EPA-66 0/2-74-925
     May 1974
                        Environmental Protection Technology Series
      Protein  Production
      From
      Acid Whey Via Fermentation
                                  Office of Research and Development

                                  U.S. Environmental Protection Agency
                                  Washington, D.C. 20460

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            RESEARCH REPORTING SERIES
Research reports of the  Office  of  Research  and
Monitoring,  Environmental Protection Agency, have
been grouped into five series.  These  five  broad
categories  were established to facilitate further
development  and  application   of   environmental
technology.   Elimination  of traditional grouping
was  consciously  planned  to  foster   technology
transfer   and  a  maximum  interface  in  related
fields.  The five series are:

   1.  Environmental Health Effects Research
   2.  Environmental Protection Technology
   3.  Ecological Research
   4.  Environmental Monitoring
   5.  Socioeconomic Environmental Studies

This report has been assigned to the ENVIRONMENTAL
PROTECTION   TECHNOLOGY   series.    This   series
describes   research   performed  to  develop  and
demonstrate   instrumentation,    equipment    and
methodology  to  repair  or  prevent environmental
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.

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                                           EPA-660/2-74-025
                                           May 1974
    PROTEIN PRODUCTION FROM ACID WHEY

             VIA FERMENTATION
                    By

         Sheldon Bernstein/ Ph.D.
         Thonas C. Everson, Ph.D.
              Grant #8-800747
          Program Element 1BB037
              Project Officer

             Kenneth A. Dostal
National Waste Treatment Research Program
 National Environmental Research Center
  U.S. Environmental Protection Agency
         Corvallis, Oregon 97330
               Prepared for

    OFFICE OF RESEARCH AND DEVELOPMENT
  U.S. ENVIRONMENTAL PROTECTION AGENCY
         WASHINGTON  D. C. 20460
For sale by the Superintendent of Documents, U.S. Government Printing Office
            Washington, D.C. 2M02 - Price $1.25

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           EPA Review Notice

This report has been reviewed by the Office
of Research and Development, EPA, and
approved for publication.  Approval does not
signify that the contents necessarily reflect
the views and policies of the Environmental
Protection Agency, nor does mention of trade
names or commercial products constitute
endorsement or recommendation for use.
                      ii

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                        ABSTRACT

Prom the operation of a demonstration pilot plant over
extended periods of time, it has been shown that yeast may
be grown on an acid whey or sweet whey medium in a contin-
uous, deep tank aerated fermentor.  Variations in
fermentation conditions, strain selection, and medium com-
position produced cell concentrations of several billion
cells per milllliter.  By a process of evaporation and spray
drying the whole fermented whey mass and the utilization of
the evaporator condensate to dilute Incoming condensed whey,
a high grade, non-toxic, protein feed material may be produced
without any effluent streams.  Amlno acid analyses and protein
efficiency ratios are presented for this feed material.
Economic estimates show that while a large capital investment
and low cost raw material are required for the commercial
feasibility of this fermentation process, it will be compet-
itive with other methods for the manufacture of single cell
protein.  This whey fermentation is one means of converting
large quantities of a potential environmental pollutant into
a useful and needed product.
                           ill

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                        CONTENTS
Section
                                             Page
  II

  III

  IV

  V

  VI

  VII

  VIII
  IX

  X
  XI
  XII
  XIII
Summary and Conclusions - International
  Minerals Corporation Preliminary
  Studies
Conclusions & Recommendations
  Amber Laboratories
Introduction
  International Minerals Corporation
Materials and Methods
  International Minerals Corporation
Experimental Results
  International Minerals Corporation
Introduction
  Amber Laboratories
Materials and Methods
  Amber Laboratories
Results and Discussion
  Amber Laboratories
Summary
  Amber Laboratories
Acknowledgements
References
Glossary
Appendices
6
11
28
29
35
58
63
64
65
67

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                       FIGURES


                                                        PAGE

     GROWTH OP SACCHAROMYCES FRAGILIS
     AT DIFFERENT INOCULUM LEVELS UNDER
     CARBOHYDRATE FEED CONDITIONS MAIN-
     TAINING 5-6# CHO IN THE BROTH                       18

     GROWTH OF SACCHAROMYCES LACTIS FROM
     VARIOUS INOCULUM LEVELS, GROWN UNDER
     CONDITIONS OF MAINTENANCE OF 5-6# CHO
     IN THE BROTH                                        20
3    SEMI -CONTINUOUS OPERATION, SOP, 0
     NH^OH, SACCHAROMYCES FRAGILIS                       22

4    CONTINUOUS OPERATION OF SACCHAROMYCBS
     FRAGILIS WHEY FERMENTATION IN A 3 STAGE
     SYSTEM

5    BLOCK DIAGRAM OF CLOSED-LOOP SYSTEM FOR
     MINIMIZING FERMENTATION EFFLUENTS

6    SEMI -CONTINUOUS GROWTH OF SACCHARQMYCES
     FRAGILIS USING S0P MEDIUM-2

7    CONTINUOUS GROWTH OF SACCHAROMYCES
     FRAGILIS USING SOP MEDIUM-2 (59 HR)                 46

8    CONTINUOUS GROWTH OF SACCHAROMYCES
     FRAGILIS USING SOP MEDIUM-2 (220 HR)               -4$

9    RAT GROWTH RATES ON CASEIN AND FERMENTED
     WHEY PRODUCTS                                       53
                          vi

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                         TABLES


No.                                                     Page

1    Summary of Experimental Data                        12

2   . Experiment #18.  Continuous operation of
       a Whey Fermentation with Saccharomyces
       fragilis in a 3-Stage System                      24

3    Experiment #4.  Amino Acid Analyses,
       Inoculum Level Effect                             2!?

4 ~  Summary of Fermentation Experiments
       EPA Project S-800747                              36

5    Comparison of Gross Composition and Amino
       Acid Content of Various Single-Cell Proteins      50

6    Amino Acid Content of Whole Wheat, Commercial
       Brewers Yeast and Saccharomyces fragilis
       Yeast compared to FAO Profile                     5!

7    Protein Efficiency Ration (PER) Assays of
       Several Typical Saccharomyces fragilis
       Yeasts                                            52

8    Annual Production of Fermented Whey Mass
       Solids vs Fermentor Size                          55

9    Production Cost Estimates of Various Single-
       Cell Proteins                                     57
                          vii

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                       SECTION I
                SUMMARY AND CONCLUSIONS
 INTERNATIONAL MINERALS CORPORATIONrPRELIMINARY STUDIES
1.  A aeries of experiments were conducted in 44 liter, pilot
scale fermentors using several strains of lactose-fermenting
Saccharomyces.  The process of conversion of lactose in whey
to yeast cellular material was reasonably efficient with con-
version rates of 45-55# recorded.  Therefore, the literature
conversion rate of 55# was attained (ll).
2.  The fermentation process was completed in 8-10 hours and
could be operated either in a serai-continuous manner with
minimum "downtime" or continuous manner without "downtime".
3.  Cell yields, cell counts, and crude protein concentrations
were approximately equivalent for the various fermentation
processes.
4.  From a fermentation standpoint the process is simple to
operate and maintain (non-sterile) and should become an
effective means of converting whey to a marketable commodity.

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                       SECTION II
            CONCLUSIONS AND RECOMMENDATIONS
                   AMBER LABORATORIES
1.  Saccharomyces fragilia can be produced at growth levels
of several billion cells per milliliter using a deep tank
aerated fermentor operated in a continuous manner on acid or
sweet whey.
2.  The complete whole fermented whey mass may be dried
eliminating effluents and produce a satisfactory feed
supplement.  The yield of fermented whey mass (PWM) was 0.675
to 0.75 pounds per pound of whey solids.
3.  A superior quality yeast, high in protein and low in ash
content, may be produced by harvesting the yeast cells.  The
product should find commercial applications in specialty feeds
and in foods as a food additive.
4.  The fermentation could be carried out on permeates from
ultraflltratlon (UF) of acid or sweet whey.  However, numerous
problems were encountered using the UF unit such as low flux
rates due to membrane fouling, inability to properly sanitize
the UF unit, and extremely low solids in the lactose permeate.
Therefore, the fermentation of lactose permeate from the UF
system studied does not look economically feasible.
5.  A large capital Investment and low cost, raw material are
required for the commercial feasibility of the fermentation
process.  These studies were limited to the laboratory and
pilot development of protein production from acid and sweet

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whey via yeast fermentation.  It is recommended that the
sealed-up testing of protein production from whey fermentation
in commercial sized fermentora be examined which would permit
full-scale, long term evaluation of the chemical, physical,
biological and economic aspects of the process.

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                      SECTION III
                      INTRODUCTION
           INTERNATIONAL MINERALS CORPORATION
The preliminary investigation and shake flask studies were
done by International Minerals Corporation, Libertyville,
Illinois.  Various Standard Operating Procedures were devel-
oped and used by Amber Laboratories in the operation of the
500 gallon, Demonstration Pilot Plant fermentor.  The results
of the Amber Laboratories studies are presented in Sections
VI-IX.

The carbon of whey lactose can be converted by a variety of
lactose-fermenting yeasts to yeast cellular components of
which 50$ are protein.  An exogenous source of inorganic
nitrogen is usually required as a supplement since whey pro-
tein is not a readily available N-source for these saccharo-
lytic strains.  It has been considered that if fermentation
processes were devised which could result in near theoretical
conversion of lactose to yeast protein in a suitably short
fermentation cycle then such a process might prove a feasible
means of producing a low cost protein feed supplement from
the high tonnage of whey which is currently considered waste
material.
As discussed in a preliminary report (international Minerals
Corporation, Section XIII Appendices) shake flask experiments
demonstrated that sufficient doublings of yeast populations
in short fermentation cycles could be obtained on simple whey

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media to warrant further investigation of the process in pilot
fermentors (15 gallon).  Plant throughputs of 2-3X fermentor
capacity per 24 hour period appeared feasible.  The shake
flask experiments also demonstrated that such a process might
be run under non-sterile conditions.

The following series of experiments was carried out in fer-
mentors as an extension of this work.  A progression from
batch to semi-continuous to continuous fermentor operation was
carried out to demonstrate the optimum method for use of
fermentor capacity and feasibility of tshe whey to yeast con-
version process.

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                       SECTION IV
                 MATERIALS AND METHODS
           INTERNATIONAL MINERALS CORPORATION
Two yeast strains, Saccharomyces fragilis (NURL, Y-1109) and
S.. lac t is (NURL, Y-1140), were used throughout the ferment or
experiments.  The strains were maintained on a weekly transfer
schedule on sterile whey agar.
Whey concentrate as received contained 35-40# total solids and
a lactose content of 35-38#.  The high lactose concentrate Has
prepared from locally collected raw acid whey by Amber Lab-
oratories at Juneau, Wisconsin, and transported hot to the IMC
fermentation pilot plant.  Whey for the experiments described
later was diluted to appropriate levels of lactose with tap
water and then used to prepare Medium 5102-A or Medium 5102-B.
Prior to dilution, whey concentrate was heated to Just below
boiling to insure complete solubillzation of lactose.
Whey agar was prepared by diluting whey concentrate with tap
water to 5% - Q.5% reducing sugar content and adding the com-
ponents of 5102-A Medium described later.  The pH was adjusted
to 7-0 with 10$ XaOH and agar added to a 2£ concentration.
The medium was heated to melt the agar, tubed in suitable
aliquots and sterilized for 10 minutes at 120°C.

