8%

2 .1

0 .1

17 . 1

0.5

0 . 9

Proximate Composition of White Potatoes

(Dry Basis)

Compone nt

Water

Protein

Fat

Carbohydrates

Ash

Total

Fiber

Ref (36) .

Percentage

7. 6

8 . 0

0 . 8

79. 9

1 . 6

3.7

Page 18

bles the larger amylase chains, but the molecules resem

are joined together in such a way that the free reducing

P of the end gluc0se unit glucosidically linked through grou

the sixth glucose carbon in an adjoining chain.

The hydrolysis reaction of amylase may be followed

with iodine according to the scheme:

Iodine reaction Course of hydrolysis

Blue Starch

l ! Blue Soluble starch

! l Purple Amylo dextrin

! ! Red

! Erythrodextrin

! Colorless Achrodextrin

! Ma ltose

Sugar.- The sugar content of potatoes may vary from " only trace amounts to as much as 10% of the dry weight

of the tuber. The two main factors which influence sugar

content during the post-harvest storage are variety and

temperature.

Non- Starch Polysaccharides.- Small amounts of the

following occur in potatoes primarily in the cell walls

l::tl 0

0 C\I l::tl 0

l::tl

0 l::tl IJ:l 0 0

C\J IJ:l !::ti 0

0 0

C\I l::tl IX:

l::tl 0

0 ::r: l::tl

0

l::tl 0

C\I l::tl 0 l::tl

0 0

l::tl ::ti IJ:l 0 0 0 l::tl

C\J 0 IJ:l ::I:! 0 0

ti::: C\I ::r: IJ:1 ::r: 0 0

<( m FIG. 5 .- Starch Structure (A) Amylose

(B) Amylopect in

Pag e 20

I

0

l::tl 0

0

l::tl 0

l::tl

0

l::tl

::ti 0

::ti

b tween cell walls of adjoining cells: (1) crude and e

Page 2'1

. (2) hemicellulose, (3) cellulose , ( 4) pectic sub-f1ber,

stances and other polysaccharides .

Potatoes also have lipids, minerals, vitamins and

proteins which can serve as nutrients for microorganisms

during fermentation.

PLEUROTUS OSTREATUS

Pleurotus ostreatus (ATCC # 9515) , a white-spored

species, is commonly called the oyster mushroom. f. 0stre

atus and related forms occur in nature on deciduous and

coniferous woods (65) and is widespread in temperate zones.

The spores of the ~· ostreatus germinate quite easily

in liquids and on moist surfaces. During the rainy or

foggy season the germination and growth occur on the

bark of a tree trunk.

At the beginning of this century several people star

ted cultivation of P. ostreatus on tree stumps and logs

(18, 45, 55). Presently , it can be grown on a variety

of agricultural and industrial waste products (5, 26 ,

38) and requires small amounts of supplementary nutrients.

~· ostreatus can grow on living or dead plants, typi

cally poor in nutrients and vitamins and can fix nitrogen

from the air (23 , 24, 59 , 12) .

~. ostreatus is among the parasites or primary agents

of decomposition. It has the ability to directly break

ellulose and lignin-bearing materials without chemdown c ical or biological preparations. The metabolism and growth

d ·tions in solid state fermentation of cellulosic matercon i

ials has been studmed by several researchers (38, 40, 75 ,

) However, £. ostreatus grows well and rapidly in 76 • submerged culture and was found to produce a high concen-

tration level of the extracellular enzyme lactase when

cellulosic materials were used as a substrate (63).

P. ostreatus has several notable features for a human

food: good taste, nice odor, texture, digestibility and

nutritive value. ( 22, 2 5, 51 ) • Its nutritive value is

associated with its high protein content on a dry bas is,

and complete amino acid distribution (22) compared with

milk and beef (Table 4). It constitutes a good source

of niacin, riboflavin, vitamin C, folic acid, calcium,

phosphorus and potassium (51).

Table 4. - Essential amino acid distribution in

protein sources , expre ssed as per cent of total protein

(22).

Amino ac id P. ostrea tus Bee f Cow ' s milk

Isoleuc ine 4.3 6. 0 7. 8

Leu cine 7 . 7 8 . 0 11 . 0

Lysine 6. 0 1o. 0 8 . 7

Phenylalanine 3 . 4 5. 0 5. 5

Cyste i ne 0. 7 1 . 2 1. 0

Methionine 1.1 3. 2 3. 2

Threoni ne 5. 8 5. 0 4. 7

Tryptophan 1. 0 1. 4 1. 5

Valine 6. 4 5. 5 7. 1

Argenine 4 . 0 . 7 . 7 4. 2

Source (22).

P.

Table 4 (b ) .- Amino acid distribution of

ostr eatus expressed as per cent of total protein.

Amino aci£ P. ostrea tus FAO standard -Aspa.rt ic acid 7 . 17 -. -Threon i ne 3 . 57 2 . 80

Serine 4 . 50 - *-

Glutamic acid 19 . 48 - . -Pro l ine 4 . 71 - . -Glycine 3 . 80 -. -Alanine 4 . 72 -. -Val i ne 1. 60 4 . 20

Methi onine 2 . 33 2 . 20

Leu ci ne 4 . 50 4 . 80

Isoleuc ine 3 . 13 4 . 20

Phenylalanine 2 . 90 2 . 80

Tyrosine 1. 83 2 . 80

Histidine 5. 91 - -. Lys ine 4. 94 4 . 20

Trypt ophan 0 . 57 -. -Argenine 14 . 4 .. - -.

Re f . ( 63) .

MATERIALS ANTI METHOTIS

Microorganism.- All experiments were carried out

wi th Pleurotus ostreatus (ATCC 9415) . A stock culture

was ma intained on po ta to dextrose agar (Tiifco) at 5° c .

Substrate. - Raw potatoes (cultivated in Russet Bur

bank, Idaho) were dried at 100 °c and ground i nto an

average size of 100 me sh (Tyler Scale) powder .

Gr owth media .- The medium composition was modified

from t hat proposed by Hofsten and Ryden (27) .

Component Amount (g/l)

Ammonium sulfate 4 . 3

L-Asparagine 1. 0

KH2PO 4 1. 0

Mgso4. 7H2o 0. 5

cac12. 2H2o 0. 1

Na Cl 0. 1

Znso4. 1H2o 0. 005

Feso 4. 6H20 0. 005

Yeas t extract 1. 0

Pota to as substrate.

pre~ration of Inoculum.- For most of the experiments

the flasks containing the sterile medium were inoculated

by transferring the microorganism directly from the pota

to dextrose agar with a loop into 100 ml of the media

contained in 250 ml flasks.

For experiments in the 5-liter fermenter an indirect

inoculation was made to obtain a uniform inoculum. The

microorganism was transferred directly into 300 ml of

media conta ining 1% potato flour in a 500 ml Pyrex flask,

using the loop method . After inoculation, flasks and

~heir contents were incubated at 27 °c in an environmental

shaker (New Brunswick Scientifi c model # G 26) at 125 RPM

for 4 to 5 da ys . This 300 ml of solution, the microorga-

nism, was transferred into 5 liter of media. The initial

pH was adjusted to 5.5 by using 0 .1 N NaOH or 0 .1 N HC l

solution.

Effec ts of Substrate Concentra tion££ Fungal Growth

Protein Content and Alpha-Amylase Activity.

Potato flour at concentrations of 1%, 2%, 3% , and

4% was used. Al l other conditions remained unchanged

(pH=5.5, RPM=125 , ammonium sulfate as Nitro ge n source ,

t:27 °c).

Effect of pH on Fungal Growth, Protein Content, and

Alpha-Amylase Ac t ivity . - Effects of pH of the growth medium were observed over

the range of 4.5, 5.5, 6, 8 using 0.1 N HCl or 0.1N Na OH .

other conditions: Temperature 27 °c, 125 RPM, ammonium

sulfate as nitrogen source, 1 % po ta to flour.

Effect of Nitrogen Sources on Fungal Growth, Protein

Content, and Alpha-Amylase Activity .

The nitrogen source was varied by using ammonium

sulfate, urea, ammonium nitrate, urea-sulfuric acid, ammo-

nium nitrate-sulfuric acid. Other conditions: substrate

concentration 1%, pH= 5.5, 125 RPM, temperature 27°c .

Effect of Sodium Bisulfi t e on Fungal Growth, Protein

content and Alpha-Amylase Activity.

Sodium bisulf ite solution is used in potato s tarch

ma.nufactuiFe. I n order - to determine~ ostrea tus growth,

medium solution containing 50, 100 and 150 ppm was used

maintaining all other conditions constant. Temperature

27 °c, 125 RPM , pH = 5.5, and 1% substrate concentration,

ammonium sulf ate as nitrogen source.

Effect of Temperature on Fungal Growth, Protein

.£.9ntent and Al pha-Amylase Activit~.

Growth temperature was considered at 20, 27, and

30 °C. All other conditions remained constant. Substrate

concentration 1%, 125 RPM , pH=5.5, ammonium sulfate as

nitrogen source.

Contro_l.-

A control using 1% D- glucose as substitu~e to pota-

to flour for the carbon source was made.

ANALYTICAL ME THODS .

