EPA-600/2-77-133
July 1977
Environmental Protection Technology Series
COMMERCIAL PRODUCTION OF PROTEIN BY THE
FERMENTATION OF ACID AND/OR SWEET WHEY
industrial Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL PROTECTION TECH-
NOLOGY series. This series describes research performed to develop and dem-
onstrate instrumentation, equipment, and methodology to repair or prevent en-
vironmental degradation from point and non-point sources of pollution. This work
provides the new or improved technology required for the control and treatment
of pollution sources to meet environmental quality standards.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
-------
EPA-600/2-77-133
July 1977
COMMERCIAL PRODUCTION OF PROTEIN BY
THE FERMENTATION OF ACID AND/OR SWEET WHEY
by
Sheldon Bernstein
Chu H. Tzeng
Amber Laboratories
Juneau, Wisconsin 53039
Grant No. S-800747
Project Officers
Max W. Cochrane
Industrial Pollution Control Division
Industrial Environmental Research Laboratory-Cincinnati
Corvallis, Oregon 97330
Larry Dempsey
Industrial Pollution Control Division
Industrial Environmental Research Laboratory
Cincinnati, Ohio 45268
INDUSTRIAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
-------
DISCLAIMER
This report has been reviewed by the Industrial
Environmental Research Laboratory—Ci, U.S. Environmental
Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection
Agency, nor does the mention of trade names or commercial
products constitute endorsement or recommendation for use.
11
-------
FOREWORD
When energy and material resources are extracted, processed,
converted, and used, the related pollutional impacts on our
environment and even on our health often require that new
and increasingly more efficient pollution control methods be
used. The Industrial Environmental Research Laboratory -
Cincinnati (lERL-Ci) assists in developing and demonstrating
new and improved methodologies that will meet these needs
both efficiently and economically.
••Commercial Production of Protein By the Fermentation of
Acid/Or Sweet Whey" is a product of the above efforts.
Currently the cheese industry in the United States is
producing approximately 30 billion pounds of liquid whey
annually. For each pound of cheese produced, approximately
9 pounds of whey is obtained. This report describes the
commercial conversion of the lactose in whey into high
quality protein via fermentation. The project shows how
this conversion can be accomplished in a unique closed-loop
system producing zero effluents. This process can utilize
large amounts of whey and thereby convert this potential
environmental contaminant to a useful product that can also
be used in large amounts by the animal feed industry.
For further information regarding this report contact the
Food and Wood Products Branch, Industrial Pollution Control
Division, Industrial Environmental Research Laboratory—Ci,
Cincinnati, Ohio 45268.
David G. Stephan
Director
Industrial Environmental Research Laboratory—Ci
Cincinnati, Ohio
Hi
-------
ABSTRACT
Saccharomy_ces fragilis may be grown on acid or sweet cheese
whey in a deep-tank, aerated ferraentor in a continuous
manner on a commercial scale. Operations in a 15,000-gallon
fermentor at low pH and high cell counts experience no
contamination during extended periods of time under sanitary
but not sterile conditions. Media additions to the raw or
diluted condensed whey include anyhdrous ammonia, phosphoric
acid, and yeast extract. The production of a condensed or
dried whole fermented whey mass (yeast fermentation
solubles), which is an acceptable high protein feed
ingredient, eliminates additional processing of waste
streams from yeast separators, increases fermentation
yields, and utilizes the unfermented whey proteins
originally present. Evaporation of the whole fermented whey
mass produces condensate water that can be used to dilute
incoming condensed whey and thereby operate a unique closed
loop system with no effluents.
Economic calculations indicate that the process can be
viable commercially, provided that a number of necessary
conditions are met. First, large amounts of raw material
(whey) must be available nearby at a low cost. Second,
sufficient capital investment must be made to build a highly
automated and controlled plant so that labor costs are
minimized. And third, the operation and process should be
versatile enough to change product mix (e.g. to food grade
yeast, ethanol production, new products, etc.) if future
energy or market considerations warrant it.
This project shows that the commercial fermentation of
cheese whey is another means of converting large amounts of
a potential environmental pollutant into useful and needed
products.
iv
-------
CONTENTS
I Introduction 1
II Conclusions and Recommendations 3
III Materials and Methods 5
IV Process 7
V Analysis and Characterization of
Process Streams 18
VI Production Operation and Material Balance 21
VII Equipment Description 25
VIII Economics 27
IX Discussion 29
X References 31
XI Glossary 33
-------
LIST OF FIGURES
Number
1 Schematic for Whey Fermentation Process 8
2 Semi-Continuous Fermentation in
15,000-Gallon Fermentor 9
3 Continuous Fermentation in 15,000-Gallon
Fermentor 10
4 Ethanol Production by Anaerobic Fermentation 15
5 Closed-Loop System for Fermentation with
Zero Effluents 16
6 Schematic Showing Monitoring Points 20
7 Material Balance for Whey Fermentation 22
8 Schematic for Whey Fermentation Process 24
vl
-------
LIST OF TABLES
Number
1
2
3
4
5
6
7
Pag
Proximate Analysis of Fermented Whey Products
Protein Efficiency Ratio (PER) Assays of
Amino Acid Content of Various Single-Cell
Analyses of Fractions Obtained During
Calculated Production Cost for Dried Yeast
[e
12
12
14
19
23
23
Fermentation Solubles from Whey Fermentation 28
vii
-------
ACKNOWLEDGMENTS
The following personnel are acknowledged for their valuable
assistance in performing parts of the laboratory and plant
studies involved in this work: Mrs. Leslie Oberts,
Mr. Douglas Sisson, Mr. Oliver Justman, and Mr- Percy Love.