Two basal media were used throughout these studies.  Medium
5102-A consisted of whey concentrate that was adjusted to
lactose concentration.  The diluted concentrate was made to

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contain (W/V) 0.5$ (NH^)2SOK; 0.5$ K2HPO^ and 0.1$ Amber  BYP-
300.  The pH was adjusted to lj.. 5 for  S. fragills  and 5-5  for £>.
laotis with 10$ H3POK.  The medium was not  sterilized for
fermentor use.  Medium 5102- B was prepared  from whey concen-
trate adjusted to "-6$ lactose but made to contain (W/V) 0.9$
NH^OH; 0.3$ Amber BYF-300; 0.001$ PD-82 an t if o am  (Hodag).
A series of nine 15-gallon  (l^ liter) fermentation vessels was
used throughout these experiments.  The fermentors were of the
jacketed type equipped with automatic pH, antifoam, temperature
and nutrient feed controls.  They were aerated by compressed
sterile air entering through a bottom sparger and agitated by
a standard shaft and impeller system powered hydraulic ally
through magnetic shaft couplings.  Steam could be directly in-
jected into the vessel or circulated through the jacket.  Cool-
ing water could also be circulated through the jacket.  The
vessels were fitted with various inoculating and sampling ports
and could be fitted to handle oxygen or carbon dioxide sensors.
The series of fermentors were inter-connected by header lines
allowing complete flexibility of material transfer from vessel
to vessel.  The series of 15-gallon fermentors was served by
three 15-liter New Brunswick fermentors which served  as seed
vessels.  The New Brunswick fermentors were of standard in-
ternal baffle design and were connected to the 15-gallon
fermentor header line.
Two procedures were used for inoculum and are designated
appropriately in the experimental summary (Table 1).  The

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procedures are outlined below:

Inoculum Buildup Procedures:

         I
    Stock Slant
                               II
                           Stock Slant
                   Wash growth off with
                   10 ml medium, inoculate
                   into
  liters 5102-A in 15 liter ferraentor
  liters air/m; 200 rpra; 0.5 PSIG
back pressure; 30°C
                                    1
    8 hours  J.

Add 6 liters 5102-A

   16 hours  I    1 gal to:

9 gal 5102-B in 15 gal seed fermentor
200 rpm; 2.32 V/V air/m; 10 PSIG
back pressure; 30°C
  7-8 hours

Use as seed as indicated
I
                           500 ml 5102-A In 2
                               liters
                               Incubator on
                               rotary shaker at
                               30°C for 2t| hours

                           8 gal 5102-A in 15 gal
                           seed fermentor 200 rpm;
                           2.32 V/V air/m; 30°C;
                           1U-16 hr
Use as seed at 1-2x10
cells/ml as indicated
Fifteen gallon fermentors were inoculated from either of the

trains indicated above as noted for individual experiments.

The 10$ level was standard.  Fermentor operating conditions

unless otherwise noted were 200 rpm; 2.32 V/V air/min; 10 PSIG

back pressure; 30°C.  The fermentors were not sterilized nor

was pH controlled,  Fermentors were loaded with 9 gal of medium

and inoculum transferred from a seed vessel to bring the total

fermentor volume to 10 gal (10$ inoculum).


A three stage continuous fermentation procedure in the 15

gallon fermentors utilized an inoculum development as in I

above.  Three fermentors were simultaneously loaded and
                            8

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inoculated.  After  six  hours  of  inoculum development the fer-
mentors were loaded  to  9  gallon  volume  with 5102-B medium and
a continuous feed of 5102-B medium to the first stage initiated.
The rate of feed was calculated  to accomplish total utilization
of lactose by completion  of the  third stage.   The  rate of con-
tinuous feed was ^3.18  gal/hr (lLj.,0 liters/hr).

Solids determinations were  made  as described  below:   A sample
representing approximately  1  gram of dried  solids was   weighed
into  a previously dried,  cooled,  and weighed.5 cm  circular
aluminum flat-bottomed  dish containing  reagent grade  seasand
(20/lj.O mesh).   The dish and contents were heated at 60°C over-
night at full vacuum (-^30").   The  dish  was  cooled  in  a dessi-
cator and then  reweighed.   Protein was  determined  in  accord
with  the A.O. A.C. method (llth  Ed.  1970  #16.035 ref  2.651).
A factor of 6,38 x percent-N  was  used as  recommended  for dairy
product protein.  Fat was determined in accord  with the  A.O.A.C.
method (llth Ed. 1970 #16.052) for dairy  products.  Ash was
determined in accord with the A.O.A.C.  method  (llth Ed.  1970
#16.031^) for dairy products.   Free and  total  reducing sugar
was determined  using a  Technioon Autoanalyzer.  The Technicon
Ferricyanide colorimetric method was used to  determine reducing
sugars before and after hydrolysis.  Total  amino acids were
determined by over-night hydrolysis  in  6N HC1  followed by
column chromatography in a  Beckman 120C Amino  Acid Analyzer.
Cell  count analyses  on  samples of  appropriate  dilutions  of
whey  fermentor broth were made in  0.1 M phosphate buffer,  pH

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7.0.  Both chambers of a hemooytometer (Spencer Bright Line
Improved Neubauer 1/10 mm deep) were loaded with the same
diluted sample and only the large subdivided center square was
used In counting.
                        Q
If cell numbers were <10 /ml the entire square was counted,
the microscopic counts from both chambers were averaged and
cells/ml calculated.  If the two counts deviated from each
other, the hemooytometer was reloaded, two more counts taken
and the four counts averaged.  In counting samples with high
cell numbers, three rows of five squares each were counted for
both chambers, the six counts averaged and cells/ml calculated
in accord with the following equation:
     cells/ml   =   count x dilution x k x 10         o
          '                             ^            -
                    # small squares counted
Cell volumes were determined by centrifuglng a 10 ml broth
sample in a conical, graduated centrifuge tube at 25>0 rpm for
30 minutes in an International PR-2 centrifuge equipped with
a fixed rotor.  Volume of the pellet was read, in ml, directly
from the tube.
                           10

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                       SECTION V
                  EXPERIMENTAL RESULTS
           INTERNATIONAL MINERALS CORPORATION
The series of experiments described below were performed to
determine the effects of inoculum size (Bxpt's 4,5), medium
Ingredients (Expt's 6-14), carbohydrate level (Expt's 12-1?)
and physical conditions (Expt's 16-17) on the two strains of
yeast under study.  In certain of the experiments operation
of the fermentation under semi-continuous (Expt's 11,12,15)
or continuous (Expt 18) conditions was examined.  Experimental
conditions and parameters are summarized In Table 1.  The data
obtained for maximum cell yield, total solids, dry weight of
protein (as is), dry weight of washed protein, % conversion
and % alcohol (ethanol) are provided where such determinations
were made on a particular run.
The effect of amount of Inoculum transferred to the fermentor
on cell growth and yield were determined in Expts 4 and 5 for
S. frag11is and S. lactls. respectively.  Growth of S. frag11is
_               T*  ————'                          —
under different inoculum size conditions is summarized graphi-
cally in Figure 1.  The experiments were conducted under con-
ditions in which carbohydrate level was continuously maintained
at 5-6£.  The data obtained were interpreted as reflecting the
inhibitory effect of excess whey on growth of the yeasts which
had been observed previously in shake flasks.  The effect was
not grossly apparent during the first four hours of fermenta-
tion.  Doubling times for the yeasts calculated for this time
                            11

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                                 Table 1.   SUMMARY OP EXPERIMENTAL DATA
ro
Max, Pop. %
cells/ral
T.S.
Condition Medium xlCP "as is"
Experiment #4. Effect of Inoculum
5-6$ CHO
W-10
*
44$ Inoculum
25$
15$
5$
10$ >



t
10$ Whey only
4
10

10
10
8
1.155
1.0?
1.07
.885
.795
.805
Experiment #5- As in 4 S. lactis
W-10
44$ Inoculum
25$
15$
5$
10$ 4



p
2$ Whey only
10
10
10
10
10
10
8.75
6-5
9.5
3.2
8.4
3.3
size on



13.05
11.30
10.40
8.9
8.8
8.2


12.7
10.5
9.5
9.1
8.1
8.9
Dry Weight
Protein Protein $ $
''as is" washed Conversion Alcohol
S. fragllis fermentation



18.03 41.99
19.08
19.84
20.42
22.22 43.60
15.51








         "Numbers in parentheses refer to hoars of fermentation at which maximum cell  population
          was reached.

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Table  l.  (eont.)
Experiment #6.  Effect of level of BYP and ammonium salts on S.. fragilis fermentation
           W-10
In
 BYP
0.1*
0.3
0.5
0.1
0.3
0.5
1.0*
1.0
1.0
0.5
0.5
0.5
10
10
Q
Q
ft
Q
1.31
1.78
1.40
1.56
1.30
1.03
6.15
6.45
6.55
5.51
6.00
6.25

36.42

27.95

28.13

43.58

42.49


23.74
29.15
28.34
30.88
27.33
36.23
Experiment #7. As in 6 S. lactis
BYP
0.1*
0.3
0.5
0.1
0.33
0.5
(MH4)2S04
1.0*
1.0
1.0
0.5
0.5
0.5
10
10
10
10
10
10
2.25
1.97
1.97
2.37
2.32
2.10
Experiment #8. Effect of N-source
BYP
0.3*




4r
-NltyOH-KHgPO^-NS
0.5* 0.5*
0.7 0.5
1.0 0.5
0.5 1.0
0.45* 0.5
0.90 0.5
10
10
10
10
10
8
2.91
1.97
2.35
1.90
1.64
(1.57)

6.15
6.55
5.30
5.20
5.40
5.40
and P- level

6.15
6.00
6.00
5.35
5.05
6.00

33.44
32.88
35.00
26.44
26.62
28.00
on S.

26.25
27.50
26.25
38.31
27.81
34.50

45.36
45.74
44.78
46.93
48.23
46.45
fragilis whey

45.74



37.51
W.09

42.75
45.38
46.25
46.84
37.77
39.26
fermentation

36.83



61.03
56.15

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Table 1. (cont.)
Experiment #9. Aa in 8 S. lactia
N-OH
-
-
-
•
0^90
W T)f^» UQ
njLinf\Jh HO
. 5* 0 . 5*
.7 0.5
1.0 0.5
0.5 1.0
0.5
0.5 - 1
Experiment #11. If fed


10
10
10
10
8
1 8
3.15
3.11
3.08
3.26
(2.17)
(2.39)
t of NH^OH/ft
,J
5.4o
5.95
6.20
5.55
5*80
PO. medium

29.67
25.01
27.88
36.43
28.97
34.32
on S.

41.09

36.11

37.13
fragilis

53

55

54

.72

.66

.36
fermentation
NH^OH adj. HgPO^
1 0.
1A
IB
2
2A
2B N
3 o.
3A
3B
4
4A
4B >
5 0.
9t




v
45




f
9
5A 1
5B +
8) 1.10
10 1.08
9
10
10
9
8
10
9
8
10
Q
10
10
Q
1.22
1.12
1.31
1.08
1.23
1.08
1.29
1.05
1.01
1.22
1.03
1.10
1.20
_


6.80


6.65


6.40


7.40

_


31.58


25.47


24.88


28.96

_


33.30


33.56


33.56


36.17




50


55


52


52




.09


.5


.42


.11


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Table I. (oont.)
Experiment f 12
. As In 11 S. lactis







MH^OH
1 0.
1A
IB
2
2A
2B >
3 0.
3A
3B
4
4A
4B >
9*




f
45




f












5 0.9
5A
5B ^
\
,
Experiment #13
«v°*
0.5*
:
-
-
MH^OH
0.9*
0.45
0.90
0.90
0.45
0.90


94
9
9
Q
Q
9
9
Q
Q
9
9
9
0
Q
Q
3.09
3.65
3.30
3.01
3.40
3.00
2.57
3.10
3.00
2.98
2.75
2.80
3.00
3.20
3.00
6.95


6.50


6.30


7.05


7.00


_. Effect of K* "source and
Lactose
5-6 1
5-6
5-6
10-11
10-11
10-11
[10
10
14
14
14
1.49
1.32
1.29
1.23
1.23
1.25

7.90
6.25
7.15
11.75
10.40
10.60
31.