;Q!Y Weight Determination of Fungal Mass and Pata to

Flour Residue

The following procedure was used:

1.0 - Dry filter paper (Whatman # 2) in an air oven at

105 °c for 24 hours , then place in desicator.

2.0 - Place a filter paper sheet, previously weighed,

in a funnel (w1 ).

3.0 - Filter the culture medium through this filter paper

(save filtrate solution for enz yme activity and

reducing sugar assays).

4.0 - Wash the residue and filter paper with distil led

water several times.

5.0 - Place the filter paper and its content in a watch

glass and dry at 105 °c f or 24 hours .

6.o - Cool at room temperature in a desicator and we igh

(W2 ).

7.o - Place t he fi lter paper in a flask, pour 30 ml of

2. 5% Na OH solution , mix completely and digest for

24 hours in a rotary shaker at 125 RPM .

s.o --- ="Use another filter paper sheet previously weighed

(W3

) and place it in a funne l . Filter the solution

(save the solution for protein determination -

Bi ure t method) .

0 _ wash the filter paper and its content completely 9. and dry in an air ove n fo r 24 hours at 105 °c .

10•0 _ Cool at room temperature and weigh (w 4 ) .

The cell weight will be ca l culated by :

w1 = filte r pape r weight

w2 = pape r plus r e sidue weight

w2 = w1 + fungal mass + residue we ight

w3 = pape r weight

w4

= w1 ~ w3

+ r e sidue weight

Fungal mass = w2 - w4

However this me thod i s not a ccurate for dete rmining

fungal mass , since potato s tarch (main compone nt of pota

to) is soluble in 2 . 5% NaOH solution. On the other hand ,

1. ostreatus is not comple t e ly s oluble in 2 . 5% NaOH solu

tion, almost 2 . 3 to 10% remains insoluble . The solubi

lity is directly relate d t o the fungi maturity. P. ostre

atus gr own from 4 to 14 days showe d t o be more soluble

in 2.5% NaOH solution , afte r 14 days of growth the insolu

bility increa sed .

I t is important to notice that after 4 or 5 days of

fermenta tion almost 98 to 99% of the potato flour is

d the fungal mass ma y be estimated without behydrolize ,

ing t r eated with 2 . 5% NaOH solution.

Biuret Protein Determination . ~ (11) • .;...--- ·. .

Protein determination was carried out as follows :

Preparation of Biuret Rea~ent :

1. 0 - Place 1.5 g Cuso4. 5H20 , 60 g sodium potassium

tartrate (NaKC4H4o6 . 4H20) , and a stirring bar

in a 1 liter volumetric flask .

2. 0 - Add 500 ml glass- distilled water to the flask

and dissolve above solids .

3 . 0 - While stirring the contents of the flask vigor

ously , add 300 ml 10% (w/v) NaOH .

4. 0 - Remove the stirring bar from the flask and

bring the volume of liquid to 1 liter with

glass- distilled water and mix completely .

Determination of Protein

1. 0 - Place filter paper containing fungal mass in a

250 ml flask and pour 30 ml of 2 . 5% Na OH solu-

tion , mix completely and digest for 24 hours

at room temperature , and 125 RPM .

2. 0 - Filter the solution using Whatman #2 filter

paper.

3. 0 - Pipette duplicate portion of 1 ml of sample

solution into a clean test tube . ~ Add 4 . 0 ml

biuret reagent to each tube and vortex the

mixture for a few seconds to effect thorough

mixing of the solut i on .

4.o - Incubate the tubes for 30 minutes at room temp

erature .

5. 0 - Measure the color in a Bausch & Lomb Spectronic

21 Spectrophotome ter (Bausch & Lomb Co ., Roches

ter, N. Y. ) at 540 nm .

Calculate the absorbance using the equation :

A = 2 - Log ( % T )

Standards were prepared by using bovine serum albumin

and fi t to a straight line by the least square rule (see

Append ix A, Fig. A. 1) . The protein value of each sam-

ple was calculated from this calibration curve .

Protein Determination.- Modified Kjeldahl .

Reagents .-

Sodium sulfate- anhydrous ,low in nitrogen.

- Mercuric sulfate: Dilute 12 ml of con

centrated sulfuric acid to 100 ml with

distilled water and dissolve 10 grams

red mercuric oxide .

- Sulfuric acid , concentrated (95 - 98%) .

- Sodium hydroxide 0 . 4 M, prepared by dilu-

ting 40 ml of 10 M NaOH to 1 liter or by

dissolving 16 g in 600 ml of distilled

water and brought to 1 liter with dis

tilled water.

- Sodium Hydroxide-sodium iodide prepared

by adding 15 g reagent-grade NaI to one

liter of 0.4 M of NaOH .

- 1000 ppm NH3 as N standard.

Digestion.

1.0 - Place samples ranging from 0.1 to 0.7 g into

a dry Kj eldahl flask, add 3.0 g of anhydrous

sodium sulfate, 4 ml of mercuric sulfate solu

tion and 20 ml concentrated sulfuric acid to

each sample.

2.0 - Boil gently on a digestion rack until the water

is boiled off, then slowly increase the heat

until the solution is completely clear.

3.0 - Cool the flask and add 5 ml of distilled water

to rinse the neck before the contents solidify.

4. 0 - Transfer the solution to a 100 ml vd>l'umetric

flask rinsing the original flask several times

with small portion of water and bring to 100 ml

with distilled water.

5. 0 - Following the same procedure prepare the blanks

and ammonium sulfate for checking the percent

recovery of nitrogen.

6• 0 _ Read t he nitrogen content using Orion Research

Microprocessor Ionalyzer Mo.del 901 (Orion Re

search I ncorporated , Cambridge , Massachusetts)

and t he 95- 10 ammonia electrode (Orion Research

Incorporated , Cambridge , MA . ) according to

directions given in the manufacturer ' s instruc

tion manual .

Total Reducing Sugar Determination.

Total reduc i ng sugar is measured using the dinitros

alycyl ic acid (DNS ) method of Miller (50) as modified

by Ma ndels (47).

Rea gen t

- Mix :

Distilled water

3 , 5- Dinitrosalycylic acid

Na OH

- Dissolve a bove , then add :

Rochelle salt (Na , K tartra te)

Phenol (melt at 50 °c)

Na me tabisulf ite Na 2s2o5

141 6. 0 ml

1o . 6 g

19. 8 g

306 . 0 g

7 . 6 g

8 .3 g

Filtered sample is analyzed following this proce

dure :

- Add 3 ml of DNS to 1 ml of properly diluted sampl e .

- Heat t he mixture i n a boi ling water bath f or 5 min. ,

f ollowe d by cooling to room temperature using

tap water . After co©ling , add 15 ml of dis

tilled wate r to each test tube .

_ Following the same steps , pre pare glucose stan

dards and a blank.

- Read the pe rcentage t ransmittance (%T) on a

:Bausch & Lomb Spectronic 21 Spe ctrophotomete r

(:Bausch & Lomb Co ., Roche ster , N. Y. ) at 550

nm . (See Appe nd i x A; Fig. A. 2) .

Glucose Concentra t ion De termination.

Glucose concentration was de t e rmine d with a Y S I

Model 23 A Glucose Analyze r ( Yellow Spring Instrument ,

Co. Yellow Spring , Ohio . ) (6) .

- Dilute sample s to the range conta i ning 0 . 1 -

5 mg glucose per ml.

- Prepare a buffer s olution f or the Glucose Ana

l yzer by diluting a 5 g vial of YSI 2357 Buffer

7 G concentra te (Yellow Spring Co . Inc ., Ye llow

Spring , Ohio ) into 450 ± 25 ml distille d wate r.

- Inject sa mple or standard solution into the

glucose ana lyze r us i ng a YSI 2704 10 1 s yringe

pet (YSI Co., Inc . Yellow Spring , Ohio) . The

result will be read in uni t s of mg/dl .

- Prior to r eading , ca l i brate t he glucose analyze r

with a standard 500 mg/ dl glucose solution

(YSI Co . Inc ., Yellow Spring , Ohio) .

Alpha- Amylase Activity Determination.-

Al pha- Amylase (1 , 4 - alpha- D-Glucanohydrolase) act i

vity wa s de termined using t he me thod of Bernfeld (7) where

in t he r educing groups liberated from starch are measured

by t he reduction of 3 , 5- dinitrosalycylic acid~ One unit

of enzyme act i vity was defined as the a mount of micro-

mole of maltose liberated per minute from soluble starch

at 25 °c and pH 6. 9 under optimum conditions .

Reagents :

- 0. 02 M sodium phosphate buffer , pH 6 . 9 with 0 . 006

M sodium chloride .

- 2 N s odium hydroxide .

- Dinitrosalyclic acid color r eagent . Prepare by

dissolv ing 1 . 0 g of 3 , 5- dinitrosalycylic acid in

20 ml 2N Na OH . Add slowly 30 g sodium potassium

tartrate tetrehydrate . Dilute to a final volume

of 100 ml with glass distilled water . Protect

from carbon dioxide .