These persons as well as general laboratory and plant
personnel of Amber Laboratories Division of Milbrew, Inc.
supported the work directed by Dr. Chu H. Tzeng and
Dr. Sheldon Bernstein.
The support of the project by the Environmental Protection
Agency, and, especially, Mr- Max Cochrane, the Grant Project
Officer, Mr. Kenneth Dostal, and Mr. Larry Dempsey is
acknowledged with sincere thanks.
vlii
-------
SECTION I
INTRODUCTION
Currently the cheese industry in the United States is
producing approximately 30 billion pounds of liquid whey
annually. As recently as 1972, it was estimated that only
about half of the whey produced was utilized.^ For each
pound of cheese produced, approximately 9 pounds of whey is
obtained. In 1974, the last year for which statistics are
available, 820 million pounds of dry whey was produced (425
million pounds for human consumption 395 million pounds for
animal feeds).2 Other amounts of whey were converted to
lactose, whey blends, protein fractions, etc. But if it is
recognized that the 30 billion pounds of whey previously
mentioned represents about 2 billion pounds of whey solids,
it is easily seen that a tremendous amount of this material
is being discarded.
Whey is the serum of milk resulting from the removal of fat
and casein from the whole milk during the cheese-making
process. It is a greenish-yellow liquid containing 6% to
6.5$ solids and most of the water soluble vitamins and
minerals of the whole milk from which it was derived. A
typical analysis of the whey solids would be: lactose 64$
to 72$, protein 11$ to 13$, minerals 8$ to 9$, plus small
amounts of fat and lactic acid. Practically all the whey
produced in this country is of two types: low acid (sweet
whey) from Cheddar, Swiss or Italian cheeses, and high acid
(acid whey) from cottage or cream cheese.
The problem of disposing of or utilizing whey is compounded
by a number of factors. The material is 93-5$ to 94$ water,
which makes hauling any distance extremely expensive. It is
perishable and will spoil easily, so it cannot be stored for
any length of time. Evaporation and drying of whey requires
a large capital investment, and the market for dried whey is
such that often the cost of processing is just barely
recovered. Also, acid whey is extremely difficult to dry at
all, and it can only be done with specialized equipment.
Such processing may nevertheless be cheaper than trying to
-------
dispose of the liquid. The BOD of raw whey is about 30,000
to 50,000 ppra, and since it is not unusual for a cheese
plant to produce 500,000 to 1,000,000 pounds of whey per
day, it would require a large disposal or treatment plant to
handle this volume.
One of the few industries capable of utilizing the large
volumes of whey available is the animal feed industry.
Unfortunately, the price that can be paid for dried whey for
feeding purposes is low, often just about equal to the
processing costs. The reason is that the major constituent
of dried whey is lactose, which can be used only in limited
amounts in most feeds and must compete in price with other
inexpensive carbohydrate and energy sources such as molasses
and corn. There is a much greater demand for high-protein
ingredients in this area.
This report describes the commercial conversion of the
lactose in whey into high quality protein via fermentation.
The project shows how this conversion can be accomplished in
a unique closed-loop system producing zero effluents. This
process can utilize large amounts of whey and thereby
convert this potential environmental contaminant to a useful
product that can also be used in large amounts by the animal
feed industry.
-------
SECTION II
CONCLUSIONS AND RECOMMENDATIONS
1 • Saccharomjrces fragilis can be grown on acid or sweet
whey in a deep-tank, aerated fermentor in a continuous
manner on a commercial scale-
2. Growth levels of several billion cells per milliliter
were reached and maintained for up to 2 weeks of time
without any contamination while operating a 15,000-gallon
fermentor under sanitary, but not sterile conditions.
3- The entire fermented whey mass may be condensed and/or
dried to produce an acceptable and satisfactory high-protein
(35% to 50$) feed ingredient (yeast fermentation solubles,
YFS). The yield of such YFS material is between 0.65 and
0.75 pounds per pound of whey solids.
4. The evaporation of the whole fermented whey mass
eliminates additional processing of waste streams, increases
fermentation yields, utilizes unfermented whey proteins
present in the media, and produces condensate water that can
be used to dilute incoming condensed whey. This allows the
fermentation to be operated in a unique closed-loop system
producing zero effluents.
5- A superior quality yeast, higher in protein (45$ to 55$)
and lower in ash (6% to 10$) content, may be produced by
centrifugal harvesting of the yeast cells. This product
will find commercial applications as a food additive for its
nutritional and flavor characteristics.
6. Supernatant effluents from yeast separators may be
incorporated into the YFS feed material and thus utilize the
soluble, unfermented whey proteins that would otherwise be
lost.
7. By changing the conditions of the fermentation, up to 9$
ethyl alcohol may be produced which can be recovered during
the evaporation stage.
-------
8. The process is commercially viable, provided large
amounts of low- or no-cost whey are available and the
production is sufficiently automated and controlled to
minimize labor costs.
9. Further studies should investigate making the process
versatile enough to vary the product mix to meet cost and
market fluctuations. Special emphasis of future work should
include study of the optimal production of ethanol as a
possible alternate fuel source.