31.


29.


30.


33.


lactose

29.
32.
32.
26.
25.
27.
33


96


92


11


30


level

54
98
98
16
01
88
35.91


34.46


39.08


39.08


37.50


on S.

34.83
40.38
38.73
31.64
36.24
34.13
49


48


40


40


45


fragilis

37
28
33
26
21
24
.05


.72


.10


.24


.07

















fermentation

.07
.10
.60
.80
.34
.30

2.05 PWC
2.33
2.63
1.75
0.78
1.05

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          Table 1.  (eont.)
a\
Experiment #14. As in 13 S. lactis
KHgPO^ HH^OH Lactose
0.5#



v



f
1.0* 5
0.5
1.0 x
I
1.0 10
0.5
1.0
\
I
10
10
10
12
14
12
2.85
2.88
4!oo
4.70
3.30
Experiment #15. Effect of lactose
6.76
5.66
6.86
10.43
10.15
10.43
level on
35.80
36.04
34.40
31.16
26.40
31.16
35.91
29.50
25.93
36.53
37.47
36.56
39
27
29
35
24
27
.45
.83
.74
.56
.12
.91
semi-continuous operation of S.
1.25
2.55
0.68
0.33
0.53
0.20
fragilis
fermentation
Lactose
1
1A
IB
2
2A
W-l
W-3
W-5
W-2
W-4
2B
2C
2D
2E
2P
W-l
W-2
w-3
W-4]
5
-6
Sub-1 1
Sub -2 J,
10-11
Sub-1
Sub-2
Sub-3
| Sub-4
> Sub-5
» Sub-6 N





v
8?
c
12
10
11
10
9^F
•• i
12
0.98
1.11
0.92
1.26
1.15
1.37
1.45
1.40
1.44
1.40

5.48

9.87
9.12





46.53

37.48
39.91





40.00

34.14
33.16





56

54
50





.04

.00
.63




1.42
1.53
1.76
2.86
3.38
4.40
3.93
4.30
4.85^
(0.65

-------
Table 1. (cont )
Experiments
(16)






(17)





CHO
5-6*





10-11





#16
AIR
50*
60
80
30
40
0
50
60
80
30
40
0
and #17. Effect
RPM
200
300
350
100
150
50
200
300
350
100
150
50
10) 4.1
10%) 1.19
10*A5 1.31
10) 0.73
10
10
13
11 '
10
1?
1 9
12
0.88
0.16
1.24
1.70
1.23
0.6?
0.89
0.15
of aeration

6.93
7.51
7.70
7.15
7.44
6.61
9.08
9v34
9.04
8.36
8.92
-
conditions

37.80
38.21
38.96
37.48
38.57
39.63
33.04
32.12
33.18
35.88
33.63

at 2

35.91
37.27
41.48
34.16
37.07
31.47
34.83
32.9
39.51
33.72
32.70

carbohydrate

36.25
42.06
41.56
29.72
26.63
13.6
30.2
36.02
35.47
21.56
25.34

levels

2
1
1
3
3

4
3
2
4
4
2

.28
.76
.36
.26
.26
.65
.02
.26
.68
.55
.17
.48

-------
        Figure 1,
Growth of S. fragills at different
inoculum levels under carbohydrate
feed conditions maintaining $-<  "
OHO in the broth.
1 x 10
1 x 10
      8
       0
                             Time (Hours)
                          18

-------
period Indicated that doubling time decreased as inoculum
size decreased.  Thus, Inoculum at 44#, 25#, 15#, 10#, 5# had
doubling times of 100, 86, 55, 50 and 35 minutes, respectively.
Extrapolating from these growth rates indicated that normal
plant inoculum levels of 10-20 percent would give the cell
yields desired (>10^/ml) In a 8 hour period or 3 batches per
24 hour period.  Subsequent experiments without maintenance
of 5-6# carbohydrate gave no evidence of inhibition due to
excess whey when the 10$ inoculum level was adopted for routine
use.  Whey inhibition was not observed with S_. lactls (Figure
2)»  S_» laetis also responded differently in initial growth
rate.  A lag phase in growth of S_. lactia was obvious.
The effects of yeast supplementation (Amber BYP) and different
sources and levels of N and P were Investigated (Expt's 6-14,
Table l).  Several possible trends were extrapolated from the
data.  First "As Is" proteins appeared higher when higher
levels of N were included in the medium.  When NHjjOH was used
as source of N, the percent conversion of lactose to yeast
cellular material appeared higher than when (Nlty^SO^ was used
at equivalent N levels.  With NH^OH as 17 source higher conver-
sions were apparently obtained at the higher N-levels (Expt1 s
8,9,12.13,14).  The higher level,, 0.9# NlfyOH was then used as
SOP for the semicontlnuous and continuous fermentation opera-
tion.
Effftota of variation of aeration and agitation conditions in
                           19

-------
        Figure 2.   Growth of S.  1actIs from various
                   inoculum levels,  grown under
                   conditions of maintenance of 5-
                   GHO in the broth.
1 x 10
1 x 10
 1  x 10
       8
         0
                              Time  (Hours)
                          20

-------
the fermentor were Investigated at standard  (6$)  and high
(11$) carbohydrate levels in the medium with S. fragilis
(Expt's 16 and 17, Table 1).  These data indicate that the
higher aeration conditions £0$ Scale Air &2.3>2 V/V/mln), 200
rpm to 80$ Scale Air, 3f>0 rpm Increase conversion efficiency
and perhaps % protein in the washed product.  They have little
beneficial effect on total solids or cell numbers.  Conditions
below experimental SOP (f>0$ Scale, 2£0 rpm)  result in lower
growth and cell yield.  The 60$ Scale Air, 300 rpm condition
would appear optimum and the proper departure point for scaleup
to larger equipment.
In order to avoid costly fermentor "downtime" in a short cycle
process, the fermentation was tested under semi-continuous
(Expt's 11, 15) and continuous (Expt 18) operating conditions.
In semi-continuous operation the fermentors  were allowed to
run until carbohydrate level reached or dropped below 0.5$.
At this point 90 percent of the beer was pumped into a holding
vessel and an equal amount of fresh medium pumped Into the
fermentor.  In short, 10$ of the fermentor beer was retained
as Inoculum for the next batch.  Continuous  operation of the
fermentation was carried out as described under Materials and
Methods.  Figure 3 provides data for serai-continuous operation
of the process under a high (0.9$ NH^OH) and low (0.1^5$ NH^OH)
nitrogen condition.  Performance for all conditions is for all
practical purposes identical and maximum cell growth is obtained
In all vessels by 10 hours.  In regard to this point, It should
                            21

-------
ro
ro
            Figure 3.  Semioontlnuous operation,  SOP, 0.9$  NH^OH,  S.  fragllis
       10
                                                                                            0
          0  2
                                                 (Hours)

-------
be notea that the trend for  the  Initial vessel  set  in  each
series (2,1,1|,3) was to reach maximum cell growth at 8 hours
while the subcultures were a little  slower.   This was  not due
to initial lag.  Slightly lower  cell levels were reached at
8 hours and the maximum reached  between 8 and 10 hours at a
lower growth rate.  The reason for this is not  obvious, but
there are sufficient differences in  individual  subculture
growth patterns to prohibit  drawing  conclusions until  the re-
producibility of this type of operation is explored further.
The fermentation was observed to operate well in a  three-
stage continuous system.  Data taken on operation of a 60-
hour run are given in Table  2 and Figure 14..  Medium was fed
at a rate of^3 gal/hr into  the  first fermentor.  This rate
is equivalent to 2? gal/9 hr, thus 9 gal/fermentor/9 hr.  This
approximates batch fermentation  time per unit fermentor, but
eliminates the fermentor downtime which would have  to  be taken
in batch fermentation operation. The system  equilibrated 12
                    9
hours after a 1 x 10  cell population was reached and  the
initial dilution effect was  overcome (22 hours).  At equilib-
rium time the equivalent of  Ij. batch  fermentors  had  already
been harvested.  Cell yield  out  of the third  stage  fermentor
                                         Q
was equivalent to batch level, oa. 1 x 10  cells/ml, with only
minor deviation from this level. About 1-1.9$  of ethyl alcohol
were accumulated in the third stage  of this process.   This is
lower than the 2-1$ levels obtained  in batch  or semi-continuous
operation (Table 1).  The effect of  process changes on alcohol

-------
           TABLE 2.
to
Experiment #18.   Continuous operation  of a whey fermentation with
S. fragilis in a 3-stage system

Vessel
Hours of
Operation
AI 0
4.5
6.0
10.0
14.0
iQ.o
21.0
22.0
23.0
31.0
33-0
35.0
37.0
39.0
41.0
43.0
45.o
47.o
49.0
53.0
55.o
59.0
TERM59.7

#1


Stage
#2
Cells/mlxlO0 % Cells/mlxlC
Alcohol
1.26
3.6
7.0
9.7
9.1
4.7
5.o
3.5
fc?
3.8
3.8
3.4
3.2
3.0
2.6
2.9
2.8
3.1
3.5
3.4
4.2
4.8


0.33
0.43
0.54
0.34
0.26
0.14
0.11
0.15
0.20
0.73
-
1.09
1.23
1.55
0.94
3.7
6.5
9.2
9.2
8.6
6,8
6.2
5.7
6.8
6.2
6.3
4.7
5.1
4.2
4-2
3.9
4.6
5.7
5.1
6.5
6.4
7.3


)° %
Alcohol


1.0
1.09
1.18
.91
.77
• 30
• 41
• 45

1.05
-
148
1.83
1.84

#3


Cells/mlxlO0 %
Alcohol
0.81
3.5
5.5
10.5
10.9
12.8
12.8
11.5
13.6
11.4
n.5

11.8
12.0
lk.O
8.3
9.2
8.6
10.6
9.6
11.2
11.5
11.3


1.27
1.45

1.36
1.31
1.30
1.10
1.07
1.05
1.90
•
1.79
1.97
1.97

-------
           Figure U..   Continuous operation of S. fra
IV)
VJl
gills whey fermentation  In  a 3-stage system.

-------
accumulation were not investigated.  Total solids and protein
analyses performed on broths from batch and semi-continuous
operations were not determined on samples from Experiment 18.
Amber Laboratories will pursue single stage continuous opera-
tion and provide chemical and bacteriological data as required
on product quality.