- 1% Starch .- Prepare by dissolving 1 . 0 g soluble

sta rch in 100 ml 0 . 002 M sodium phosphate buff er ,

pH 6 . 9 with 0.006 M sodium chloride . Bring to

a gentle boil to dissolve , cool and bring volume

to 100 ml with water if necessary . Incubate at

25 °c for 4 - 5 minutes prior to assay .

_ Maltose stock solution , 5 micromoles/ml . Pre

pare by dissolving 100 mg maltose (MW 360. 3)

in 100 ml glass distilled water . Incubate at

25° C for 4 - 5 minutes prior to assa y.

Procedure.-

Us ing t he maltose stock solution prepare a maltose

standard curve by pouring into a serie s of numbered tubes

1 ml of maltose di lutions ranging from 0 . 3 to 5 micromoles

per ml. Include two blanks with distilled water only .

Add 1 ml of dini trosalycylic acid c olor r eagent . Incubate

in a boiling water bath for 5 minutes and cool to room

tempera ture . Add 10 ml distille d water to each tube and

mix well. Read in a Bausch & Lomb Spectronic 21 Spectro

photome ter (Bausch & Lomb Co ., Rochester , N. Y. ) at 540

nm. Ca lculate t he absorbance and fit to a straight line

by t he least square rule or plot a bsorbance versus micro

moles maltose .

Enz yme Assay . -

1. 0 - Pipette 0 . 5 ml of sample solutions (or diluted

samples into two series of numbered test tubes

(include a blank with 0 . 5 ml distilled water) .

2.0 - I ncubate tubes at 25 °c for 3 - 4 minutes to

achieve temperature equilibration. At timed

intervals , add o. 5 ml of .s t arch solution at

25 °c to one s e ries of test tubes and 0 . 5 ml

of distille d wate r to the other series to com

plete 1 ml.

3. 0 - Incubate exactly 3 minute s and add 1 ml dini

trosalycyl ic acid color reagent to each tube .

4. 0 - Incubate tube s in a boil ing wate r bath for 5

minutes . Cool to ro om t emperature and add 10

ml glass distilled wate r . Mix well and read

in a spectrophotometer at 540 nm . Difference

between two se rie s of r eading in maltose con

tent will give alpha- amylase activity . (See

Appendix A, Fig . A. 3) .

Be ta- Amyl ase Activity Dete rmination.

Be ta- Amylase (1,4- Be ta - D- Glucan maltohydrolase)

was de t ermined using the method of Be rnfeld (8) whe rein

the rate at wh ich maltose is libe rated from starch is

measure d by its abil ity to reduce 3 , 5- dinitrosalycylic

acid.One unit liberates one micromole of beta- maltose

per minute at 25 °c and pH 4 . 8 under the specified con

ditions.

Reagents :

- 0.016 M Sodium acetate , pH 4 . 8

- 2 N Sodium hydroxide

- Dinitrosalycylic acid color reagent (the same

as alpha- amyl ase )

Pa ge 38

_ 1% Starch solution . Prepare by dissolving

1.0 g of soluble starch in 100 ml of 0 . 016 M

s odium acetate bu ffer pH 4 . 8 . Bring to a ge n

tle boil to dissolve. Cool and , if nece s sary ,

dilute to 100 ml with distilled water . Incu

bate at 25 °c for 4 - 5 minutes prior to assay .

Enz yme Assa y .-

- Follow the same steps as far the alpha-amylase

determinat i on using proper 1 % starch solution .

IV . ~ RESULTS AND DISCUSSION

Submerged Fermentation of Pleurotus Ostreatus .

Genera.1 Observations .

This study was carried out using 1% potato waste

flour a s substrate , except when the effect of concentra

tion on P. ostreatus growth was determined , and ammonium

sul fate a s the nitrogen source , except when the nitrogen

source was to be studied .

The fermentation broth changed color from clear yel

low to brown . This brown color may be attributed to en-

zymatic reaction rather than to the sugar- ammonia , sugar

amine (Maillard browning) reactions. It is known that

there are: ammonium ions in the solution due to ammoni-

um sulfate addition , or amine radicals due to water solu

ble prote ins of potato ~ or ammonium formation by£. ostre

atus, but the pH and temperature are very low to bring

Ma illard browning reaction. Color change was directly

related to alpha- amylase activity , reducing sugar content

and microorganism age.

The pH wa s observed to rise in value throughout each

experi me nt from 5. 5 (initial) to 6 . 8 or 7 . 2 in some cases . R' . is1ng pH was attributed to urea and ammonium formation

P os treatu~. This change in pH was not observed un

bY -· -til the death phase in all the experiments . This agreed

. h tudies done with mushrooms grown in solid state wit s

ntat ion (1 , 13 , 61 , 67) where pH change was directl y ferme related t o mycelium maturity and the selected nitrogen

source.

P. ostreatus produces specific extracellular enzymes

according to the substrates. Production of alpha-amylase

was observed in all of these experiments but beta-amylase

was not de tected . The amount of alpha- amylase varied

experimental conditions .

Whe n P. ostreatus was grown on cellulosic materials

it produced cellulase which hydrolize cellulose; when

lignin was used as substrate it produced laccase (63)

and using starch as a substrate it produced alpha- amylase .

Assays fo r cellulase and l accase were done on broth from

fermenta t i on of starch by f . ostreatus but the results

were ne ga tive . This means that some substrates have an

induct i ve effect on enzyme production.

Gluc ose as Carbon Source .- When P. ostreatus was

grown on 1% glucose solution as carbon source , at 27 °c ,

pH 5.5 (initial) , using ammonium sulfate as nitrogen

source, 125 RPM, the lag phase was increased considerably

compared to 1 % of potato flour solution as carbon source

and the other conditions r emaining the same ; the growth

rate a nd yield of cell mass de crease d r e lative to potato

P age 41

as Shown in Figure 6 . The maximum gr owth of the flour,

. r ga nism was reached after 16 days . 011 croo

Alpha- amylase

was not present in the fermentation broth due to the re -

pressive e ffect of glucose on enzyme production. The

prote i n content determined by the modified Kjeldahl method ,

varied f rom 6 . 05% dr y weight at 8 .days to 37 . 75°/o dry weight

at 16 days .

Effect of Substrate Concentration on Fungal Growth , Protein

Content and Enzyme Production .

Thi s portion of the study was carried out using 1% ,

2%, 3%, and 4% potato flour solution as substrate for P .

ostreatus ATC C 9415 . The fermentation was carried out in a

250 ml flask , at 27 °c , pH 5. 5 , in an incubator shaker at

125 RPM .

Results obtained using 1% of the substrate are shown

in Fi gure 7 . Notice that the highest cell mass yield

occurred at 14 da ys of fermentation . The crude protein

content, dry content% in cell varied from 38 . 18% after

4 days to 48 . 75% after 14 days , decreasing during the

death phase to 39 . 9°/o after 18 days . Protein content is

reported as dry basis of eell mass . The net protein (pro

tein de termined by biuret method) varied from 35 . 97°/o after

4 days t o 43 . 2°/o after 14 da ys . At the end of the process ,

after 18 days , the net protein content was 32 . 2°/o .

The highest value of alpha- amylase activity was ob-

ed a fter 4 da ys . Highest values of reducing sugar · se:rv

obtained at da y seventh (See Fig . 7) . Alpha- amylase were activ i t y decreased after 4 da ys • This may be due to com-

petitive inhibition by glucose which by that time reached

73 .6% of the total reducing sugar content . Finally, the

pH cha nge d f rom 5. 5 (initial) to 6 . 2 after 18 days of

fermenta tion .

Results of fermentation using 2% of smbstrate con-

centrat ion are s hown in Figure 8 . Maximum cell mass yield

was at the eleventh da y . Crude protein content varied

from 39. 9% to 47% and net protein content from 37 . 3% to

42.3%.

Al pha- amylase activity and reducing sugar content

reached their highest values after 5 da ys (See Fig. 8 )

and t he pH value changed from 5. 5 (initial) to 6 . 5 after

18 days of fermentation .

Figure 9 shows t he highest yield of cell mass using

3% substrate concentration and was obtained on the twelveth 1

day while observed protein content changed in similar

fashi on to experiments 1%, 2%, of substrate concentration.

Ma ximum alpha-amylase activity was observed on the

fi f t h da y and , for reducing sugar content , was on the

sixt h da y . The pH value changed ' from 5 . 5 (initial) to

6.4 a f te r 18 days of fermentation .

Results obtained using 4% of substrate concentration

are shown in Figure 10 .

4

,......,, r-f s

3 .......... QD s

'-../

E-1

~

CJ 2

H

µ:j

;::;:

H

H µ:j

1

0

0 . I 12 1b 4 8 20

T I M E ( days )

Fig . 6 .- Growth of £. ostreatus on 1 % glucose at 27 °c, pH 5. 5; ammonium

sulfate was used as nitrogen s ource . f-d Jg

CD

+=\.N

O CELL WEIGHT 0 RED. SUGAR 1.5-, ,......_

I I I 6 CRUDE PROTEI N Q GLUCOSE .--I

' s )\ 0 NET P ROTEIN -+- AMYLASE ACTIVITY .........