-------
SECTION III
MATERIALS AND METHODS
Since the work reported here is a continuation of extended
preliminary and pilot experiments reported elsewhere,3 the
materials and analytical methods remained the same. Minor
variations in procedures were experienced in the scale-up
from a 500-gallon operation to working in a 15,000-gallon
fermentor. For example, anhydrous ammonia was used as the
exogenous nitrogen source instead of aqueous ammonium
hydroxide to avoid medium dilution because of large volumes,
to decrease operating costs, and to enhance automatic pH
control.
Incoming condensed whey (either acid and/or sweet) was
diluted to the appropriate lactose concentration (equivalent
to 10? to 15? whey solids) with water, raw whey or
condensate water as described in the process section. Other
additions included, phosphoric acid, yeast extract,
anyhdrous ammonia, and hydrochloric acid to give a typical
medium composition as follows:
Whey solids 12.0?
Phosphoric Acid 0.1?
Yeast Extract
(Amber BYF Series 100) 0.13?
Ammonia 0.3? to 0.5?
Hydrochloric Acid 0.5? to .0.2? (to pH
4.5)
The organism used was Saccharomy_ces fragilis (N.U.R.L.,
Y-1109). Inoculum buildup was accomplished as reported
previously from stock slant to shake-flasks to 10 liters in
a 14-liter fermentor to 60 gallons and to 300 gallons in a
500-gallon seed fermentor. When growth reached a minimum of
-------
1 x 109 viable cells/ml, this seed was used to inoculate
first 3,000 gallons and then 10,000 gallons of medium in the
15,000-gallon production tank. The progress of the
fermentation was followed by lactose concentration, cell
count and cell pack volume.
Analyses were made on samples of all fractions obtained
during the fermentations, especially effluents (see Section
V and tabulated results) . These varied from 4 to 12 in
number, depending on the degree of processing of the
fermentation broth and included such tests as Kjeldahl
nitrogen, ammonia nitrogen, lactose, ash, protein, pH,
temperature, solids (or moisture), BOD, suspended solids,
and ethanol.3 Since the vast majority of the final series
of fermentations were run using the closed-loop system
producing no effluents, the number of monitoring points were
eventually reduced to 6 in number.
-------
SECTION IV
PROCESS
The fermentation of whey by various microorganisms has been
known and studied for years.^>5 Our own preliminary work,
reported elsewhere,3,6 showed that the organism of choice in
our process is a strain of Saccharomy_ces fragilis. The
conversion of lactose into cellular material is efficient
and in the range of 455& to 52% under nonsterile conditions.
Successive scale-up has been accomplished from shake-flasks
to 14-liter fermentors to 500-gallon and 3,000-gallon
fermentors to our present operation in a 15,000-gallon tank.
Whey (acid and/or sweet) is obtained in concentrated form
(455& to 50/6 solids) from cheese manufacturers. This is
diluted with water, raw whey or condensate water (as in
"closed-loop" operations, to be described shortly) to the
appropriate lactose concentration. Other medium additions
include: anhydrous ammonia as the primary exogenous
nitrogen source, yeast extract, phosphoric acid and some
hydrochloric acid to adjust the pH to 4.5. The medium is
heated to 80° for 45 minutes and then cooled. The
fermentation is carried out in a 15,000 gallon stainless
steel deep-tank fermentor that is fully aerated and
jacketed. Automatic instrumentation controls pH (4.5),
temperature (90-92°F), aeration (1.0 ± 0.2 vol. air per vol.
medium) and foaming, as well as levels and volumes in and
out. The fermentor may be operated in a batch,
semi-continuous or continuous manner. After fermentation,
the fermented whey mass is collected and processed further -
An overall schematic of the fermentation process may be seen
in Figure 1.
In a batch fermentation, starting with a seed with a viable
cell count of 1 x 10^ cells per milliter and an inoculum
level of 10/&, all the lactose is utilized in 8 hours under
appropriate conditions with an increase in cell
concentration of ten to twenty fold. This represents a
generation time of approximately 2.0 hours and 4 doublings
within an 8 hour period.
-------
Cond. Raw
Whey Whey yeast pxtra
SEED
FERMENTOR
> COOLING
WATER
ETHANOL
AIR
oo
MAIN
FERMENTOR
CENTRIFUGES
YEAST PRODUCT
To Alcohol Recovery
and/or
Waste Treatment
Figure 1 Schematic for Whey Fermentation Process
-------
10-
HI
g
C
I
io
8
Batch #1
Batch #2
10-
Hl
6
o
o
(D
O
Cell Count
Lactose
10
8
8 10
246
HOURS
8 10
10
8
6 a)
(0
o
4 s
fd
8 10
Figure 2 Semi-continuous Fermentation
-------
Begin Continuous Fermentation
40
HOURS
Figure 3 Continuous Fermentation in 15,000-gallon fermentor
-------
The semi-continuous runs were made by drawing off 90% of the
fermentation broth and using the 10% remaining to seed the
next fermentation batch. A number of consecutive draw down
batches could be run in this manner (Figure 2). The only
"down-time" experienced in such a procedure is the time it
takes to pump off 90% of the fermented broth and to pump in
fresh medium.