Amino acid analyses were performed on samples from various
experiments.  Data is shown for Experiment 4, in Table 3*
Since less than 30£ of the protein values were recovered as
amino acids some N-containing entity other than amino acid is
present in the broth.  The N-containlng entity could be excess
medium nitrogen that was not incorporated into the cell or
amide by-products from the yeast fermentation.
                           26

-------
              TABLE 3.  Experiment #k.  Amlno Acid Analyses, Inoculum Level Effect
ro
Experimental _,_.., _ _
Conditions *°% Inoculum
10$ Inoculum
25/o I«wuulum As ls Washed Enzyme hydrolyzed
Lysine
Histidlne
Arginine
Aspartic Acid
Threonine
Serine
Glut ami c Acid
Proline
1/2 Cystine
Glyoine
Alanine
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
1.00
0.25
0.36
1.37
0.77
0.79
3.71
1.01
0.15
O.k9
                           0.66
                           0.76
                           0.67
                           2.22
                           0.37
                           0.51
1.09
0.25
O.k2
1.58
0.87
0.85
2.69
1.13
O.Ik
O.kk
0.80
o.7k
0.28
0.61}.
1.27
0.14.1
o.55
Amino Acid %

     1.27
     0.31
     0.51
     1.70
     0.96
     0.93
     3.05
     1.2k.
     0.16
     0.50
     0.93
     0.80
     0.32
     0.71
     I.k3
     O.k8
     0.62
3.22
0.77
I.k8
0.96
O.k8
0.51
1.95
2,16
0.11
0.55
0.69
0.60
0.27
0.58
1.70
0.71*
1.00
                                                                                    3.17
                                                                                    0.77
                                                                                    1.76
                                                                                    3.12
                                                                                    1.61
0.09
1.25
2.0k
l.kl
0.36
1.08
2.31
0.92
1.26
                          16.
                                         . 15
                             15.92
                       17.77
                               29.12

-------
                       SECTION VI
                      INTRODUCTION
                   AMBER LABORATORIES
The primary goal of the fermentation studies was to develop a
method for the production of low cost, single cell protein
(SCP).  The SCP was produced by Saccharomyces fragilis grown
on lactose from whey and to be used for animal feed supplemen-
tation.  The specific objective of the yeast fermentation
studies conducted in the Demonstration Pilot Plant was to
scale-up a process Standard-Operating-Procedure (SOP) devel-
oped by the subcontractor, International Minerals Corporation
(IMC).
The preliminary study conducted by IMC using shake flasks and
pilot fermentors produced several conclusions and SOP's for
the Amber Laboratories studies.  The most encouraging rec-
ommendation indicated that the whey fermentation process could
be run under non-sterile conditions.  In addition preliminary
results indicated that operation of the process in a continuous
manner might be feasible.

However, the detailed SOP for the continuous process, Includ-
ing product quality, yield and cost estimations required
study at Amber Laboratories.  The following experiments were
conducted to demonstrate a method for simple and economical
conversion of lactose from whey into a high quality feed
supplement.
                           28

-------
                      SECTION VII
                 MATERIALS AND METHODS
                   AMBER LABORATORIES
Saccharomyces fragilis (NURL, Y-1109) was used in the majority
of experiments except for two preliminary studies involving
Saccharomyces lactis (NURL, Y-1140).  Morphologically S_. lactis
was smaller in size than S.. fragilis and it was found that the
fermentation broths of ,S. lactis did not separate efficiently
in commercial sized, yeast separators.  Therefore, S_. lactis
was not included in further studies.

In general, concentrated, acid whey was obtained from the
manufacture of cream cheese and condensed to 45-50$ total
solids (T.S.).  Acid whey from cream and cottage cheese has pH
values in the range of 4.2-4.9 in comparison to sweet whey
from Cheddar, Italian, and Swiss cheese with pH values of 5«7-
6.2.  It was found that the organic nitrogen content of cream
cheese whey was lower than other wheys tested.  Wasserman (12)
found that only 25# of the organic nitrogen could be utilized
by S_« fragilis, therefore, we assumed that the fermentation
characteristics of acid and sweet whey would be similar as
long as a slight excess of inorganic nitrogen was added to the
media.
The whey was diluted to appropriate levels of lactose with tap
water and then used to prepare the various media described
below:
                           29

-------
Whey agar was prepared for sterile transfers of S..  lactls  and
S.' fragllls.  Acid whey was diluted to  10# T.S. and the  follow-
ing ingredients added so the media contained (w/v)  0.5#  (Nltyte
304, 0.5$ f^HPOip and 0.3# Amber BYF 100  (see Glossary).   Agar
was added to the whey media at a 4# level and the mixture  of
whey and agar dispensed in suitable aliquots to 20  x  150 mm
test tubes.  The whey agar tubes were pasteurized at  85-90C
for 20 minutes.  Saccharomyces lactis and S.. fFagilia were
aseptically transferred on whey agar slants at weekly inter-
vals.
Two basal media were used throughout these studies.   SOP
Medium-1 was used for inoculum build-up in shake flasks.   SOP
Medlum-2 was used for Inoculum build-up In the New  Brunswick
fenaentors and for studies utilizing a 500 gallon,  pilot fer-
ment or.
One part whey concentrate was diluted with three parts tap
water and the following Ingredients added so the medium  con-
tained (w/v) 0.5# (NH4)2 SOjp 0,5# KgHPOj^ and 0.3#  Amber BYP
100.  The pH was adjusted to 5.5 for Si. lactis and  4.5 for S_.
fragilis with 8556 H^PO^.  The SOP Medlum-1 was not  sterilized
for shake flask use.

Whey concentrate was adjusted to various  lactose concentra-
tions by dilution with tap water.  The diluted whey was made
to contain (w/v) 0.9# NH^OH, 0.3# Amber BYP 100,0.Q5#

-------
and adjusted to pH 5.5  for  S_.  lactls  and  4.5  for J.  fragilla

with 30# HC1.  The medium was  heated  to 800,  held  for 45 min-

utes and cooled.  Deviations in  the SOP Medlum-2 are described

in the Experiment Condition for  each  Experiment Number, (Table

4).


A 500 lb/hour, ultrafiltration (UP) pilot plant manufactured

by Abcor.  Inc. was utilized to fractionate acid or sweet whey

into a protein concentrate  and lactose permeate.  The lactose

permeates  were prepared for the  fermentation  studies by

following  the SOP Medium-2  format.  The permeates were sub-

stituted for diluted whey in preparing the media.  Analyses

of ultrafiltration permeates from both acid and sweet whey

were as follows:

                                      Ultrafiltration
        Gross Composition               Permeate

                                      Average   Range
      % Total Solids                    4      3-5
      % Lactose (dry basis)           88     86-90
      % Protein (dry basis)            4      3-5
      % Ash (dry basis)                8      7-9

The whey permeate was produced from acid  or sweet whey using

the Abcor  ultrafiltration unit (model #UF-8o-S) operated under

the following conditions:


  Gross Composition
     of Raw Whey               Acid Whey       Sweet Whey

pH (as is)                     4.6-4.9         5.7-6.0
% Lactic Acid (as is)          0.50-0.65       0.12-0.19
% Total Solids                 6.0-7.0         5.5-6.5
% Protein  (dry basis)          5.0-9.0        12.5-14.0
% Ash (dry basis)              8.0-12.5        6.0-8.0
                           31

-------
                  Operating Conditions
      Inlet Temp, of Whey              120-124°F
      Inlet Pressure                   55 PSIG
      Outlet Pressure                  25 PSIG
      Flux Rate                        2.0-3,0 gal/hr
      Membrane Surface Area            70 ft2
The closed-loop fermentation studies were made utilizing media
prepared according to the SOP Medium-2 format.  However  coa-
densate water was substituted for tap water to dilute the whey
concentrate.  The condensate water, derived from the evapora-
tion process of fermentation broths, contained up to k% alcohol
(w/v) and less than one part per million nitrogen.
A 500 gallon, fully baffled, deep tank fermentor was used
for the semi-continuous and continuous studies.  The media
were aerated by compressed air entering through a bottom
sparger and agitated by a standard shaft and impeller system.
(See Appendices for photographs of equipment).  Constant
temperature control of the media was obtained by circulation
of water and steam through the jacket of the fermentor.
Three 15-liter New Brunswick fermentors served as the seed
vessels for the 500 gallon fermentor.  The New Brunswick
fermentors were manufactured to standard design and contained
air  agitation  and temperature controls.

Inoculum for the 500 gallon fermentor was prepared according
to the following procedure:
                           32

-------
\f
                        Stock Slant
                                  Wash growth off with 5 ml
                                  medium, Inoculate into:
                    200 ml SOP Medium-1
                                  Shake Flasks-Ambient con-
                                  ditions for 16 hours.  In-
                                  oculate 1,000 ml into:
                 10 liters SOP Medlum-2
                                  New Brunswick fermentor-
                                  10 liters air/rain, 30C.
                                  Inoculate 30 liters into:
               60 gallons SOP Medium-2
                                  500 gallon ferraentor - One
                                  volume air /volume medium,
                                  30 C.  Inoculate 60 gallons
                                  into:
                240 gallons SOP Medlum-2

The semi-continuous fermentations were made by drawing off 9O£
of the fermentation broth and using the remaining 10$ to seed
the next fermentation batch.  The number of consecutive draw-
down batches are indicated in each experiment (Table 4).  The
continuous fermentations were begun when the cell count of the
fermentation broth reached 1 x lOVml and the lactose concen-
tration, 0.50-0.755^ (w/v).  The rate of continuous feed and
removal was maintained at 37 gallons/hour which corresponded
33

-------
to a batch fermentation cycle of about 300 gallons/8 hours
(dilution rate 0.125 hrT ).  The number of hoars the fer-
mentation was maintained continuous Is shown In each experi-
ment (Table 4).
The conditions of operation for the 500 gallon fermentor were
0.5-1.0 volume of air/volume of SOP Medi«ni-2j agitator speed
200 rpra; 30°C; pH 5.5 for S.. lactis and pH 4.5 for S. fragilis.
The fermentor was not sterilized however, the SOP Medlum-2
was heated to 80C for 45 min as described earlier.
Solids determinations were made according to the Mojonnier
Method (?).  Protein was determined in accordance with the
A.O.A.C. Method (l).  A factor of 6.25 x percent - M was used
as recommended for yeast protein.  Ash was determined In
accordance with the A.O.A.C. Method (l).  Lactose concentra-
tion was determined using the Anthrone procedure described
by Umbreit, et al. (9).  Aralno Acid analyses performed by
Wisconsin Alumni Research Foundation using the method des-
cribed by Moore, et al. (8).  Official rat Protein Efficiency
Ration assay performed by Wisconsin Alumni Research Foundation
using the A.O.A.C. procedure (l).  Cell count analyses made
according to International Mineral Corporation Method (3).
Results reported as yeast cells/ml of fermentation broth.

-------
                      SECTION VIII
                 RESULTS AND DISCUSSION
                   AMBER LABORATORIES
The effects of variation in the  type of acid  (phosphoric or
hydrochloric) used to adjust the SOP Medium-2  to pH 4.5 on
yeast cell population, gross composition  of yeast and conver-
sion of lactose to yeast material are  shown in Experiment
Numbers 1-7,  (Table 4).  The use of phosphoric acid to adjust
the pH of the medium prior to and during  fermentation pro-
duced yeasts with ash levels of  35-43$.   The  inclusion of
hydrochloric acid (HCl) in the medium  resulted in a lower ash
content that approximated the 9-13$ ash level  typical of
commercial yeasts.  The use of low cost HCl did not adversely
affect the fermentation and produced a more economical product
than that obtained with phosphoric acid.
The 0 = 9$ (w/v) ammonium hydroxide level recommended by IMC
investigators and used in Experiments  1-7, 15  and 18, (Table
4) as a supplementary nitrogen source, resulted in S_. fragilis
yeasts with crude protein levels that  were abnormally high.
The effects of 0.3-0.7$ (w/v) ammonium hydroxide concentra-
tions on growth of ;S. fragilis are shown  in Experiments 8-13,
(Table 4).  It was found that the crude protein levels approx-
imated commercial yeasts with ammonium hydroxide levels of
0.3-0.7$ and the conversion of lactose to yeast material re-
mained constant with varied ammonium hydroxide levels.  A
comparison of net protein concentrations  (($  crude nitrogen-
                           35

-------
                            Table  U.   Summary  of  Fermentation  Experiments
                                            EPA  Project  S-8007U7
Number
1 (4?