I QO 0 s ' '-"' ,......_

3 ~3, ~6 .--I s l=:r:l

w. r-1 ""'-""'- 0 S OD (f} 0 ""'- s

.µ 1 ~ OD

·r-1 H s '-"' >=:: 0 ~ '-"' '-"' I

8

:>-I p:: 3 z21 @4 8 «: H 0 H i:£1 t> ~ :s: H w. µ:i 8 0 0 8 J H

«: z . :;-- H o H fXl 0 µ:i w. ~ p:: 0 «: ~ 1 P-1 1 2 H µ:i :>-I p::

~

0 -- o- 0 L~ 8 12 16 20

T I M E ( d a y s ) f-cJ Pl

Fig . 7.- P. ostreatus gr owth on 1 % substrate concentration , at 27°c , aq (j)

pH 5. 5, ammonium sulfate as nitrogen s ource . +=-+=-

5

Notice t hat the highest cell mass yield occurred at

13 da ys of fermentation . Crude protein and net protein

content varied i n similar form to experiments 1% , 2% ,

and 3% of subs trate concentration.

The highest value of alpha-amylase activity was ob

served at 7 da ys and the highe st value of reducing sugar

content wa s obtained at 8 da ys of fermentation .

A summary of results using different substrate con-

centrat ion is given in Table 5. Optimum substra te con

centrat ions are 1 % or 2% for cell mass y ield, reducing

sugar and alpha- amyl ase activity per gram of substrate;

however, at 2% subs trate concentration the cell mass doub-

ling time is a minimum, and alpha- amylase activity is the

highest at 5 days . Hi gh substrate concentrations seem

to have a ne ga tive eff ect on P . ostrea tus growth and the

fermentat ion products : cell mass , reducing sugar , and

alpha-amylase activity . (See appendix . B. Figures B.1,

B. 2, and B. 3) .

When the substrate concentration was increased and

the other nutrient concentration remained constant, cell

mass also increased due to the increase in total level

of nutrients and energy sources . Howeve r , yield results

indicate the e ff iciency of substrate utilization decreased .

The likely explanations are : (1) a inhibition of micro-

organism growth at high glucose or reducing sugar concen-

3

' I 0

' ~

r1 s

-......... 2 U)

+:> ·rl ~

:=i '-/

:>-i 8 H :::> H E-l

~ 1 f:il w ~ H :>-i 8 ~

0 CELL WEIGHT + R£D . SUGAR

'1 '1 2~ I \ ~ CRUDE P ROTEIN D NET P ROTEIN

'· 0 AMYLASE ACTIVITY

~

r1 s

-......... ~ ~ till r-1 r1 s 8 s

-......... -......... '-/

till till s s

'-/

~ j 8 ~ c.':J

p::j Z H ~ f:il §5 5 H 5 ~ 1 w f:il

c.':J [-i

1 ~ z H 0 i::i-~ 0 0 :=i p::j q !J:1 P--i p::j

O--'- 0

4 8 12 16

T I M E ( days ) Fig. 8.- P. ostreatus growth on 2 % substrate concentration, at 27° c,

pH 5. 5, ammonium sulfate as nitrogen source .

20

ftj

~ <D

+()\

3

'

I I

0

' r-. .-l s

"-.. en .µ ·rl >::: :::i 2 '-"

;,..; E-1 H :.> H E-1 0 <r: i:x:i w. <r: 1 H :>-i .,,,~

"""' <r:

10) 1~ 2~ I\ 0 CELL WEIGHT 0 NET P ROT-EIN

6 CRUDE PROTEIN O RED . SUGAR

+ AMYLASE ACTIVITY

r-. .-l s r-.

"-.. .-1 00 s s· "-..

'-" r-. 00 r-1 s s '-"

i::r:i "-.. <r: 00 CJ E-1 :::i lil Cf) CJ

H CJ 5 z :5 ~ ~l1

,.$ H 0 :::i ~ i:x:i p::

H i:x:i

I E-1 H 0 H i::r:i i:x:i ~ 0

0 ---~~~~-.-~~~~--~~~~~....--~~~~--~~~~---.

4 8 12 16

T I M E ( days )

Fig . 9.- P. ostreatus growth on 3 % substrate concentration, at 27 °c, pH 5. 5, ammonium sulfate as nitrogen source.

20 ru Pl oq CD

..j:::' --J

' I 0

' ,...... rl s

.......... 2 tfl

..µ ·rl ~ p

"-..../

?-I E-1 H :::::. H E-l 0 «: 1 µ:i Cfl

~ ~

0

,

1 1

,...... rl s

.......... QD s ,......

"-..../ rl s p::: ~ 0

,...... rl s

.......... QD s

"-..../

E-1 P:1 0

2

0 CELL WEIGHT 6 CRUDE PROTEIN

0 NET 'PROTEIN 0 RED . SUGAR

-t- AMYLASE ACTIVITY

~ 5 ~ ~ H1 ~

0 z H 0 p i:::::i µ:i ~

z H µ:i

I H

E-1 1-=l 0 1:£1 p::: 0 p,

0

4 8 12 16

T I M E ( days)

Fig . 10.- P. ostreatus growth on 4 % substrate concentration, at 27 °c , pH 5. 5, ammonium sulfate as nitrogen source .

20

1-rj

~ <D

ffi

TABLE 5.- Results of substrate concentration variation

studies of P. ostreatus grown on potato

waste at 27 °c, pH 5.5, 125 RPM , ammonium sulfate

as nitrogen source .

Substrate concentration ( % )

Max . Specific Growth Rate ( day- 1 )

Max . Cell Growth ( mg/ml)

Max. Amylase Activity (unit /ml) 10-1

Max . Crude Protein Prod . (mg/ml)

Max. Net Protein ( mg/ml)

Cell Mass Doubling Time (day )

Max . Red . Sugar Cone. (mg/ml)

Max. Cell Mass Yield ( g/ g of potato)

1

0. 26

6 .5

1.3

3.2

2.8

2.7

3. 5

0. 61

2

0. 37

12 .1

2.9

5 . 7

5.1

1 .9

9.0

0 . 60

3

0.33

16 . 9

2 . 2

8 . 2

7.4

2 .1

11.3

0 .56

4

0. 27

18 .5

2. 7

s .5 7. 6

2.5

8 . 4

0 .46

t-d Pl oq (D

~ \..0

t . n which ma y decrease wate r activity a nd consequently t:rB. lO

drat ion of cells in such concentrate d solution. dehY (2

) The :ra tio nitrogen of carbon ma y not be at the op-

. m relationship since nitrogen source salt concentrat1mu tion rema ined constant . (3) Deficiency of any other nutri -

ts or toxi c or inhibitory effects _of specific compounds en ,

on ke y enz ymes or structural ce ll components and; (4) Dif-

fUsional or kinetic limitations that will be seen with

temperature e ffects on funga l growth.

At high substrate concentration alpha- amylase acti

vity per gram of substrate decreased due to a competitive

inhibition of glucose and other r e ducing sugars produced

by hydrolysis of starch.

Effect of Nitro~en Source s

For t his part of the study a potato concentration

of 1% wa s alwa ys used at 27 °c , 125 RPM and initial pH

was ad justed to 5. 5 with 0. 1N NaOH or 0 . 1N HC l .

Ammonium sulfate wa s compa re d with ammonium nitr ate

and urea to determine the e ffe ct of nitrogen source .

Results with ammonium sulfa te we r e shown in Figure 7 .

Results with ammonium nitrate are shown in Figure 11 .

The hi ghe st cell mass y i eld per gram of substrate was

obta ined after 11 da ys . The best yie l d of reducing sugar

was obta ined a fter 8 days .

Results of P. ostreatus growing with urea a s nitrogen

source a re s hown in Figure 12 .

The amount of ammonium nitrate and urea was in stoich

re lationship to nitrogen contained in ammonium iometri c

t a s proposed in the basic media , pg . 27 . A comsulfa e of cell mass yield using the different nitrogen parison

is shown in Fig. 13 and Table 6 . sources

Ammo nium sulfate as nitrogen source maximized cell

mass yield compared to urea and ammonium nitrate . Results

were similar to those in solid state fermentation of P.

ostreatus (73 , 74) in that whe n ammonium nitrate was used ,

degradati on of cellulosic materials and production of

cell mass decreased with increasing ammonium nitrate le

vels. This phenomenon indicate s a toxicologic effect

of nitra t e s .

Sulfuric acid was added to ammonium nitrate and urea

solutions in a stoichiometric relationship to provide the

same amount of sulfate radicals as ammonium sulfate in

the basi c media. Results using a mmonium . nitrate and sul

furic a ci d for P. os trea tus growth are shown in Figure 14

and resul ts using urea plus sulfuric acid are shown in

Figure 15.

Sul fa te ions have a positive effect on growth of

~· ostreatus. A comparison of results are shown in Table

6 and Appendix B, Figures B. 4 , B.5 , and B. 6. When ammo-

nium nitrate and sulfuric acid w-ere used , the cell mass

Yield was higher than for ammonium sulfate alone or urea

sul furic a cid (See Appe nd i x B, Fi g . B. 4) . For re plUS

. sugar pr odu ction and alpha- amylase activity , ammoduc1ng

. sulfa t e alone was the be st nitrogen source. The n1um

growth ra t e with a mmonium nitrate plus sulfuric acid is

higher t han t ha t f or ammonium sulfate or urea plus sul-

fUric ac i d.