The ''down-time" can be eliminated if the fermentation can be
run continuously, i.e., with the continuous addition of
fresh medium and the removal of the fermented mass. This
proved to be applicable in our yeast-whey fermentation. The
continuous fermentation is begun when the cell count in the
fermentation broth reaches 1 x 1Q9 cells per milliliter and
the lactose concentration falls to 0.50% to 0.75% (W/V). At
this time fresh medium is added and the fermented whey mass
removed at an equal rate. The fermentor may be operated in
this manner for up to two-three weeks of time, maintaining
both cell count and lactose concentration at a relatively
constant level, without any indication of contamination or
build-up of metabolic products that would interfere with the
fermentation. In the 500 gallon and 3000 gallon tanks,
continuous runs of several weeks have been accomplished.
The 15,000-gallon fermentor has been operated in this manner
for up to 15 days with no difficulty. One such run in the
large tank is shown in Figure 3- After initial cell growth,
fresh-medium is added and the fermented whey mass removed at
the rate of 1,250 gallons per hour, which corresponds to a
batch fermentation cycle of 10,000 gallons per 8 hours
(dilution rate of 0.125 hours -1) .
The production of Saccharomy_ces fragilis on whey has been
done in the United States on a small scale 7 and is approved
for use in feeds and foods.° The whole fermented whey mass
(called AMBER Yeast Fermentation Solubles, YFS) can be used
as feed ingredient. This simplifies the processing for it
entails only the concentration of the fermentation broth and
spray drying. If the yeast cells are harvested by
centrifugation and washed, a food grade, primary grown yeast
is obtained (AMBER Nutrex). The proximate analysis of these
products are shown in Table 1.
11
-------
TABLE 1.
PROXIMATE ANALYSIS OF
FERMENTED WHEY PRODUCTS
Constituent Yeast Ferm. Sol. Cent. Yeast
Amber_YFS_Hl Amber_Nutrex(_ll
Crude protein 35-50 45-55
Ash 12-20 6-10
Fat 2-3 2
Moisture 3-4 3-4
The use of these products as feeds or foods and the quality
of the protein they contain are indicated by rat feeding
tests. Rat growth rates using YFS and centrifuged YFS as
the sole protein source in the diet were compared with those
using casein standard. No evidence of toxicity was noted
during this period of time. From these experiments, Protein
Efficiency Ratios (PER) were calculated and found to be 1.72
for the whole Yeast Fermentation Solubles and 2.26 for the
centrifuged yeast9 (Table 2). These values are 69% and 91%
respectively of that of the casein standard. Actual feeding
tests conducted with larger animals determine specific
values. Such tests with cattle have proved successful and
the YFS material is being used in a number of commercial
feed formulations at the present time.
TABLE 2.
PROTEIN EFFICIENCY RATION (PER)
ASSAYS OF FERMENTED WHEY PRODUCTS
PRODUCT Average per Value % of ANRC
_for_4_Weeks Casein
Amber YFS 1.72 69
Amber Nutrex 2.26 91
ANRC Casein 2.50 100
12
-------
The quality of the protein produced by this fermentation of
whey is further indicated by the amino acid analysis. In
Table 3 the amino acid composition of this yeast is compared
to other yeasts used in feeds and foods (Brewers Yeast and
Torula Yeast) and to yeast produced by the fermentation of
hydrocarbons and ethanol. The standard amino acid FAO
profile is also listed for comparison.
During the fermentation, one of the metabolic products
formed is ethyl alcohol. By changing conditions of
fermentation (operating under anaerobic conditions, 0.1 to
0.3 volume of air per volume of medium) one can increase the
amount of this material formed. The results of one such run
in our 15,000-gallon fermentor is shown in Figure 4. After
an initial period of aerobic operation to build up cell
concentration, the fermentation broth was "spiked" with
additional lactose in the form of condensed whey and the
fermentor was run under anaerobic conditions. The lactose
was utilized at a continuous steady rate. The cell population
no longer increased, but remained at a constant level. By
adding condensed whey incrementally, ethyl alcohol was
produced in larger amounts, and better than 90/£ of the
lactose was converted to this material, the rest being
metabolized for cell maintenance. The production of over 9$
ethanol in this preliminary experiment indicates the
potential of the production of this important chemical,
simultaneous with the production of single cell protein.
Complicated economic factors, including energy, production
and recovery costs, as well as market values of the
products, will determine the optimum operation of the
process.
In an effort to minimize waste streams from the fermentation
operation, a "closed-loop1' system was designed. In this
process the concentrated whey was diluted with condensate
water derived from the evaporation of the fermentation
broths, and this was followed by the fermentation of the
whey. Theoretically, this closed-loop could be repeated as
often as new whey was added to the cycle (Figure 5).
13
-------
TABLE 3- AMINO ACID CONTENT OF VARIOUS SINGLE-CELL PROTEINS COMPARED TO FAO PROFILE
H
4=-
AMINO ACID
Lysine
Methionine
Valine
Leucine
Isoleucine
Tyrosine
Phenylalanine
Tryptophan
Histidine
Threonine
FAO
profile
4
2
4
4
4
2
2
1
-
2
.2
.2
.2
.8
.2
.8
.8
.4
—
.8
Amber
Nutrex
6.9
1.6
5.4
7.0
4.0
2.5
3.4
1.4
2.1
5.8
lof
Brewers
yeast
6
1
4
5
3
2
3
1
2
5
.8
.5
.7
.8
.6
.7
.4
.1
.1
.9
total
protein
Torula
yeast
8.
1.