2 (5)
\ ^ f





3 (2)





Experiment
Condition
S. fragilis
Semi- continuous
SOP Medium-2
Phosphoric Acid
Acid Whey
6-300 gal.
Tank Perm.
S. fragilis
"Semi- continuous
SOP Medium-2
Phosphoric Acid
Acid Whey
8-300 gal.
Tank Perm.
S. fragilis
Batch
SOP Medium-2
Acid Whey
1-300 gal.
Tank Perm.
Lactose
Cone.
7-Qfo





7-Qfo






6-7$





Max . Pop .
Cells/ml
X 10V
o.55





o.75






o.3o





T.S.
PWM
7.1





7.9






5.5





fo Dry Basis
Crude
Protein Ash
kl*k





1*8.6






56.8





35.0





lj.2.6






20.9





% Conversion
gm T.S./
gm lactose
0.96





1.01






0.85





UJ
         a
          Numbers  in parenthesis refer to raw data experiments

-------
      Table Ij. (cont.)
Number
M6)




5 (13)





6 (15)





7 (16)




Experiment
Condition
S. fragilia
"Semi-continuous
SOP Medium-2
Acid Whey
7-300 gal.
Tank Perm.
S. fragilis
Continuous
SOP Medium-2
Acid Whey.
Perm. Operated
59 k*1. cont.
S. fragilis
Continuous
SOP Medium-2
Acid Whey
Perm. Operated
90 hr. cont.
_S. fragilis
Continuous
SOP Medium-2
Acid Whey
Perm. Operated
220 hr. oont.
Lactose
Cone.
8-9$




5-6$£





5-6*





5-6^




Max. Pop.
Cells/ml
X 10V
1.52




2.50





2.50





2.2l|




T.S.
PWM
8.2




5.1





5.19



% Dry Basis
Crude
Protein Ash
73.5




76.9





69.7



*

5.89





68.5




15.6




17.2





15.2





17.6




% Conversion
gm T.S./
gm lactose
0.96




0.92





0.99





0.93




U)

-------
      Table l|  (oont.)
Exp.
Number
8 (9)






9 (10)




10 (7)
» * /





Experiment
Condition
S. fragilis
"Semi-continuous
SOP Medium-2
0.7$ NHhOH
Acid Whey
8-300 gal.
Tank Perm.
S. fragilis
Semi-continuous
SOP Medlum-2
Acid Whly
6-300 gal.
Tank Perm.
S. fragilis
"Semi - oo lit" i nuo us
SOP Medium-2
Acid Whiy
8-300 gal.
Tank Perm.
•
Lactose
Cone .
5-6$






8-9$




l|-5$






Max. Pop.
Cells/ml
X 10
1.22






1.57




0.88






T.S.
PWM
5.38






8.3l




U.98






% Dry Basis
Crud e
Protein Ash
66.1






51.7




1|7.9






13.2






13.9




22.8






$ Conversion
gm T.S./
gm Lactose
1.08






0.96




1.15






oo
GO

-------
      Table ij.  (cont.)
Number
11 (8






12 (21






13 (26)





Experiment
Condition
S. fragilis
"Semi-continuous
SOP Medlum-2
0.5$ NB.OH
Acid Whey
10-300 gal.
Tank Perm.
J3. fragilis
Continuous
SOP Medium-2
0.3$ NB.OH
Acid Whly
Perm. Operated
61$ hr. cont.
S. fragilis
Continuous
SOP Medium-2
0.3$ NHLOH
Acid Whty
Perm. Operated
56 hr. cont.
Lactose
Cone.
lj.-5$






lj.-5$






7-8$





Max . Pop .
Cells /ml
X 10 v
1.21






2.17






2.16





T.S.
FWM
k*&






5.13






U.9U





$ Dry Basis
Crude
Protein Ash
5U.2






Ij4.6






32.8





11^.0
i





11.2






25.9





$ Conversion
gm T.S./
gm lactose
0.91






1.11






0.68





U)
VO

-------
Tgble ^ (cont.)
Humber
U (25)






15 (l^)





16 (22)






Experiment
Condition
S. fragilis
Continuous
SOP Medium-2
Acid Whey -
Permeate
0,3$ NHi.OH
Perm. Operated
50 hr. cont.
S. fragilis
"Semi-continuous
SOP Medium-2
Sweet Whey
7-300 gal.
Tank Perm.
j3. fragilis
Continuous
SOP Medium-2
Sweet Whey
0.3$ NHL OH
Perm. Operated
76 hr. cont.
Lactose
Cone .
3-li$






ll- 5$





6-7$






Max. Pop.
Cells/ml
X 10 v
2.20






1.5l|





2.1*3






T.S.
PWM
3. Ok






5.38





5.38






$ Dry Basis
Crude
Protein Ash
51.6






81.0





52.2






16.6






11.8





11^.5






$ Conversion
gm T.S./
gm lactose
0.83






1.26





0.82







-------
Table LJ. (oont.)
Number
17 (24)







18 (12)








19 (27)









Experiment
Condition
S. fragllls
"Continuous
SOP Medium- 2
Sweet Whey-
Permeate
0.3$ NHL OH
Perm. Operated
32 hr. cont.
S. fragllls
"Semi-continuous
SOP Medlum-2
Acid Whey
Closed Loop
Condensate -
lj..l$ w/v
Ethyl Alcohol
5-300 gal.
Tank Perm.
S. fragllls
"Continuous
SOP Medium-2
Acid Whey
0.3$ NHL OH
Closed Loop
^^ ~st
Condensate-0 . o>
w/v Ethyl Alcohol
Perm. Operated
38 hr. cont.
Lactose
Cone.
3-1$







IH*








6-7 fo









Max. Pop.
Cells/ml
X 1
-------
% ammoniacal nitrogen) x 6.25)) was made between the whole
fermented whey mass from a media with 0.3-0.7$ ammonium hy-
droxide and a media with 0.9# ammonium hydroxide.  The net
protein values were 30-35# for both levels of supplemented
nitrogen.  It was apparent that the 0.9# ammonium hydroxide
level did not increase the nitrogen up-take into cellular
protein.  Therefore, ammonium hydroxide levels of 0*3-0.7#
(w/v) were used for production of S>. fragilis yeast on whey.
Although lactose derived from acid whey was used throughout
the studies as the main carbon source for yeast fermentation
experiments were performed using lactose from sweet whey
(Experiments 15-16, & 17, Table 4).  No significant differ-
ences in fermentation characteristics were noted between acid
and sweet whey (compare Experiment 4 (Acid Whey) with Experi-
ment 15 (Sweet Whey) and Experiment 12 (Acid Whey) with Ex-
periment 16 (Sweet Whey)).
A corollary to the fermentation study of acid and sweet whey,
was to study the fermentation characteristics of deproteinated
acid or sweet whey from an UP unit.  The results shown in Ex-
periments 14 and 17, Table 4, indicated that yeast would fer-
ment lactose from an ultraflltration permeate and produce
similar cell counts and crude protein concentrations as yeast
propagated on whole acid or sweet whey.  Under the conditions
of these experiments a number of difficulties were experienced
in using ultrafiltration as a pre-treatment for acid or sweet

-------
whey to be used for fermentation.   The  raw material  used  for
the UP unit was whey  (acid  or  sweet)  having a  solids content
of 5.5-7.0^.  The resultant permeate  was  very  low  in solids
(total solids 3-5#) which would  be  uneconomical  to ferment.
If the solids of the  raw whey  were  raised by evaporation  or
partial dilution of condensed  whey, considerable membrane
fouling and clean-up  problems  were  experienced.  Operation of
the UF unit under these conditions  reduced flux rates by 50-
75#.  In such a situation,  mechanical cleaning (with a sponge
ball) of the membranes was  necessary  which probably  decreased
membrane life.  Moreover, the  inability to adequately sanitize
the UP unit resulted  in extremely high  plate counts  after
completion of each experiment.   Therefore,  It was  concluded
that the use of UP pre-treatment of whey  prior to  fermentation
would be unattractive.
A study of the feasibility  of  using a method for minimizing
waste streams from the fermentation operation was  conducted
in Experiments 18 and 19, (Table 4).  The block diagram des-
cribing the closed-loop system is shown in Figure  5.  A closed
loop design was used  which  consisted  of dilution of  acid whey
concentrate with condensate water derived from the evaporation
process of fermentation broths followed by fermentation of
the whey.  Theoretically, the  closed-loop could be repeated
as often as new whey  concentrate was  added to  the  cycle.
Since the entire condensed  fermentation broths were  spray
dried, no waste streams would  be obtained from the process.

-------
Condensed Acid
Whey @ \£>% T.S.
                                  Diluted Whey
                                Medium @ 12$ T.S,
                                  Fermentation
                                    Process
Condensate
  Water
                                   Evaporation
                                     Process
                                   Spray Dry
                                    Process
                                    Product
      BLOCK DIAGRAM OF CLOSED-LOOP SYSTEM FOR MINIMIZING
      FERMENTATION EFFLUENTS

-------
The fermentation results were  not  significantly different
from acid whey fermentations using tap water  for dilution
although ethyl alcohol  concentrations as high as 4#  (w/v)
were present in the  condensate water.

Although the emphasis of the studies was to develop  a fermen-
tation method for  production of high quality  feed supplements,
several yeasts were  washed  to  produce food yeasts.   A bland
food yeast was obtained with crude protein concentrations of
52-55# and ash concentrations  of 6-10$.  If the higher grades
of yeast products  are produced, the additional processing
through centrifugation  produced a  supernatant effluent that
would have to be processed  further by some form of waste
treatment.  These  supernatant  streams from the centrifuges
contained 5 day BOD  values  that averaged 10,100 mg/1 (average
for 16 experiments,  range 5100-12,700 mg/l).  The principal
organic materials  in these  streams were ethyl alcohol (average
less than 2# w/v)  and lactose  (average 0.5#).  These
supernates could not be used to dilute Incoming condensed
whey (as in the closed-loop process) because  high concentra-
tions of inorganic salts were  present in the  supernatant
which interfered with the fermentation.  If a centrifuged
product was desired, it was found  that yeast  slurries could
be centrifuged successfully after  partial evaporation (to 15-
\1% T.S.) which reduced supernate  volume substantially with -
out raising the BOD  value (still 10,000 mg/l).  The  BOD value
did not increase because some  of the ethanol  was removed

-------
during partial evaporation.