The maximum redu c ing sugar production was obta i ned

at 6 days us ing a mmonium nitrate plus su l furi c acid , at

7 days us i ng a mmon ium sulfa t e alone, and at 9 da ys u s ing

urea.

Alpha- a myla s e activ ity peak was obse rved at 4 da ys

using ammonium sulfate alone , a t 5 da ys using ammonium

nitrate plus su l fu ric acid , and a t 6 da ys fo r u r ea plus

sulfuric ac id . The most suitable ni trogen source f or

reducing sugar production and alpha- amylase activ ity t hen

was ammoni um sulfate , and wh ile ammonium nitrate with

sulfuri c acid is t he mos t suitabl e for ce ll mass yi e ld .

The crude prote i n content of the drye d cell mass col lecte d

at the maximum l eve l us i ng ammonium sul fate a s the ni t ro

gen source wa s 50 . 27%; with ammonium nitra te plu s sulfur

ic acid it was 45 . 67%, a nd wi t h u rea plus sul fur i c acid

was 50. 01%. This means ammonium sul fate is the most sui t

able and conven i e nt nitr oge n s ou rce to produ ce prote i n

Using po tato a s substra te for P. os treatus .

During t he f ermenta_tion , the pH cha nge d as follows :

1. 0 - Ammonium sulfa t e: pH increased fr om 5. 5

,.,---,, ,...-j

s .........

QD s

..__,,

p:; <t:l 0 p w. 0 z H 0 p i::::l f:t=I Pei

3 1 ~3 ,...-j

s .........

QD s

2 ..__,, 2

z H f:t=I 8 0 p:; p,

1 1

,.,---,, ,...-j

s .........

QD s

..__,,

E-l4 :::c: 0 H

~ H H f:t=I 0

2

4

0 CELL WEIGHT

0 REDUCING SUGAR

0 PROTEIN

8

T I M E

12

(days)

16

Fi g . 11.- R· ostreatus growth using ammonium nitrat e as nit r ogen s ource , at 27° c, pH 5. 5, 1 % substrate concentration .

20

f-d P' oq (])

\J1 \}J

0 CELL WEIGHT

0 REDUCI NG SUGAR

0 P RO'rEI N

7---' 7, 1 hi

"" rl s "" ............ rl OD s s ............

"" OD ......_,, rl s s P=< ............ ......_,,

<G OD 0 s p ...._,, I E-i rJJ. p:::

0 H

0

J I i:r.:i

z z ::s: H H 0

~ J p H r=:i H ril i:£l P=< 0

0 1

T I M E ( days)

Fig . 12 .- P . ostreatus growth using urea as nitrogen s ource , at 27 °c, pH 5. 5, 1 % substr at e concentration .

1 0 I-cl Pl oq (])

\Jl +=-

"""' rl

~ bO a

'..../

frj 0 H ~~ '.3

~ ~ 0

0 NH4N0'3

0 Urea

6. e:,, ( NH4

) 2so

4 6

4

2

0

4 8 12 1

T I M E ( days)

Fi g . 13.- Comparis on of c e ll weight obtained u s ing different

nitrog en sources.

20

1-tj Pl oq (!)

\J1 \J1

' I 0

' ,,......_ r-1 s

........... Cl)

.µ ·rl

>::: p

.......,,

;>-1 E-l H ~ H E-l 0 <c: f.r.4 U) <c: H ;>-1 8 <c:

1., I I

I 3--f 3--i 6

1~~ ~ ~4 s ,,......_

.......,, r-1 s

"" 0:: bO <c: s c..'J p .......,, E-l U) iI1

0

. 5 0 H z z ~ H H 0 f.r.4 p E-l 1 H 2 ~ 0 H f.r.4 0:: f.r.4 p:::: P-1 0

0 0 0

I

4

0 CELL WEIGHT 0 REDUCING SUGAR

~ PROTEIN KJELDAHL DET . '\! GLUCOSE D PROTEIN BIURET DET . + AMYLASE ACTIVITY

12 16 8

T I M E ( days )

Fig . 14. - P . ostreatus growth on 1 % substrate concentration, at 27 °c, pH 5. 5, using ammonium nitrate plus sulfuric acid as nitrogen

source .

20 1-d PJ oq <D

\Jl 01

1.51

' I 0

' ,..--.._ rl s

............ 1 en -P ·rl

s:::: p '-../

:>-i E-1 H > H E-1 o .r ~

fil rJ) ~ H :>-i :s ~

0

0 CELL WEIGHT 0 RETIUCING SUGAR

' f r A CRUDE PROTEIN 'V GLUCOSE

I I D NET PROTEIN + AMYLASE Acr:J:IVITY ,..--.._

rl s

............

3 OD 3 -I s 6 '-../

fil ,..--.._ rJ)

,..--.._ 0 rl rl 0 s s p ............

H OD ............ 0 s W2 - H 2 '-../ 4 s z '-../ ~

E-1 p:; ::r:1

z ~ 0 0 H

H p ~ rJJ. fil

E-1 1 ~ 11 2 H H 0 o H

p fil p:; H O

~ ~

0 0 0 l~ 8 1 16

T I M E ( days ) Fi g . 15.- P . ostreatus growth on 1 % substrate concentration, at 27° c,

pH 5. 5, using urea as nitrogen source plus su l furic acid.

20

f-d P' oq <D

\J1 -..J

TABLE 6.- Results of different nitrogen source variation

studies of P. ostreatus grown on potato waste

at 27 °c, pH 5.5, 1 % s ubstrate c oncentration.

Nitrogen Source

Max . Specific Growth Rate (day-1 )

Cell Mass Doubling Time ( day)

Max. Cell Growth ( mg/ml)

Max. Crude Prot ein Production (mg/ml)

MaxQ Net Protein Production (mg/ml)

Max. Amylase Activity (Units/ml) 10-1

( NH4 ) 2so4 (alone)

0 . 26

2.7

6.5

3.2

2 .. 5

1.3

Max . Redfilcing Sugar Concentration (mg/ml) 3.5

Max . Cell Mass Yield ( g/ g of potato) o.65

NH4No3 Ure a

( alone) (alone )

0.26

2 . 6

4.4

-.-1.6

-.-1.9

o.43

0.26

2.6

5.0

-.-2.0

-.-2.4

0.51

NH4No3 +

H2so4

o.26

2.6

7.3

3.3

3.0

1.2

3. 2

0.7

Urea +

H2so4

0.24

2.9

5.9

3.0

2 .8

1.2

2.9

0.6

1-d

~ <D

\J1 co

Pag e 59

~ t0 6. 2

2. 0 - Urea : pH increased f r om 5 . 5 to 6 . 8 and ,

3. 0 - Ammonium nitrate : pH incr eased from 5. 5

to 7. 2

afte r 18 da ys mf fermentation in all experiments .

Effe c t of Te mpe r ature

The e ffect of t e mperature on gr owth of P. ostreatus

was studied using a 1% po t ato substra te concen t ration ,

ammonium sulfate as nitroge n source, and shaker speed

at 125 RPM . Results obtaine d at 20 °c , 27 °c , and 30 °c

are shown in Figur es 16 , 7 , and 17 re spe ctive ly .

~· ostreatus grows well at 20 °c , 27 °c , and 30 °c

in subme rged culture, but at 20 °c and 30°c the growth

rate is higher tha n at 27 °c . This doe s not agree with

results obtained by Zadra zil (77) in solid state fermen

tation whe re the optimum tempe ratures .we re reported be ~

tween 15 °c to 20 °c . Za drazil , however does not r e port

the particular s t rain u s ed i n h i s expe r iment .

Results for comparison a re s hown in Table 7 and Ap

pendix B, Figure s B. 7 , B. 8 , and B. 9 .

The maximum cell mass yie ld was obse rve d after 13

days a t 20 °c , at 27 °c it was a t 14 da ys and at 30 °c

it was at 10 da ys . The highest cell mass yield per gram

0 ~ subs trate was at 27 °c and t he protein content of the

cell ma ss was in the sa me p~oport ion fo r three cases .

Th e maximum amount of reducing sugar was obtained

after 9 days at 20 ° c;and after 7 days at 27°c and 30°c .

The r educ ing sug ar content was directly related to the

amount of cell mass and it disappe ars as the microorga-

nism grows .

Alpha-amylase activity increased with increasing

temperature as seen in Appendix B, Fig . B. 9 . Not only

was t he maximum amount of alpha- amylase increased with

increas i ng temperature , but the rate of amylase produc-

tion wa s also increased .

To determine s t oichiometric or kinetic limitation ,

Arrhenius equation has t o be used as f ollows :

).I = A e-Ea/ RT

lnµ = ln A - Ea/Rt

Plot ing lnµ vs 1/T , Ea/ R will be the slop e of the

straight line,where:

Ea= activation energy

R = gas c onstant ( 1 . 987 cal/ g - mole K)

A = Arrhenius constant

µ = maximum specific gr ow rate .