5.
8.
6.
4.
5.
—
2.
5.
5
5
6
0
4
3
1
-
2
1
Brit. Pet.'0 Amo
yeast y
7
1
5
7
5
3
4
1
2
4
.5
.8
.8
.8
.3
.6
.3
.4
.1
.9
6
1
5
7
4
3
4
1
2
5
CO"
east
.6
4
.7
.1
.5
• 3
.1
.2
. 1
.5
-------
in
(0
O
4J
O
10
8
Add
Condensed
Whey
20
40
60
HOURS
80
100
10
8
120
•8
o
r-t
at
4 %
•P
W
Figure 4 Ethanol production by anaerobic fermentation
-------
M
CTv
Raw Whey
Condensed
Product
e mentation
Process
10% T.S.
Diluted
Whey Medium
vaporation
Process
30% T.S.
15% T.S.
Condensed
Whey
45% T.S.
Dry
Product
Ethyl alcohol
Figure 5 Closed-loop system for fermentation with zero effluents
-------
Since the entire fermentation broth is spray dried, no waste
streams are obtained. Effectively, therefore, this process
has zero effluents. The fermentation results do not differ
significantly whether tap water, raw whey or condensate
water is used to dilute the incoming condensed whey. The
process has been run over extended period of time, using
this closed-loop system, with no apparent build-up of any
metabolic toxic product to inhibit the fermentation. In the
overall schematic shown in Figure 1, one can see the return
of the condensate water to the medium mixing tank.
17
-------
SECTION V
ANALYSES AND CHARACTERIZATION OF PROCESS STREAMS
The results of fermentation of whey by S^. fragilis are
reported in the "Process" section where they pertain to
cellular growth and product characterization and use.
Additional tests were run to monitor and characterize the
various streams and fractions produced. These are
summarized in Table 4 and represent a summary of the range
of analyses obtained over many months of operation and many
fermentations run in various ways. Samples were taken
during the fermentations at a number of monitoring points in
the process (Points A - L, Figure 6) (also, see 'Methods and
Materials) .
Condensed whey (either acid or sweet) had initial BOD values
from 350,000 to 800,000 mg/1 compared to those of raw whey,
which were in the 37,000 to 50,000 mg/1 range.
If the fermentation was run to produce YFS material, the
whole fermented whey mass was evaporated and spray dried.
Evaporator condensate contained some ethyl alcohol. If
possible, this alcohol should be recovered by stripping in a
distillation column. The condensate, with or without the
alcohol removed, can be recycled and used to dilute incoming
condensed whey.
If the yeast cells are harvested for food grade material,
additional fractions from the centrifuges are obtained. The
first supernatants have a high BOD (60,000 to 104,000 mg/1)
because of the ethanol and soluble protein and other organic
material they contain. This stream may be added to raw YFS
material before evaporation and thus recover these
constituents. The final supernatants (washings) contain
such low levels of soluble materials (0.5% to 1.8$) that it
may not be economical to recover them in this manner. In
such a case, the final streams (containing BOD of 10,000 to
28,000 mg/1) would have to go to some further form of waste
treatment.
18
-------
TABLE 4 ANALYSES OF FRACTIONS OBTAINED
DURING FERMENTATION AND PROCESSING
vo
Fraction Mont
pt.
Cond . whey
Raw whey
1*0/1 i iim
ncoiuin
YFS (whole broth)
Evap. condensate
Condensate after
alcohol recovery
Condensed YFS
First creams
First supernatant
Final creams
Final effluent
supernatant
Powder
A
A
C
D
E
F
G
H
J
K
L
. Kj'el.
Prot.
8.7-
9.4
11.9-
14.4
Oft f|_
JV/ » V
37.0
35.0-
62.0
___
35.0-
62.0
40.0-
55.0
5.0-
30.0
40.0-
55.0
0.0-
6.0
35.0-
60.0
Dry
Basis
%NH3-N
Kjel. N,
23
23
fi^
O •}
5
—
-
5
5
10
5
0
<5
.0-
27.
.5-
26.
0_
74.
.0-
30.
.0-
30.
.0-
15.
.0-
75.
.0-
10.
.0-
80.
.0-
20.
0
5
0
0
0
0
0
0
0
0
% % pH °F. % mg/1 mg/1
Ash Lactose Temp. Solids BOD S.S.
7.3- 58.0-
9.2 71.0
5.0 69.0-
8.2 78.0
4O- ">7 O-
. w ^ / . V
6.5 68.0
7.0-
16.0
___
7.0-
16.0
6.0-
10.0
10.0-
20.0
6.0-
8.0
4.0-
8.0
7.0-
16.0
0.0-
14.0
0.0-
14.0
0.0-
5.0
0.0-
18.0
0.0-
2.0
<2.0
0.0-
15.0
4.8
5.7
5.0-.