Throughout the entire study, non-sterile, semi-continuous,
and continuous operation of the fermentation process was dem-
onstrated.  The results shown in Figures 6, 7, and 8 are
typical of the high cell populations (l x 10^ cells/ml) ob-
tained with S. fragilis using the SOP Medium-2.
A comparison of the gross composition and amino adld content
of several single-cell proteins is presented in Table 5«
Another comparison of the amino acid composition of several
yeasts, whole wheat, and the FAO (PAO Committee, World Health
Organization, United Nations) profile Is shown in Table 6.
The high lysine and threonlne contents of S.. fragilis and
commercial Brewers' yeast and low values for wheat make the
yeasts valuable supplements for cereal diets.  The low con-
tent of sulfur amino acids, especially methionine, in S_.
fragilis and commercial Brewers' yeast is an obvious deficit
for a feed supplement.  It has been reported by Klhlberg (4)
that yeast with added methionine produced a high quality feed
supplement.
Feeding studies with growing rats were conducted on centri-
fuged (commercial, nozzle type, yeast centrifuges) fermented
whey mass (FWM) and whole FWM which was fed as the only
nitrogen source in an otherwise adequate diet.  The results
are reported in Table 7 and plotted in Figure 9.  The Protein
Efficiency Ratio (PER) of centrlfuged, FWM was greater than

-------
             3ELL COUNT

              ACTOSE,
10
  8
0   2

Figure
     6   8  10

6.  Semi-continuous
 0   2  l|
     TIME
Growth of
                                                                           8  10
  6   8  10       0   2   l±   6
(Hours)
Saooharomyoes fragllis using SOP Medlum-2.

-------
(DO
                   0
u
                               10         20          30
                                            TIME (Hours)
                     Figure ?,  Continuous Growth of Saooharomyoes fragllis Using  SOP
                                Medium-2 (£9 Hr.).

-------
•IS-
vo
                 0  It
                         20
60
80
160   180   200   220
                             100   120
                             TIME  (Hours)
Figure 8.  Continuous Growth of .Sacoharomyoes  fragllis Using SOP
           Medium-2 (220 Hr.).

-------
                              Table 5.  Comparison of Gross Composition and
                           Amlno Acid Content of Various Single-Cell Proteins
VJI
o

Typical Analysis
Protein
Pat
Ash
Moisture
Amlno Acid
Lysine
Threonine
Methionine
Valine
Leucine
Isoleucine
Tyros ine
Phenylalanine
Tryptophan
Esso
Yeast (10)

54.0
10.0
10.0
a
7.0
3.9
1.2
4.0
5.9
3.6
a
3.7
0.5
Wheast (5)

57.2
1.1
8.5
5.0
?
7.4
5.2
1.5
6.3
7.6
5.2
3.1
3.7
1.4
Brewers
Yeast
% (As Is)
44.9
0.7
6.9
3.0
i of Total 1
6.8
5.9
1.5
4.7
5.8
3.6
2.7
3.4
1.1
Torula
Yeast

47.0
1.2
6.9
5.0
!»T»ot:e1n
8.5
5.1
1.5
5.6
8.0
6.4
4.3
5.1
1.1
S. fragilis
FWM

44.6
1.1
11.2
3.5
6.9
5.8
1.9
5.4
6.1
4.0
2.4
2.8
1.0





b(8.8)
(5.5)
(1.5)
(6.6)
(9.9)
(5.5)
a
(3.9)
(1.5)
           a Not Reported
           b Dried Pragllis Yeast, Standard Brands Inc., 1967  (6)

-------
       Table 6.  Amino Acid  Content  of Whole Wheat,
Commercial Brewers
fragilis Yeast
PAO
Amino Acid (7) Profile


Lysine 14.. 2
Threonine 2.8
Methionine 2.2
Valine l±.2
Leucine [4.. 8
Isoleucine i|.2
Tyro sine 2.8
Phenylalanine 2.8
Tryptophan 1.4
Histidine
Arginine
Aspartic Acid

Serine
Glut ami c Acid
Proline
Glycine
Alanine

Cysteine 2.0
Yeast and Saocharomyoes
compared to PAO Profile
Whole Brewers S.
Wheat (4) Yeast
_ df, n-P Tn-l-nT Pv»n4-nii-i ___ .

2.8 6.8
2.9 5.9
1.5 1-5
If. 4 l|..7
6.7 5-8
3.3 3.6
2.7
if.5 34
1.1 1*1
2.1
74
8.3
\ i i
T 4«-'
12.1
U.2
3.9
5-7
a
t \
7
fragilis
Expt. £5


6.9
5-7
1.9
54
6.1
l±.Q
24
2.8
1.0
2.1
3.7
8.6
• ,
M-.
134
if.i
3.6
5.7
a

a
  Not Reported
                            51

-------
 Table 7.  Protein Efficiency Ratio (PER) Assays
of Several Typical
Sample
Fermented Whey Mass
Experiment #16
Centrlfuged,
Fermented Whey Mass
Experiment #12
ANRC Casein

Sample
Fermented Whey Mass
Experiment #16
Centrlfuged,
Fermented Whey Mass
Experiment #12
ANRC Casein


Sample
Fermented Whey Mass
Experiment #16
Centrifuged,
Fermented Whey Mass
Experiment #12
ANRC Casein
Saccharomyces fragilis Yeasts.
Average
1 Week 2 week
3 15

18 25

20 24
Average
1 week 2 week
3-6 7-1

5-2 7-7

5-4 7.7
Average

As is
1.97

2.59

2.86
Weight Gain - gm
3 week 4 week
18 17

22 27

25 31
Protein Consumed
3 week 4 week
8.1 8.1

10-5 12 1

9-7 12.2
Total
53

92

100
- gm
Total
26.9

35 5

35-0
PER Value - 4 week
1
Corrected
1.72

2.26

2.50







Corrected to 2.50 as PER value of Casein
(as is value) x (2-50/2.86) = Corrected

-------
100
 90
                AKRC Casein
                Centrlfuged, Fermented
                  Whey Mass
                Fermented Whey Mass
    Figure  9.
1           2  Weeks
 Rat Growth Rates on Casein and Fermented
 Whey Products.
                           53

-------
    of the PER value for the standard casein (ANRC-Animal
Nutrition Research Council).  Thus, it appeared that £3. fragllis
yeast was utilized efficiently by the animal without methlonine
supplementation.  However, further studies are necessary with
each animal in question to estimate the true effectiveness of
§.- fragilis single cell protein as a feed supplement.
Prom the data derived in the pilot plant operations, it is
possible to calculate preliminary costs of producing yeast for
food or feed purposes by the fermentation of whey.  Certain
assumptions must be made as to substrate cost, size of equip-
ment, hours of operation, and capital investment.  However,
from these assumptions, it is possible to examine the commer-
cial feasibility of such a process.
Prom the experimental results in the 500 gal fermentor, run
continuously for extended periods of time, the conversion rate
is 0.9 to 1.0 pounds of solids in the whole fermented whey
mass (PWM) per pound of lactose in the original whey.  Since
the lactose content of the whey solids is approximately 75#
one would therefore realize 0.68 to 0.75 pounds of PWM (dry
basis) from each pound of whey solids in the original medium.
Assuming fermentation vessels of various size operated con-
tinuously, as in the pilot plant, and using a 300 day produc-
tion year, the production capacities of different size commer-
cial operations may be seen in Table 8.

-------
Table 8,  Annual Production  of Fermented Whey Mass  Solids vs
                           Fermentor Size
Size of Fermentor                 Fermented Whey Mass  Solids
    (gal Ions)                      	(Tons/yr)	
      5,000                                    900
     10,000                                  1,800
     20,000                                  3,600
     30,000                                  5,*00
     40,000                                  7,200
 The cost of the medium for the fermentation Is based on  a  10-
 12J* total solids level and will be primarily determined  by
 the price of whey.  While it is not difficult to foresee a
 time when this material will have a negative value due to  the
 necessary cost of waste treatment and pollution abatement, at
 the present time it may be possible to obtain the needed whey
 for no cost or for fche cost of transportation only.  Using a
 cost of whey of zero as a low value and 2|/pound solids  as a
 high value, medium costs will be in the range of 2.5-5.7^/
 pound of product.
 The cost of production of FWM material is directly affected
 by the size of the operation.  The cost of labor and opera-
 tion decreases on a per pound, finished product basis as the
                             55

-------
size of the equipment and Its degree of sophistication in-
creases.  However, as the capacity increases so does the
capital investment with its connected charges for deprecia-
tion, taxes, insurance and physical facilities.

A plant capable of an annual production of 4,000 to 10,000
tons per year would cost 5 to 15 million dollars depending on
the design.  A plant this size is considered a small fermenta-
tion plant, in light of those being designed for the produc-
tion of single cell protein from hydrocarbons (100,000 ton,
annual capacity).

The operating costs of a yeast-whey fermentation, including
those for utilities, power, aeration, recovery and processing
are lower than other processes for producing similar materials
from other substrates.  The aeration requirements for the fer-
mentation of hydrocarbons is 2.5 to 5.0 times that of the
fermentation of a carbohydrate such as lactose (10).  Because
of the solubility of the whey substrate, agitation and power
needs are lower, as Is the amount of cooling.  A cost compar-
ison between acid whey, hydrocarbon, and cellulose fermenta-
tions Is shown in Table 9.  The basis of the production
capacities are 100,000 tons per year for the products from
hydrocarbons or cellulose raw material and 4,000-8,000 tons
per year for PWM from whey.
                           56

-------
      Table 9.  Production Cost Estimates of Various Single-Cell Proteins
                                   Acid Whey     Hydrocarbon (10)    Cellulose (2)
                                              Annual Capacity (Tons)
                                  4,000-8,000       100,000
                     100,000
     Type of Cost
Medium
Operating & Utilities
   Aeration & Agitation
   Cooling
   Recovery
   Dryi ng
   Other
Capital Investment
Labor
Total
Cents per pound
2.





2.

9.
5-5.7
o.5o
0.50
0.30
0.90
0.60
0-4.0
1.90
2- 14.4
2.0-4.0
1.25-2.00
1.00
0.25-0.50
0.40-0.60
0.20-0.40
1.20-2.10
0.60
6.9-11.2
3-0
0.50
0.10
0.20
0.50
0.30
1.60
0.60
6.8

-------
                       SECTION IX
                        SUMMARY
                   AMBER LABORATORIES
The experimental data and experience obtained operating the
demonstration pilot plant over an extended period of time,
leads to the following observations:

Saccharomycea fragllls may be grown on an acid (or sweet) whey
medium In a continuous, deep tank, aerated, fermentor.  While
similar fermentations have been described and demonstrated In
the literature for some time, this study has shown that varia-
tions In fermentation conditions, strain selection and medium
composition produced cell counts of several billion cells per
millillter, that may be maintained for extended periods of
time.

The fermentation Itself has many advantages easily recognized
by the experienced Investigator.  By operating at a low pH
(4.5) and with a large seed size and a high cell count, con-
tamination Is no problem and therefore, sterile or special
aseptic equipment or techniques are not necessary.  The aera-
tion requirements are not excessive with adequate agitation
and efficient baffling, nor Is there any problem In foam con-
trol.  Wang (10) reported that hydrocarbon fermentations re-
quired 2.5-5.0 times the amount of oxygen as a carbohydrate
fermentation.  Temperature control, despite the rapid growth
                     *f
rate, was surprisingly easy and a low level of cooling water
was needed.  The medium is simple In composition and, at the
                           58

-------
concentrations tested,  the  carbohydrate  (lactose)  Is  completely
soluble.  The absence of  potential  toxic substances in the
medium eliminates the necessity  of  harvesting  the  cells by
centrifugation.  Thus the production  of  a dried whole fer-
mented mass precludes additional processing  of waste  streams
from yeast separators and increases the  yield  of the  fermenta-
tion.  As a result  of the evaporation process  of the whole fer-
mented mass prior to drying,  condensate  water  is obtained that
can be used to dilute Incoming condensed whey  and  thereby
operate a completely closed system  with  no effluents.

However, if a high  protein,  food grade yeast is desired, the
cells may be harvested  from the  medium by centrif ugation.
The yeast cells  are sufficiently large for efficient  centrif-
ugatlon on standard yeast separators.