Wi th results obtained at different temperatures Ea

was determined to be - 6 . 757 cal/ mole which is very

low f or a chemical reaction, indic ating a p ossible dif

fusional limitation in these experimentso

0 CELL WEIGHT 1.;, I • I 0 REDUCING SUGAR

D PROTEI N

' I I I I + AMYLASE ACTIVITY I 0

' I ~ ~ 6 "' r-l s ..........

1,~ U2 .µ

~ J~ 2~ ~ 4

·.-1 ~ 0 .......,,.

b.O s s i>--1 p:; .......,,.

8 <i:! .......,,. H 0

P:1 :> 0 H UJ. 8

b .5 0 0 H

<i:! z z ~

ti 1 H '.3 1

~ ~ 1 UJ. 0 8 H <i:! i:=:i 0 H H ~ p:; ~ i>--1 p:; ~ 0 ?-: <i:!

0 0 0 0 8 '12 '16 20 1-tJ 4

p.J oq co T I M E ( days )

pH 5. 5, ()\ ~

Fi g . '16 .- P. ostreatus growth on '1 % substrat e concentrat i on , 20°c , ammonium sulfate as nitrogen source .

" I 0 '\ ,.... r-1 s

..........

u:i ..µ ·rl ~

:=i '1 '-.../

:>; E-1 H !> H E-1 0 <!!

f:Lj • 5 U2 <!! H ?--! :8 <!!

6~ I \ 0 CELL WEIGHT

0 REDUCI NG SUGAR

0 P ROTEI N 3 3

,.... + AMYLASE ACTIVITY r-1 ,.... s r-1

.......... s QO .......... s ,.... QO

r-1 s '-.../ s

.......... '-.../

QO p::j 2 82 4 <!! E-1 0 '-.../ ~ :=i 0 U2 H

f:Lj 0 ::;;: z z H H H 0 f:Lj H :=i E-1 f:Lj ~ 0 0

~ '1 ~ '1 2

0 0 0 4 8 '12 '16

T I M E ( days ) Fig . '17.- P . ostreatus gr owth on '1 % substrate c oncentration, pH 5. 5,

at 30° c, ammonium sulfate as nitrogen s ource .

20 f-d PJ oq <D

en N

TABLE 7.- Results of temperature variation studie s of

P. ostreatus grown on potato waste at 125 RPM ,

pH 5. 5 and 1% substrate concentration; ammoni -

um sulfate as nitrogen source

Tempera tu re 20 °c 27 °c 30 ° c

Max. Specific Growth Rate (day- 1 ) 0. 31 0. 26 o;.30

Cell Mass Doubling Time (day ) 2. 2 2. 7 2. 3

Max. Cell Growth (mg/ ml ) 5.4 6. 5 4. 6

Max. Net Protein Production (mg/ ml) 2. 1 2. 5 1. 7

Ma x . Amylase Activity (Unitsim1) 10-1 1 • 1 1. 3 1. 5

Max . Reduc i ng Sugar Cone . (mg/ ml) 2.4 3. 5 2. 5

JV!a x . Cell Mass Yield (g/ g potato) 0. 54 0. 61 0.46 I

1-d P'

aq CD

(J) \}J

Effe c t of Sodium Bisulfite (Na HS03 )

This study was carried out using a 1% potato substrate

solution, pH 5. 5 , temperature at 27 °c , shaker speed at

125 RPM , a nd ammonium sulfate as nitrogen source . I t

was found t hat in the presence of low concent:rations of

sodium bi sulf ite , E· ostreatus grew well but at increasing

concent:rat i ons , the growth rate decreased considerabl y .

Results at 50 ppm , 100 ppm and 150 ppm of sodium bisul-

fite are sh own in Figures 18 , 19 , and 20 , respectively ,

and Table 8 . Notice t hat when P. ostreatus was grown

in 50 ppm of sodium bisulfite the growth rate is h i gher

tban in 100 ppm and 150 ppm ( See Appendix B, Fig . B. 10) .

Protein cont ent in all of these experiments was in the

same proportion , varying from 37% to 49% or 50% on dry

cell mass basis .

However , t here was a marked difference in alpha

amylase act ivity and reducing sugar content . At 150 ppm

NaHso3 the maximum amylase activity was 1 . 69 times less

than t hat produ ced at 50 ppm Na Hso3 and 1 . 85 times less

than t hat produced at 100 ppm Na Hso3

•

Alpha - amyl ase activity was a maximum f or this micro-

organism at 100 ppm sodium bisulfite.

Fig. B.1 2)

(See Appendix B,

' I 0

' ~ r-1 El

'-.... Cl)

..µ ·rl q :::i .........,

?-1 E-l H ~ H E-l 0 <11

Fl w <I-1 H :>-; "'~

""' <I-1

1.5 0 CELL WEIGHT 0 RBDUCI NG SUGAR 6 CRUDE PROTEI N

3 3 6 D NET PROTEI N

+ AMYLASE ACTIVITY ~ {":\ __ r-1 El ~

'1 '-.... r-1 QO El El '-.... ........., QO

~ El r-1 .........,

P=i2 S2 4 <I-1 '-.... E-l 0 QO ::i:1 :::i El 0 w ........., H

0 ~ :z; :z;

, 5 H H 0 Fl :::i

~ '1 ~ H

r=1 '1 ~ 2 ~ 0

0 0 0 -

4 8 '12 '16 20

T I M E ( days ) Fig . '1 8 .- P . ostreatus growth on '1 % substrate concentration , at 27°c , pH 5. 5,

50 ppm of sodium bisul fite and ammonium sulfate as nitrogen source .

f-cJ P' aq (])

()\ \J1

"J • :,> I I I I ...L 0 CELL WEIGHT ._---

~ ~ I \ 0 REDUCING SUGAR

I 0 6 CRUDE PROTEI N ._---

,.-.... rl

3 0 NET PROTEI N s .......... + AMYLASE ACTIVITY tQ ,.-.... +:> rl s ·rl s .......... ~ .......... QO p 1 QO s ......_,, s ,.-.... ......_,,

......_,, rl ?-i s 8 .......... H ~2 if 1 ~4 t::> H c_I) ......_,, c_I) 8 p H 0 r:JJ. µ::i «: z ::s: z H l:Ll H l:Ll cI2 0 8

I ~ «: p 0 H • i=i P::l ?-i l:Ll 1-l-i ?-: P::l 1 1 «:

0 0 ---~~~~.--~~~~.,--~~~~-..--~~~~-.-~~~~--4 12 16 20

T I M E ( days ) Fig . 19 .- P. ostreatus gr owth on 1 % substrate concentration, 0 at 27 c , pH 5. 5,

100 ppm of s odium bisulfite and ammonium sulfate as nitrogen s ourc e .

1-rj P' oq ({)

()\ ()\

1.

' 0 CELL VJEIGHT I 0

0 REDUCING SUGAR ' ,........_ r-l

~ CRUDE PROTEI N s ......... ,........_3 3 6

0 NErr PROTEI N u.i +> r-l ·rl s + AMYLASE ACTIVITY r;::: ......... ,........_ p bD rel .......,, 1 s ~ .......,, ?-..; ,........_ bD E-1 p:; r-l s H <r! s .......,, :> g2 ";;o2 84 H E-1 CD. s P:.1 0 ..._,, 0 <G H

0 Fl Fl z z ::s CD. H H <G 0

~ J H H p ?-..; • 5 i::::i o H ~ ~Lj p:; Fl <r! p:; 1 p,1 ° 2

0 0 o --~~~~.-~~~~.--~~~~,.....~~~~-.-~~~_,:_--

4 8 1·2 6 2~ 1-cJ µi

(JtJ CD T I M E (days )

0 at 27 C, pH 5.5, 01

nitrogen source . --..]

Fig . 20.- P . ostreatus gr owth on 1 % substrate concentration,

150 ppm of sodium bisulfite and ammonium sulfate as

TABLE 8 .- Results of NaHso3 content variation studies of

P. ostreatus grown on potato waste at 27 °c ,

125 RPM and 1 % substrate conce ntration

Sodium Bisulfite content

Max . Specific Growth rate (day - 1 )

Cell Mass Doubling Time (day)

Max. Cell Growth (mg/ ml)

Max . Crude Protein Production (mg/ ml)

Max . Net Protein Product ion (mg/ml)

Max . Amylase Activity (Units / ml) 10

Max. Reducing Sugar Cone . (mg/ ml)

Max . Cell Ma s s Yield (g/g of potato)

50 ppm

0.41

1. 7

5. 6

2. 9

2. 6

1. 4

3 .. 2

0 .. 56

100 ppm

0 .23

3 .. 0

4 .. 6

2. 3

2 . 1

1 .. 5

3 .. 1

0. 46

158 ppm

0 . 20

3.4

4 .. 2

1 . 6

1 . 5

0 .. 8

0. 6

0 .. 42

1-d Pl aq (j)

(}\ CXl

Effect of pH Variation

The initial growth pH was changed from 4 . 5 to5 . 5 , 6 or 8 .

Results are shown in Figure s 21 , 7 , 22 , 23 , respec

tively , and Table 9 . The highest cell mass yi eld was

obtained at pH 6. 0 and the l owest a t pH 8 . Unfortunately ,

t hese set of experiments were initiate d with a different

inno culum from that use d at pH 5. 5 , ther e fore no compar

ison of t hese r e sults can be made with pH 5. 5 .