6.2
4«_ na_
• y oo
5.5 92
4.5- 90-
5.5 94
5.2 120
6.0
5.0
5.5
4.5
5.2
4.5
5.2
5.0
6.0
5.0- 50-
6.5 80
44.8-
49.0
5.6-
6.3
1 f> fl-
X V • \J
15.5
7.0-
11.0
— _
27.0-
50.0
14.0-
20.0
3.8-
6.0
8.7-
20.0
0.5-
1.8
96.0-
98.5
OCrt M_ _
800 M
•17 M_
54 M
— — — — ____
20 M-
45 M
0.40 M
800 M-
950 M
58 M-
110 M
60 M- 900-
104 M 2000
53 M-
100 M
10 M- 150-
28 M 450
— — — _ —
%
EtOH
1.0-
9.0
5.0-
10.0
<0.2
<0.5
__ *»
3.4-
5.1
0.1-
1.0
_——
-------
Cond. Raw NH3,H3PO4,EC1
IftTKox? Who\/ •
ivjLiw y iiiiw y xjos* GT" o%
r- -i I ^TX ^1 VCCIO L pJI
[Ah-Hr fA>H, I
nA
ETHANOL
ro
o
SEED
FERMENTOR
> COOLING
WATER
MAIN
PERMENTOR
^__^x
HOLD
TANK
^ i
X-^y
1 f
k t j
Stlam i I I
f-^
EVAPORATOR
I
I
V
CENTRIFUGES
YEAST PRODUCT
To Alcohol Recovery
and/or I
Waste Treatment ** ^^-
IPigure 6 Scliematic showing monitoring points
-------
SECTION VI
PRODUCTION OPERATION AND MATERIAL BALANCE
The demonstration plant was operated as described in the
PROCESS SECTION (IV) for a continuous period of six to eight
months. During this time, standard operations included
using fresh seed each week, inoculum build-up, cell build-up
and continuous fermentation for 72 hours. During each 24
hours of continuous fermentation, the material balance is
indicated in Figure 7 and the Total Mass Balance and Water
Balance in Tables 5 and 6, respectively. Operation in this
manner was at an annual production rate of 2000 tons of feed
material (YFS) and represented the use of approximately
100,000,000 pounds of raw whey equivalent as raw material.
This level of production is 50% to 60/6 of the actual plant
capacity and was dictated by sales and new market
development and customer testing. In addition, during this
period of time, over 100 tons of food grade yeast (Nutrex)
was produced for customer evaluation.
21
-------
WATER
or
COMPENSATE WATER (182880 Ib/day)
CONDENSED WHEY
(68UOO Ib/day
45% T.S.)
(30600 Ib solids/day)
(1272 Ib/day)
NH-
1320 I '
Ib/day I
I
1560 Ib/dav)
•©2 (from
Air)
PROCESS
ro
IV)
21432
Ib/day
5088
Ib/day
EtO
YFS
solids
220248 Ib/day
8232 Ib/day
CO2 (+ AIR minus
Supernatant
(Ash & Soluble protein)
NUTREX
SALTS,ACID,
VITAMINS
WATER
Figure 7 Material balance for whey fermentation
-------
TABLE 5. TOTAL MASS BALANCE
Material Amqunt_(_lb/day_l
Input material:
Whey solids* 30,600
Water* 220,248
Salts, acid, vitamins 1,560
Ammonia (N^) 1 ,320
02 from air 1,272
Total 255,000
Output material:
YFS solids 21,432
Water 220,248
Ethyl alcohol (EtOH) 5,088
C02 8,232
Total 255,000
*Source of whey solids and water is either 39,864 Ib
condensed whey +210,984 Ib raw whey or 68,000 Ib condensed
whey + 182,848 Ib water.
TABLE 6. WATER BALANCE
Material Amount _£lb/day_l
Input water:
From:
39,894 Ib condensed whey 21,936
+
210,984 Ib raw whey 198,'312
Total 220,248
or
From:
68,000 Ib condensed whey 37,400
+
182,848 Ib water or
condensate water 182,848
Total 220,248
Output water:
From:
Evaporation of YFS 170,240
(fermentation broth)
Drying 50,008
Total 220,248
23
-------
Cond.
Whey
A
PEED
TANK
(a)
V
Raw
Whey
?EED
TANK
(b)
V
'
1
KH3,H3P04,KC1
yeast e>
rlA
SEED
FERMENTOR
(e,f)
-> COOLING
WATER
ETHANOL
ALCOHOL
RECOVERY
UNIT
(q)
ro
4=-
MAIN
FERMENTOR
YEAST PRODUCT
To Alcohol Recovery
and/or
Waste Treatment ^
Figure 8 Schematic for Whey Fermentation Process
-------
SECTION VII
EQUIPMENT DESCRIPTION
All equipment used throughout the plant is stainless steel
(304 or 316), and all piping and fittings are sanitary
design and in most instances tanks and lines are connected
to CIP systems for cleaning. Major pieces of equipment are
described below and may be identified in Figure 8. Not
included are necessary support equipment such as pumps,
mixers, acid and anhydrous ammonia storage, intake areas,
product packaging and storage, utilities, etc.
Identification and description Size
a,b Feed tanks (2) each 20,000 gal.
c Medium mix tank 15,000 gal.
g,h Hold tanks (2) each 30,000 gal.
d Main fermentor 15,000 gal.
e Seed fermentor 500 gal.
f Seed fermentor 3,000 gal.
i Plate (heater/cooler) 3,000 GPH
j Plate (heater) 2,000 GPH
k,l Air compressors, (2) each 1,300 CFM
reciprocating at 60 psi
m,n Yeast Centrifuges, (2)
continuous, nozzle type—each— 10,000 GPH
o Evaporator, triple effect, vacuum 30,000 Ib
water/hr
25.
