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University of Rhode Island University of Rhode Island
DigitalCommons@URI DigitalCommons@URI
Open Access Master's Theses
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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Recommended Citation Recommended Citation Arenas Santiago, Adilades A., "Potato Waste as a Substrate for Single Cell Protein and Enzyme Production" (1981). Open Access Master's Theses. Paper 1388. https://digitalcommons.uri.edu/theses/1388
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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
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
Page 2
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
Page 4
· 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
Page 5
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
Page 6
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
Page 8
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 --"
~
Page 15
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
Page 16
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
Page 19
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).
Page 23
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).
Page 24
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) .
Page 25
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.
Page 26
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).
Page 27
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
Page 28
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
Page 29
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
Page 30
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
Page 31
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-
Page 32
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.
Page 33
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. ,
Page 34
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) .
Page 35
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 .
Page 36
_ 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
Page 37
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
Page 40
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 .
Page 42
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 . +=-+=-
Page 4-5
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
Page 50
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 .
Page 51
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
Page 52
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 .
Page 60
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
Page 64-
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
Page 69
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 ~
Page 75
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 .
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 .
Page 79
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) .
Page 80
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 .
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Page 83
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25.-
Page 84
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Page 86
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Page 87
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.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 .
,,-....
,....; 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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