Alpha- amyl a se activity was the highest at pH 4.5 .

value s obtained at pH 8 are not shown in Figure B. 15 ,

Appendix B, because the y were insignificant . Low pH had

a pos i t ive effe ct on alpha-amyl ase activity but high pH

had a n inhibitory effe ct . Re ducing sugar content is dir

ectly related to alpha- amylase activ ity , reaching its

high values 1 day after alpha- amyl ase activity reached

its high value .

5- Liter Fermentor Study

Two runs in a 5- liter f e rmentor were carried out at

opt imum conditions determine d in 2 50 ml flask , pH 5. 5 ,

27 °c, 2% Substrate concentration , air flow rate 2 1

min. stirred at 150 RPM and ammoni um sulfate as nitrogen

source. In these expe riments the mi cr oorganism grew in

Pellet form and floated on the broth sur face , consequently ,

it wa s difficult to obtain homoge ne ous samples .

1. 0 CELL WEI GHT

' 0 REDUCING SUGAR I 0 6 CRUDE PROTEIN ' ,,......_ .-f 0 NET PROTEIN s ,......_3 3 ......... + AMYLASE ACTIVITY Ul .-f .µ s ,,......_ ·rl ......... .-f ~ bO s 81 s .........

'-" ,,......_ bO .-f s s '-"

~ P:< ......... E-l <r: bO H 0 2 s 21 84 :> p '-" ::r::: H CD. 0 8 H 0 0 f:LI <r: z z ::s

H H f:LI 0 f:LI

I ~ CD. p E-l <r: H 0 ~ .5 f:LI P:<

P:< P-1

~ 1 1

0 _._ &I- 0 --'- 0

4 8 12 16 20 !-rj

T I M E ( days ) PJ aq

ostreatus growth on 1 % substrate concentration , at 27° c, (!)

Fig . 21 .- P . pH 4 . 5 , ammonium sulfate as nit r ogen s ource . -.,J

0

=-2;-":· - - - ·- :=~

1.71 I I I 0 CELL WEIGHT

0 REDUCING SUGAR

' 3~ 3~ 6~ 6. CRUDE PROTEIN I

;/\ ~ 0 0 NET PROTEIN ' ,,....... + AMYLASE ACTIVITY rl

~ ,,....... rl ,,.......

Cl1 s rl .µ 1 ......... s ·rl bD ......... ~ s bD

:::> '-" ,,....... s '-" rl '-"

?-i ~ ~2 bD

4 8 c..'J s 8 H :::> '-" P::: :> w. c..'J H H 8 c..'J µ:i 0 iZi iZi ::s ~ H H

.5 0 µ:i H µ:i :::> 8 H w. ~ 0 µ:i ~ i:r:i1 0:: 1 0 H P=< P-l ?-i :;s «:

Q....L G-l- 0 _j_ 0 I I I I

4 8 12 16 20 f-d

T I M E ( days) ~ aq

Fi g . 22 .- P . ostreatus growt h on 1 % substrate concentr ation, at 27 °c, (1)

pH 6 and ammonium sulfate as nitrogen source . ~ _:,.

1.

' I 0

' ,,......_ r-1 s

......... (fl

..µ ·rl s:: e 1

l>-1 8 H p. H 8 0 <:r: i:i1 w. <:r: ~ • ~

0

1. 0 CELL WEIGHT

0 REDUCING SUGAR

D. CRUDE PROTEI N

0 NET PROTEIN ,,......_ 3 r-1 s r.i + AMYLASE ACTIVITY .........

~ ,,......_ r-1

'.../ s ,,......_ ......... .-t OD

~ s s ......... '.../

c_'J 002 4 p s 8 w. :::q '.../ c_'J

c_'J H :z; :z; ~ H H 0 f.ij p 8 H r=i• 0 H f.Ll o:< f.Ll o:< p., 1 02

0 0 o..__~~~~.,--~~~~...,.-~~~~---~~~~---~~~~ ...... 4 8 12 16 20

T I M E ( days ) Fig. 23 .- f • ostreatus growth on 1 % substrate concentration, at 27° c,

pH 8 , ammonium sulfate as nitrogen source.

ftj 11' aq <D

--J I\)

TABLE 9 . - Results of pH variation studies of

P. ostreatus grown on potato waste

at 27 °c , 125 RPM , 1% substrate

concentration.

pH ( i nitial ) 4. 5 5 .. 5

Max . specific Growth rate (day- 1) 0 .. 33 0. 26

Cell Mass Tioubling Time (day) 2. 1 2. 7

Max. Cell Growt h (mg/ ml) 5. 5 6. 5

Max. Crude Protein Production (mg/ ml) 2 .. 1 3. 2

Max . Net Protein Production (mg/ ml) 1 .. 9 2 .. 5

Max . Amylase Activity (Units/ml) 10 1. 3 1 .. 3

Max. Reducing Sugar Concentration (mg/ml) 3 . 7 3. 5

Max . Cell Mass Yield (g/g of potato) 0. 55 0. 65

6. 0 8 .. 0

0. 45 0. 37

1. 5 1. 9

6. 4 5. 5

2. 5 2 .. 2

2. 2 2 .. 0

1. 2 0 .. 8

3. 3 o. 6 ftj SD

aq

o .. 64 0. 55 ([)

-....) \.N

TABLE ~ ~- 5-Liter Fermenter studies using 2% substrate

concentration , pH 5 . 5 , at 27 °c , 2 1 air/ min

and stirred at 150 RPM .

Time (days ) 4 5

Cell Mass Yield (mg/ml) 1o . 0 8 . 3

Crude Prote in Production (mg/ml) 5. 0 4 . 0

Net Protein Produc tion (mg/ml) 4 . 0 3 . 3

Amylase Activity (Uni ts / ml) 10 3 . 1 3 .. 0

Reducing sugar (mg/ml) 9 . 3 8 . 8

1-tJ ~

aq (!)

---:I ~

The inoculum was pre pared in a 300 ml flask using

2% of substrate concentration at pH 5 . 5 (initial) and

27 oc . Afte r four da ys , which is the lag phase period ,

it was transferred from a 250 ml of inoculum to a 5- liter

fermentor. It was obse rved that afte r two da ys the broth

become s complete l y clear and homogeneous . This suggests

t hat the potato s t arch present in the broth was hydrolized .

Broth colo r cha nged from clear ye llow to brown but no pH

cha nges were observe d . A comparison of results for 4 days

and 5 da ys of fermentation pe riod i s given on Table 10 .

Resul ts of the 5- liter fermentor stud i e s compare WGll

to shake flask r e sults , base d on cell mass yield .

Using indirect inoculation , the l ag phase was re duced

sui tablefor scale- up for continuous culture fermentation .

Pages 76 and 77 are missing from the Santiago thesis.

P ag e 78

Conclusions

This study considered t he feasibility of potato was tes

as substrate for single cell protein and extracellular

enzyme production by E· ostreatus in submerged culture .

conclusions of this study are the following :

1. - "·A two per cent substrate concentration max-

i mize d cell mass yield per gram of substrate , alpha - amylase

activity a nd reducing sugar production in growing E· ostre-

atus . -2.- E· ostreatus batch growth was carried out using

three nitrogen sources : ammonium sulfate , ammonium nitrate ,

and urea. Cell mass yield , alpha-amylase activity and re -

duc ing sugar decreased with nitrogen source from a maxi-

mum using ammonium sulfate to a minimum using ammonium

nitrate.

3.- When sulfuric acid was added to ammonium ni-

trate or urea , the cell mass y ield and other products

were improved.

4.- Cell mass yield was 1 . 12 times higher with

ammonium nitrate plus sulfuri c acid than with ammonium

sulfate alone.

5. - The optimal temperature for E· ostreatus growth

at 1% substrate concentration was 27 °c .

6 . - At 30 °c, the production of alpha- a mylase

is 1.2 times higher t han that at 27 °c using a 1% sub

strate con centration.

7.- Optimal pH for cell mass y i eld was 5. 5 , but

alpha- a mylase and reducing sugar content were higher at

pH 4.5.

8 . - P. ostreatus grows well at low concentrations

of sodium bisulfite .

9.- I ndirect inoculation reduces the lag phase .

10 . - P. ostreatus under optimal cond i tions can

achieve 0 . 6120 g of dry cell mass per gram of substrate.

Protein content of t h is cell mass was 48% to 51 % (repor

ted as crude protein) or 43 . 2% (reported as net protein) .

RECOMMENDAT I ONS

1.- study the effect of the different variables on nucleic

ac id contained in P. ostreatus grown in submerged culture

us ing potato wastes as a substrate .

2.- Study if mycotoxins or other toxin forms are produced

growing P . ostreatus on submerged f e rmentation to be used

dire ctly a s human food , since P . ostreatus grown in solid

sta te f ermentation does not contain toxins .

3.- Study the kinetics and stability of alpha- amylase

produced by P . ostreatus which can justify this micro

organism as a source of starch hydrolizing enz ymes .