These products may  be obtained from the  fermentation  of whey
and are described as follows: A dried whole fermented whey
mass  (PWM), a dried cream obtained  from  the  centrifugation
process of the FWM, and a dried  cream from centrifuged,
washed, PWM.  A  dried whole PWM  would make a good  feed Ingre-
dient.  It has a crude  protein of 40-5<#, a  light  color, a
free flowing characteristic and  a pleasant "dairy" odor.  The
protein shows an excellent  amino acid profile, high in lysine,
although somewhat low in  the sulfur containing amino  acids.
The quality, as  indicated by PER determined  in rat feeding
                           59

-------
tests, is good (70% of casein PER) although it is much better
if the fermented whey mass is centrifuged (90% of casein PER)*

Actual feeding tests with animals would have to be run to
determine its true value as a feed ingredient.  Saccharomyces
fragilis is an accepted, non-toxic material and may be used
in feed formulations.

When the cells are harvested by centrifugation and the creams
dried or when the creams are further washed and centrifuged,
two superior products are obtained.  The dried creams have a
lower ash content than the dried FWM and a PER almost equiv-
alent to casein (PER for centrifuged FWto-2.26; PER for casein
2.50).  The uses for such a product may be in specialty feeds
that can pay for the added cost of processing.  The washed and
dried cells, give an excellent food grade yeast that compares
favorably with the protein and ash levels (If7% protein and 1%
ash) of Torula and Debittered Brewers Yeasts now being sold
commercially.

The economics of the fermentation is dependent on many factors
and should compare favorably with other procedures for the pro-
duction of single cell protein*  By beginning with a soluble,
inexpensive carbohydrate source (lactose in whey) many distinct
advantages should be recognized over those processes that  use
hydrocarbons as the carbon substrate.   A few of the advantages
are lactose is more soluble than hydrocarbons; lactose
                           60

-------
fermentations require  1/3  to  1/2 the amount of oxygen necessary
for hydrocarbon fermentation;  lactose fermentation broths do
not require separation or  solvent removal; lactose fermenta-
tions require less agitation  and cooling than hydrocarbon
fermentation*  In addition the ease of processing and its
present acceptability  as a feed and food ingredient are
valuable considerations.

The cost of production of  such an ingredient is primarily de-
pendent on the cost of whey and the capital investment.
Obviously tremendous amounts  of excess whey are available in
this country.  The figures are well known and do not  need to
be repeated.  Excess whey  must be removed as a contaminant or
a pollution source from our environment and in one way or
another the cheese producer will be required to pay the expense
of pollution abatement.  For  these reasons the cost of whey
to a whey processor will be low or non-existent.   At  the pre-
sent time, there are a number  of cheese producers who pay
others (either whey processors or municipalities) to  dispose
of their excess whey.  Therefore, the cost of whey should be
realistically set at zero  and possibly a negative value in
the future.
Another factor that will determine the cost of the final pro-
duct is the capital investment required.  To be truly efficient
and competitive, a sophisticated plant is required with a
large fermentation capacity.  A plant that produces 3,600 tons
                           61

-------
of material annually would require an investment of over J>
million dollars*  The production of the product in the nec-
essary amounts to be competitive, would require a plant with
an annual capacity of 7*000 to 10,000 tons and an investment
of 10-15 million dollars.
The economics of the market place will ultimately determine
the industrial feasibility of this process.  Several factors
favor the use of such an ingredient in animal feeds and may
be listed as follows:  The shortage of high quality protein,
the high cost and continued shortage of feed grade non-fat
dried milk, and the constant reformulation and special
vitamin requirements of the new high energy feeds.  The need
for such a valuable feed ingredient will continue in the
foreseeable future and should increase*  The use of the des-
cribed process should produce material to fill this need as
well as provide a useful outlet for a potential pollutant.
                          62

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                       SECTION X
                    ACKNOWLEDGMENTS
The support of the  sub-contractor, International Minerals
Corporation, and  In particular Dr. Ralph Anderson, Dr. Martin
Rogoff, and Mr. Doug  Slsson  is acknowledged with sincere
thanks.

The following personnel  are  acknowledged for  their valuable
assistance In performing the laboratory and pilot plant
studies at Amber  Laboratories, Mrs.  Leslie Oberts, Mr. Oliver
Justman,  and Mr.  Percy Love, who supported the work directed
by Dr. Tom Everson  and Dr. Sheldon Bernstein.
The support of  the  project by the Environmental Protection
Agency, and in  particular Mr. Kenneth Dostal, the Grant
Project Officer Is  acknowledged with,sincere  thanks.
                             63

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                       SECTION XI
 1.  Association of Official Analytical Chemists.  1970.
    Official Methods of Analysis,  llth Edition.  Published
    by the Association, Washington, D.C.

 2.  Calllhan, C.D. and Dunlop C.E.  1969.  The economics of
    Microblal proteins produced from cellulostic wastes.
    Louisiana State University.

 3.  International Mineral Corporation.  1972.  Cell count
    analyses using hemocytometer .  IMC Plaza, Llbertyvllle,
    Illinois.

 4.  Kihlberg.   1972.  The microbe as a source of food, p 437
    in Annual Review of Microbiology, C.E. Clifton, Raff el,
    S.,  and Starr, M.P., Ed., Vol. 26, Annual Reviews Inc.,
    Palo Alto,  California.

 5.  Knudsen Milk Products Co.  1970.  Analysis of yeast
    products.   Knudsen Milk Products, 715 No. Divisadero,
    Visa 1 la, California.

 6.  Mateles R.I. and Tannenbaum S.R.  1968.  Single-Cell
    Protein.  The M.I.T. Press.   Massachusetts Institute of
    Technology, Cambridge, Massachusetts, and London, England.

 7.  Mojonnler Bros. C.  1925.  Instruction Manual for setting
    up and  operating the Mojonnler milk tester. Bull. 101.
    Mojonnier Bros. Co., Chicago, Illinois.

 8.  Moore,  Stein and Spackman.   1958.  Analytical Chemistry
    30:1190-1206.

 9.  Umbereit, Burris and Stauffer.  1957.  Manoraetric Tech-
    niques.  Burgess Publishing Company, 426 S. Sixth St.,
    Minneapolis 15, Minnesota.

10.  Wang, I.C.  1968.  Proteins  from petroleum.  Che*.
    Engineering.  75:99-108.
11.  Wasserman, A.E.  1960.   Whey Utilization. II Oxygen re-
     quirements of Saecharomyces  frag 11 is  growing in whey
     med ium .  Appl . Microblol 5:291-293 •
12.  Wasserman, A.E.  I960.   Whey utilization.  IV.  Availability
     of Whey Nitrogen for The Growth of Saecharomyces fragilis.
     J. Dairy Sci. 43:123-34.
                           64

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                       SECTION XII

                        GLOSSARY

        y " The Product remaining after removal of caaein and
fat from cream in the  process  of making cream cheese, or from
skim milk in the process  of making  cottage cheese.

Amber BYF 100 - Autolyzed yeast fraction: Spray dried yeast
supernatant.

Amber BYF ^00 - Autolyzed yeast fraction: Spray dried yeast
cream.

A.N.R.C. Casein - A  standardized casein product adopted for
use by Animal Nutrition Research Council for Official Associa-
tion of Analytical Chemists rat P.E.R. assays.

B.O.D. - Five Dav -  Biological Oxygen Demand: Procedure per-
formed in accordance with methods for the Examination of Water
and Wastewater, 13th Ed., 1971, pp. 489-495.

PD-82 Hodag Antifoam - Fermentation defoamer from Hodag
Chemical Corporation,  Chicago, Illinois.

Medium 5102A - A whey  medium used for production of Sac-
charomyces fragilis  or S.. lactis yeast.  Medium consisted of
whey with a lactose  concentration of 6%; 0.5% (NHj,)2 SOh (w/v)
            (w/v) and  0.1$ Amber BYF 300.
Medium 5102B - A whey medium used for production of Sac-
charomyces fragilis or S_.  lactis yeast .  Medium consisted of
whey with a lactose concentration of 6%; 0.9% NItyOH (w/v); 0
Amber BYF 300; 0.001# FD-82 Hodag antifoam.

N.U.R.L., Y-1140 - Northern Utilization Research Laboratory,
Strain Y-1140, Saccharomyces lactis.

N.U.R.L., Y-1109 - Northern Utilization Research Laboratory,
Strain Y-1109, Saccharomyces fragilis .

Protein Efficiency Ratio - Assay Method used to determine
efficiency of utilization  of nitrogen by rats.  Single-cell
Protein fed to growing rats as only nitrogen source in an
otherwise adequate diet.

Scale Air - Arbitrary method of measuring the amount of com-
pressed air injected into  the fermentation vessel.

S.O.P. Medium-1 - A whey medium used for production of
Saccharomyces fragilis of  S_. lactis yeast, medium used for
                           65

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yeast studies in shake flasks and consisted of whey with a
solids content; 0.5# (NHii)2 SOh  (w/v); 0.5# KoHPCii (w/v); and
0.3# Amber BYP 100 (w/v).

S.O.P. Medium - 2 - A whey medium used for production  of
Saccharomyces fragilis or S_. lactis yeast.  Medium used for
yeast studies in New Brunswick and 500 gallon fermentors and
consisted of whey with a 10# solids content: 0.9# NHhOH (w/v);
0.3# Amber BYF 100 (w/v), 0.05# HoPOj, (w/v); and sufficient
30^ HC1 to adjust pH to 5.5 for S_. lactis and to 4,5 for S..
fragilis fermentation.

Sweet Whey - The product remaining after removal of casein
and fat from milk in the process of making Italian, Swiss and
Cheddar Cheese.
                           66

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                     SECTION  XIII

                      APPENDICES

Title:    Whey Fermentation     Investigators:   M. Rogoff
                                                 N. Janosko
                                                 D. Sisson

Project:  Contract Research     Location:      Libertwille
          -5000

Cost
Center:   Microbiology  )  0741  Period:        Oct-Dec 1971

Objective:

Develop an economic process to convert whey lactose to yeast
protein without deproteinlzing the whey.  A high quality pro-
tein feed supplement is the desired product.

Background

Processes based on utilization of whey lactose by yeasts on
deproteinized or whole whey substrates have been previously
described (l).  Some result in rather efficient utilization
of whey lactose, e.g. the process described by Wasserman
while others are apparently less so.  Among such processes
in the latter category are the "Wheast" process (2), that of
Metwally et al (3) and that of Amundson (4).  If Wasserman's
24 g/litre yield of yeast is  taken as approaching the
theoretically feasible yield  of yeast (55% weight conversion
from lactose) then the other  processes might be ranked Wheast,
Metwally and Amundson in  decreasing order of efficiency.

The more efficient processes  in terms of lactose conversion
are characterized by a short  cycle time, 6-8 hours, and a
single doubling of the yeast  cells in the fermentor.  This
type of process requires  a 50# inoculum to the fermentor.
Use of such a high Inoculum level has several drawbacks in
operation of an economical fermentation process as follows;

l) The seed vessel must be at least half the size of the
   fermentor.

2) Inoculum to the seed vessel must be sufficient to complete
   seed development in a  time s fermentor cycle time.  Unless
   this criterion is met, more than one seed vessel per fer-
   mentor or a semicontinuous seed operation is required.

3) The entire seed train  must be high volume or long in dura-
   tion to obtain the required number of cells.
                            67

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4) If operated as In the "Wheast" process requires a centrl-
   fuged seed which constitutes an additional operating cost
   and is equivalent to recovering half the volume of the
   ferment or twice.