-------
Spray dryer 1,500
Ib product/hr
Alcohol recovery unit including:
distillation column (3* diara., 29
bubble cap trays)
distillation column (20" diam., 15
bubble cap trays) ,
Condensers (2) (420 sq. ft. and 360
sq. ft.), reboilers, pumps, 9 tanks,
piping, fittings, etc. 600,000
gal/yr
26
-------
SECTION VIII
ECONOMICS
Cost calculations of the large scale fermentation confirm
those projected in the extended pilot fermentor operations3.
The cost of the medium for the fermentation is primarily
determined by the cost of whey. While it is possible in the
future this material may have a negative cost due to the
necessary cost of waste treatment of pollution abatement, at
the present time, it is realistic that whey may be obtained
at no cost or for the cost of evaporation and/or
transportation alone. The actual costs ranged from zero to
one cent per pound whey solids delivered for raw whey and
one to four cents per pound whey solids delivered for
condensed whey. The medium cost is 0 to 4.5 cents per pound
of finished product.
The cost of production is also affected by the size of the
operation. Labor and other costs decrease on a per pound,
finished product basis as the size of the equipment and its
degree of automation and sophistication increases. However,
as the capacity increases so does the capital investment
with its connected charges for depreciation, taxes,
insurance and physical facilities. A plant capable of an
annual production of 4,000 to 10,000 tons per year would
cost in the range of $6 to $15 million, depending on its
design, and this is still considered a small fermentation
plant. Those being designed for the production of single
cell protein from hydrocarbons are in the 100,000-ton annual
capacity range. Calculations using present labor, utility
and capital investment figures indicate that the feed grade
material can be produced in a plant with an annual capacity
of 5,000 to 10,000 tons for a total production cost of 11.8
to 16 cents per pound. This does not take into account any
credit for alcohol recovered which can be of the order of
several cents per pound, depending on the conditions of the
fermentation and the yields obtained. The process looks
economically viable at present. Its future viability will
depend on market conditions and availability and cost of
energy and expansion dollars.
27
-------
TABLE 7-
CALCULATED PRODUCTION COST FOR DRIED
YEAST FERMENTATION SOLUBLES FROM
WHEY FERMENTATION (1974-1975)
Item 0/lb. of product
Medium 0-4.5
Operating utilities:
Evaporation and drying 1.45
Aeration, cooling 1.00
Other .40
Labor 2.40
Capital investment 2.05 - 4.0
Overhead 2.40
Total 11.8-16.15
During the time of this study the actual production cost
averaged to 15.4 cents/lb. of YFS material produced. Sales
of this material were made at 180 to 240/lb.
28
-------
SECTION IX
DISCUSSION
Sacch.argray.ces fragilis may be grown on acid or sweet whey in
a deep tank, aerated fermentor in a continuous manner on a
commercial scale. The fermentation has many advantages. By
operating at a low pH and with a large seed size and high
cell count, contamination is no problem, and therefore,
sterile or special aseptic equipment or techniques are not
necessary. The aeration requirements are not excessive,
(0.8 to 1.2 volume air per volume of medium) nor is there
any problem with foam control. Temperature control, despite
the rapid growth rate, is maintained with a low level of
cooling water or by the addition of cooled medium in
continuous operation. The medium is simple in composition,
and at the concentrations used, the carbohydrate substrate
(lactose) is completely soluble. The absence of any
potentially toxic substances in the medium simplifies
harvesting. The production of a whole fermented whey mass
(Yeast Fermentation Solubles) eliminates additional
processing of waste streams and increases the yield of the
fermentation. As a result of the evaporation of the whole
fermented mass prior to drying, condensate water is obtained
that can be used to dilute incoming condensed whey and
thereby operate a completely closed-loop system with no
effluents.
Food grade yeast may be produced by harvesting the yeast by
centrifugation. The supernatant streams (except the final
washings) from this process may be used in certain animal
feed fractions. The protein quality of the finished
products is good, although slightly low in the sulfur
containing amino acids, as is true for most single cell
proteins. Rat feeding tests show no toxicity and feeding
experiments with other animals show uses in other feed
rations, especially those for cattle.
By varying the conditions of the fermentation, increased
amounts of ethyl alcohol can be produced and recovered but
at the expense of cell yield. However, as one expert has
recently stated, "Energy considerations could also affect
29
-------
the kind of SCP process. Aeration costs could be important
if energy costs continue to rise. Hence, an anaerobic
fermentation process, in which both alcohol and SCP feed are
produced, could be the process of choice".12
The cost of production is dependent primarily on two
factors — the cost of whey and the capital investment.
Efficiency demands a plant that is highly automated and
instrument controlled and of a size to have an annual
capacity of at least 4,000 - 10,000 tons of finished
product. This would represent a raw material requirements
of the equivalent of 200 to 500 million pounds of raw whey.
Therefore the location of such a facility must be near a
large cheese producing area. Such an area would have an
excess of whey at little or no cost. The capital investment
is large, but must be made to allow the product to compete
in the market place. The vast majority of the material from
such a plant must be sold as animal feed because at the
present time human food uses for yeast is not of sufficient
volume. Most of such feed products would be used in cattle
feeds (either in concentrated liquid or dried form) because
of the proximity of large numbers of these animals in this
dairy (cheese producing) area.