B I B L I 0 G R A P H Y

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7.-

8.-

Page 82

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Page 83

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2;.-

24.-

25.-

Page 84

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31.-

Page 85

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Page 86

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51.-

52 .-

Page 87

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A P P E N D I X A

.5

.4

.3

.1

2 4 6

mg . of Protein

Page 92

APPENDIX A

FIG. A.1

8 10

STANDARD FOR PROTEIN DETERMINATION : Biuret Method.

. 5

. 4

Q)

0 . 3 i:: m .0

~

0 . 2 (I}

.0

<

• 1

. 2 . 4 . 6

mg/ ml of gl u cose

l?a,ge 23

APPENDI X A

FIG . A. 2

. 8 1

STAN DARD FOR REDUCING SUGAR DETEill'IINATION WI TH

D N S.

. 6

. 4

0 1 2 3

Page 94

APPENDIX A

FIG. A.3

4 5

Mi cr omol/ml of malt ose

MALTOSE ASSAY . - STANDARD FOR ENZYME ANALYSIS .

A P P E N D I X B

,,-....

,....; s

" bD s "'-..-/

w. w. c:i:: :8

H

~ 0

0 2 % substrate Cone.

6 3 % substrate Cone .

20~ 0 4 % Bubstrate Cone .

0 -........

~

6

10

0 4 8 12 16

T I M E ( days )

Fi g . B.1.- Comparison of cell mass obt ained at di f f erent

subst rat e conc entrat ion .

20 '\j Pl

(Jq (J)

\ 0

°"

,.......,, .--l El

.......... bD s

'-.../

~ ·::C: c.'J :=i (/)

c.'J ~~ H 0 ~ 1-l f'~ ~

0 2 % Substrate Cone. 10 6 3 % Subst rate Cone.

0 4 % Sub s t rat e Cone.

5

0 L~ 8 1 2 16

T I M E ( days ) Fi g . B. 2.- Compari s on of reducing sugar content obt ai ned

at dif f erent substrat e concentration.

20 .1-d Pl

IJl:j ro

"O -.:i

3

I A 0 2 % Substrate Cone.

' I j/\ 3 % Substrate Cone . 'o /.}.

' ,,---... 0 4 % Substrate Cone. r-l s

.......... 2 ()) ..µ ·rl ~ 0

'-../

:>-i [~ H ::::-H E-l 0 ~ 1 Fl Cf). ~

~ ~ I ~ p:: p., H <Xl

0 I I ' I

4 8 12 16 20 .·'I:! Pl

T I M E ( days ) (JQ ro

F i g . B.3.- Comparison of alpha-amylase activity at different •\O o:>

substrate concentra tion.

----~_.-i

,...,, .-I

~ QO s ~

8 ::q 0 H r..LJ ~

H

~ 0

6 NH4No3

+ H2s o4

0 ( NH4 ) 2so 4 alone

6 -I 0 Urea + H2SOL~

2

0 4'

T I M E

6

8 '12 '16

( days) Fig. B.4.- Cell weight of P . 9streatus grown using different

nitrogen sourc es plus sul furic acid.

20 .:>-i::J ID

()q <1l

;\{) ;\O

31 _/f\ \ O ( NHL~ ) 2so4 a lone

,.._, r-1

6 NH4No3 + H2s o4 s .......... r.-.

QD s I I I \ \I \ 0 Urea H2s o4 +

'-.../

8 z µ:i 8 2 z 0 0

~ ~ c_I) :::i rJ2

c_I) z 1 H 0 :::i r=i l.r:J ~

0 . I ' 4 8 12 16 20 ~

ll' ()ti rt>

T I M E ( days ) f-' 0

F i g . B.5.- Reduc i ng sug a r cont ent ob tained u s ing d ifferent 0

nit rog en s ourc e s p l u s sul fur i c ac id.

" I 0

" ,----. rl s

.......... (/}

+:> ·rl s:: p

'-..../

N 8 H ::> H E-1 0 <r::

13-=l [IJ <r:: H N

~ * 22 P-i

~

1.

I 0 (NH4 ) 2s o4 alone

/AA 6 NH4No

3 + H2s o4

0 Urea H2s o4 +

1

0.5

0

4 8 12 16 T I M E ( d a y s )

Fig . B. 6 .- Alpha- amylase activity varia tion using

different ni t rog en sources a nd sulfuric

acid.

20 '"i:J Ill

01'.l ro I-' 0 I-'

,...... .--i 8

........... bO s

'-./

E-l ~ 0 H ~LI :;c;

H H ri1 0

0

6

4

2

0 o at 20 C

Cl at 30 °c

0 at 27 °c

o--~~~~--.-~~~~---..--~~~~......-~~~~--..~~~~--.

4 8 12 16

T I M E ( days)

Fig . B. '? .- Cell weight of P . ostreatus obtained at different

temperatures .

20 . '1j pl

Oti CD

~ 0 I\)

,...,, r-1 8

......... bD 8

'--'

8 /-~ ,.::. 4

:J::\ 8 ~ 0 0

p~ <~ c..'..l ;::J rJJ

c..'..l ~~ H 0 ::::> q

~

0 At 20 °c

31 I \ 6 at 30 °c

0 at 27 °c

2

1

0 4 8 1

'I' I M .t: ( days )

Fi g . B. 8 .- Reduc i ng sugar content v a r i at i on a t d i f fe rent

t emperat ure s .

2 . '<:J >ll ()Q CD

}-' ,Q w

'1. - . . . 0 at 20 °c

'\

j \ I 6 at 30 °c 0 '\

0 at 27 oC ,...... .....-l s ' (f) .µ ·r-l '1 ~ p

'-"

:>-I 8 H :.> H E-1 0 ~

µ:i 0 .5 Cf). ~

~ ~ I ~ ::r::: P-4

~ 0

I I I I I 4 8 '12 '16 20

'U (ll

T I M E (days) (JQ (1)

Fi g . B. 9.- Al pha-amyl ase act ivity vari ation at different f-J 0 +:""

t emper atures .

,...,. rl E3

......... 0.0 E3

..._,,

8 ,_,._, t-'-1

CJ H ~Li ,_S

j fI~

0

6

4

2

0

6 50 ppm

0 1 00 ppm

0 150 ppm

4 8 1 2

T I M E ( d a y s )

16 20

Fi g . B.10.- Cell we i ght v a ri a tion of P . ostrea tus grown at d ifferent conc ent r at ion l ev e ls of s od ium b isulfite.

.11:1 J\l ()'Q .(D

~ : ·O

V1

,.----.. rl 8

.......... bD 8

'--"

8 z f.:-.::j E-1 z 0 0

p:: <!! c.')

:=> CfJ.

c_'J z H 0 :=> i:=i µ:i ~

!:::. 50 ppm

0 100 ppm

I /'""'( D 1 50 ppm

3

2

1

0 -------

4 8 12 16

T I M E ( days)

Fi g . B. 11 .- Reducing sug ar c ontent vari ation at d i f fe r ent

l evels of s odium b i sulfit e

20 >tj Ill

()ti (I)

f-' 0 ()\

' 'o ' ,......

r4 s '-

U}

+:> ·.-1 ~

:_:::, .._,,

~ [-1 H ::> H [-1 0 -=r: µ:] cQ <c!

~ ~ I

~ P--l H <c!

1.5

I II \\ t::,. 50 ppm

I I \\ 0 100 ppm

I I \\ 0 150 ppm 1

I I I u '\.\_ - - ~t::,.

0.5

0

4 8 1 2 16

T I M E ( days )

Fig . B. 1 2 .- Alpha-amylase activity v a ri ation at different

concentration leve ls of sodium b isulf ite.

20 _ l-d Pl

Otl (])

I-' 0 -J

8 0

6

,--..., ..-1 s

'-.... 4 bD s '-.../

E--1 iJ::l l'.J H

s 2

1-.::i H f_:l 0

0 4 8 1 2

T I M E ( days )

Fi~ . B. 13.- Cell we i ght v a riat ion of P . ostreatus - ~ -

at dif f erent pH values .

0 pH 4.5

6 pH 6.0

0 pH 8 .0

0 pH 5.5

16 20 1-tj Pl

()Q (J)

I-' 0 co

" ..--1 8

" OD 8

'-.../

8 :z.~ r.:::i 8 ~·· ,__, 0 0

0::: ~ c...'J :=i CQ

0 --~ ?-1 H 0 :=i 1-=l r.r.:i 0:::

0 pH 4-. 5

I I ~/\ O pH 5. 5 3 I I I X\ >ti 0 pH 6 .0

2

I I II 110

1

0

4- 8 '1 2 16

T I M E ( day s ) F i g . B. "JL~.- Heducing sw~ar content vari ation at diff e r ent

pE va lues .

20 >tj p.l

Oti CD

I-' 0 \0

~

•o ' ,.......... r-l s

.......... ti) .µ ·rl ~

::::i '.._/

?-I 8 H >H [-1 0 ~

Ix~ c:J <[,

~ ~ I

< 1-rl 1 ...l-~

il1 t-::\ <.C:

1.5

1

o.

0

4

0 pH L~ .5

0 pH 5.5

0 pH 6 .0

0

8 12 T I M E ( day s )

0

16

Fi g . B. 15.- hl pha- amylas e activity vari at ion a t d i f f erent

p H val ues .

20 cu Pl

01'.l C1l

I-' I-' 0

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