The present and future shortage of protein has been reported
extensively, and many investigators feel one potential
solution is the production of single cell protein.12
Agricultural and industrial waste materials may be used as
substrates for such production thereby decreasing the
immense disposal problems at present.'3,14 whey is a clean,
wholesome, food-grade substance, in excess supply and a
potential environmental pollutant. By the process described
here, it can be converted to a useful and needed high
protein material, the demand for which is now present and
should increase in the future.
30
-------
SECTION X
REFERENCES
1. Whey Products Institute, Report 10373; "Cheese/Whey
Production for 1972."
2. U.S. Department of Agriulture; "Production of Whey
Products - 1974, U.S. Department of Agriculture."
3- Bernstein, S. and Everson, T.C.; "Protein Production
from Acid Whey via Fermentation:" Environmental Pro-
tection Technology Series; EPA-600/2-72-025 (May 1974)
4. Wasserman, A.E.; "The Rapid Conversion of Whey to
Yeast;" Dairy Engineering 11 374-379 (1960).
5. Amundson, C.H.; "Increasing the Protein Content of
Whey thru Fermentation;" Proceeding 33; Washington
State University Institute Dairying (1966).
6. Bernstein, S. and Everson, T.C.; ''Protein Production
from Acid Whey via Fermentation;" Proceedings of the
1973 Cornell Agricultural Waste Management Conference,
page 103-
7. Mayer, B.B.; "Whey Fermentation;" Proceedings, Whey
Utilization Conference; University of Maryland
(1970); page 48.
8. Federal Register, Volume 28, Number 97, 4948 (1963).
9- Wisconsin Alumni Research Foundation, Madison,
Wisconsin, (1972, 1973).
10. Courts, A.; "Recent Advances in Protein Production";
Process Biochemistry, Feb. 1973, page 31.
11. Anon.; "Torula Yeast from Petroleum"; Food Processing;
July 1974, page 28.
31
-------
12. Humphrey, Arthur, E.; "Current Developments in Fermenta-
tion;11 Chemical Engineering, (Dec. 9, 1974), page 98.
13. Seeley, R.D.; "Current Status of Single Cell Protein;11
Paper presented at American Chemical Society, 169th
National Meeting, (April 11, 1975).
14. Skinner, K.J. "Enzymes Technology - Special Report;"
Chemical and Engineering News (August 18, 1975), page 22,
32
-------
SECTION XI
GLOSSARY
Acid_Whey_ — The product remaining after removal of casein and
fat from cream in the process of making cream cheese, or
from skim milk in the process of making cottage cheese.
Amber_BYF__lQQ — Autolyzed yeast fraction: Spray-dried yeast
supernatant .
Aj.N ^R^Ci.casein — A standardized casein product adopted for
use by the Animal Nutrition Research Council for the
Official Association of Analytical Chemists rat PER assays.
BQD--Five_Day_ — Biological oxygen demand: Procedure
performed in accordance with methods for the Examination of
Water and Wastewater, 13th Ed., 1971, pp. 489-495-
Expressed as milligrams per liter (mg/1) or parts per
million (ppm) .
Clgsed-lqQp__sy_stem — Process of fermentation of whey
producing no effluents.
N^U ..RiLiJ._Y-llQ9_ — Northern Utilization Research Laboratory,
Strain Y-1109, Saccharomy_ces fragilis.
Nutrex — Food grade yeast obtained by harvesting and washing
the cells by centrifugation of the fermented whey mass,
followed by evaporation and drying.
YF_S — Yeast Fermentation Solubles: Feed grade material
obtained by the evaporation and drying the whole fermented
whey mass .
33
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/2-77-133
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
Commercial Production of Protein By The Fermentation
of Acid And/or Sweet Whey
5. REPORT DATE
July 1977 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
Sheldon Bernstein and Chu H. Tzeng
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Amber Laboratories
Juneau, Wisconsin 53039
10. PROGRAM ELEMENT NO.
1BB610
11. CONTRACT/GRANT NO.
S-800747
12. SPONSORING AGENCY NAME AND ADDRESS
Industrial Environmental Research Laboratory-Cin., OH
Office of Research and Development
U.S. Environmental Protection Agency
-Cincinnati, Ohio_ 45268
15. SUPPLEMENTARY NOTES
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/12
16. ABSTRACT
Saccharomyces fragilis may be grown on acid or sweet cheese whey in a deep-tank,
aerated fermentor in a continous manner on a commercial scale. Operations in a
15,000-gallon fermentor at low pH and high cell counts experience no contamination
during extended periods of time under sanitary but not sterile conditions. Media
additions to the raw or diluted condensed whey include anyhdrous ammonia, phosphoric
acid, and yeast extract. The production of a condensed or dried whole fermented
whey mass (yeast fermentation solubles), which is an acceptable high protein feed
ingredient, eliminates additional processing of waste streams from yeast separators,
increases fermentation yields, and utilizes the unfermented whey proteins origi-
nally present. Evaporation of the whole fermented whey mass produces condensate
water that can be used to dilute incoming condensed whey and thereby operate a
unique closed loop system with no effluents.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COS AT I Field/Group
Industrial Wastes
Cheeses
Caseins
Fermentation
fcAcid whey
fcA.N.R.C. Casein
Nutrex
13B
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)'
Unclassified
21. NO. OF PAGES
42
20. SECURITY CLASS (Thispage)
—TTnr-MreiAfiod
22. PRICE
EPA Form 2220-1 (9-73)
* US. GOWJtmiBCn'IIIKTINC OFFICE: 1977- 757-056/6482
------- |