5) The economics of operating with the fermentation /turnaround
   time ratio very low may be unfavorable.
The feasibility of operating a process based on a 5<  inoculum
level is. minimally, questionable, and not representative of
normal fermentation operations.  The latter are usually
operated at a nominal 15# maximum inoculum level.
The present study was undertaken in order to demonstrate
operation of a whey lactose to yeast conversion process which
would be commercially feasible and reproducible for ready
scaleup.

Status at Beginning of Quarter

Project initiated this quarter.

Progress During Quarter

Shake flask studies were completed for determination of the
following:

1. Strain.  Six yeast strains were screened for growth on
   diluted whey concentrate.  Two cultures Saecaromyces
   lactia NRRL Y-1140 (S^ lactis H. ) and Si. fragilis NRRL Y-
   1109 (£[. fragilis W) were retained for scaleup to pilot
   plant fermentors.

2. Medium.  Effect of N-level and Yeast Extract additions
   were tested.  SOP for these to FPP was 0.5 and 0.1#, re-
   spectively.

3> Effect of carbohydrate level.  Serial feeding of lactose
   was tested to determine whether sugar was limiting in the
   system.  An indication of increased cell numbers was
   obtained by pure lactose additions either in the batch or
   by serial feeding.

4. Growth rate.  Generation times were examined under several
   conditions.  It was found that at 10# inoculum levels one
   log increase could be obtained in 8 hours to yield maximum
   population obtainable on or 5# lactose in flasks.  These
   data indicated feasibility of an 8 hour fermentation time
   in larger equipment thus 3 cycles/24 hour period.
                           68

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Results and Discussion

Methods.  Shake  flask experiments were  carried out as follows:

l) Stock cultures were maintained on whey agar slants (5%
   lactose) pH 5.0  and transferred  at weekly  intervals.

2) Inocula were  prepared by washing the growth from a 24 hour
   whey agar  slant  into  5 ml  of whey medium and inoculating
   1.5 ml into 50 ml of  whey  medium in  a 250  ml flask.  Flasks
   were incubated on a rotary shaker at 270 rpm at 30 C.
   Media were not sterilized  in any of  the procedures used
   with the exception of whey agar  for  stock  slants.

3) Shake flasks  were charged  with 90 ml of whey medium pre-
   pared as follows: whey concentrate diluted to  5# lactose
   (or as indicated in individual experiments) and 0.5#
   (NHu)2SO&, 0.5#  K2HPOU and o.l#  yeast extract added; pH
   as infiictted  in  individual experiments.  Flasks were in-
   oculated with 10 ml of 16  hour growth from suitable in-
   oculum flasks.   All Incubations  were at 270 rpm, 30 C.
   Optimal pH for T. lactoaa  was 3.5- S. lactis 5.5 and S_.
   fragilis 4.5.  These  pHfs  were used  for the respective
   cultures.

4) Analytical,   a)  Cell  count, microscopically, by standard
   dilution technique in a blood counting hemocytometer,
   b) Cell pack  - 10 ml  of broth were spun down in volumetric
   tapered centrifuge tubes for 30  minutes at 3000 rpm.
   Volumes were  read directly.

5) Reducing sugar (lactose) values  were determined by a
   standard Technicon Autoanalyzer  colorimetric method.

6) Dry weights were determined by shell freeze drying of appro-
   priate aliquots  of broth.

Experimental

The shake flask  experiments described bel-ow were set up
following initial pure culture isolation of strains from in-
coming cultures  and preliminary demonstrations of the ability
of raw diluted whey concentrate to  support growth.  The first
five experiments were carried out using all six strains of
yeast originally obtained.  These included: Torula lactosa
NRRL Y-196; T. lactosa NRRL Y-1203; Saccharomyces lactis NRRL
Y-1140J S. fragilis ]*RRL Y-1156; S. fragilis  NRRL Y-1109, S..
fragilis""l208 (from T. Everson).  All subsequent experiments
in shake flasks  included only T. lactosa Y-196, S. lactis
Y-1140  (S.. laetis  H) and S.  fragilis Y-1109  (S.fragilis W).
                           69

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Shake flask experiments were designed to Investigate factors
limiting the growth of the yeast.  Following preliminary ex-
perlemnts to determine optimal pH for each test culture a
shake flask standard condition was set.  Carbohydrate level
was investigated first.  Experimental design was to supplement
whey concentrate diluted to a carbohydrate level of 5# with
pure lactose in five percent Increments.  Carbohydrate supple-
mentation was made by either a batch procedure (SF 2,3) or
by a serial feeding of 5 percent increments at 8 hour In-
tervals (SF 4).  An almost direct response to carbohydrate
level in terms of cell numbers obtained was observed for the
T. lactosa. S., lactla H (Figure l).  Response of IS. fragllis
to Increased carbohydrate was not observed consistently
although a response trend might be extrapolated from the data.
Z- lactoaa did not utilize all carbohydrate provided; the
other two cultures evidenced usually less than 1% residual
carbohydrate in the broth.

The effect of nitrogen level and yeast extract supplementation
was investigated in an experiment in which SOP condition was
followed and an additional 5# lactose added to the flasks
after 8 hours to allow full growth potential (SF-5).  Although
full utilization of carbohydrate was obtained no differences
in cell pack volume attributable to Increased N-levels were
observed.  Since all carbohydrate was utilized it was assumed
other factors which might be limiting on the system e.g.
dissolved oxygen, might be repressing a nitrogen-response.
Pursuance of this line of experimentation was accordingly post-
poned until operation in pilot plant fermentors was initiated
and where 02 availability might be less limiting.  Investigation
of P effect was also postponed.

A series of shake flask experiments were next carried out to
determine whether lactose in excess of 5# provided as additional
whey would evoke the same growth response as found on addition
of pure lactose (SF 6-7).  Experimental format used was dilution
of concentrated whey to desired carbohydrate level (5-10#)
with no further sugar addition.  N and Yeast Extract supple-
ment levels were varied.  The results of these experiments in-
dicated that suboptlmum growth was obtained and that Incre-
mental growth response to carbohydrate was not observed.  Pro-
viding additional carbohydrate as whey is apparently Increasing
the amount of some non-carbohydrate material in the medium
to an inhibitory level. Under this apparent inhibition no re-
sponse to adjuncts was observed.  S_, laetla was perhaps more
sensitive to the inhibitory effect than was S_. fragilis.

Generation times for the shake flask fermentation were cal-
culated from cell counts taken on flask populations under
various conditions.  Since it had been observed that carbohy-
drate depletion had taken place usually within 8 hours
                           70

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generation times calculated were  all based on an 8 hour period.
Some of these data are  summarized in Table 1.  Slightly over
3 doublings are required  to bring a 1O#  Inoculum (of maximum
cell growth) to original  cell  levels;  In short one log in-
crease in cell numbers.   Examination of  count data indicated
2.8-3.2 doublings of  populations  of S. lactis and S. fragllis
were obtained In 8 hours.  This information  indicates that
3 fermentor cycles per  day may be feasible.

The data in Table 2 are a general summary of shake flask data
obtained.  Details of Individual  experiments are recorded in
IMC Laboratory Notebook #5587  assigned to M. Janosko.

Future Plans

1.  Initiate Pilot Plant  fermentor studies on shake flask SOP.
    Characterize population response.

2.  Investigate effects of N,  Yeast or Yeast Extract, P and
    carbohydrate levels in fermentors.

3.  Investigate effects of physical parameters, e.g. , 02,
    agitation, on the fermentation.

4.  Investigate effects of Inouculum levels  on yeast yield.
    Investigate effect  of retention of an aliquot of fermentor
    final whole culture as Inoculum on semi-continuous
    operation of the  process.

5.  Initiate characterization  of  yeast or product yields in
    terms of carbohydrate, protein, fat,  ash and moisture
    under various fermentation conditions.   Carry out amino
    acid analysis on  selected  representative product samples.

References

1.  Wasserman, A.E.,  Appl. Microbial.  I960,  8. (5), 291.

2.  Robe, K., Pood Processing, Chicago,  1964, 2£ (2), 95

3.  Metwally, M.E., et  al., J. Dairy Sci., 1964, 4£ (6), 680.

4.  Amundson, C.H., Amer. Dairy Rev.,  1967,  2£ (7), 22.
                            71

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        Effect of Lactose Level on Yeast Cell Count  (SP  3)
  10-
o
tc

•=}•
CVi
a
w
u
  10
    8
                    5              10

                       TOTAL CARBOHYDRATE
15
                          72

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TABLE 1.  Yeast Population Increases During 8 Hour
          Fermentations Under Various Condtitions
Condition
SOP (see text)
SOP
SOP, Whey
adjusted to
[lactose]
= 12.6*
= 10.4
= 7.5
= 6.0
= 10.4
= 7-5
= 6.0
_ i o f-^oL
— J- d. . fJyo
* T.5
* 6.0
Initial
cells/ml
Yeast xlO8
T.
S.
S.
T.
I.
S.
T.
T.
T'.
|
S.
S.
s.
s.

lactosa
lactis
fragllis
lactosa
lactis
fragills
lac toga
lactosa
lactosa
lactosa
lactis
lactis
lactis
lactis
fragilis
fragilis
fragills
0.27
0.96
0.33
0.37
1,1
0.27
0.54
0.54
O ^SM
O ^M
1.2
1.2
1.2
1 2
0.32
0.32
0.32
AVERAGE
T.
S.
S.
Final
cells/ml
xlO65
No.
Doublings
2.04 3.0
10.75 3.4
4.75 3.8
2.3 2.6
17.5 4.0
6.7 4.5
0.85 0.6
1.10 1,0
2.0 1.9
2.15 2.0
7.05 2.5
6.45 2.3
8.35 • 2.8
7.55 2.6
3.1 3-2
4 . 05 3-6
3-25 3.3
4.85 3-9
GENERATION TIME:
lactosa =3-2 hrs
lactis s 2.4 hrs
fragllis=.2.0 hrs
                        73

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TABLE 2.  Summary of Shake Flask Experiments
# Experimental Conditions
1
2
3

4



5



6





SOP
SOP
SOP
SOP
SOP
SOP
SOP
SOP

SOP
SOP
SOP

SOP
SOP


SOP
SOP
SOP

+ 5% lactose )Batch

* ?Llacto.se Isatch
•»• 1055 lactose)
)
* 5% lactose) 5$
serial feed
+ 10?6 lactose)
- As Is H4 Whey )
c" 2x (NHjj^SO^ }
As Is
c 2x Yeast Extract)
9 Con c . whey
adjusted to 1096
CHO
+ 0.4# N .05^6 Y
* 0.5# N 0.196 Y
+ 0.6# N 0.1556 Y
T. lactosa Y-196
Cells
xlO8
2.9
2.0
1.7
2.04
8.5
11.5
2.3

10.95
18.6










Resid
CHO %


3.76
.54
4.20
3.7

4.8
4.3
0.60

0.49
0.54






p*ck#*
ml T**
0.35



.30

.62
.77
.75

.775
.80






S.
Cells
xlO8
10.5
10.2
7.6
10.8
24.0
22.5
17.5

24.7
20.1







5.6


lactis
Resid
CHO %


.37
.40
.44
1.02

.47
.34
0.56

0.40
0.42



8.1
8.9
9.6
#
" Pack*»
ml T
0.55



.36

.64
.64
1.025

1.15
1.1



0.7
.675
0.75
S.
cell
xlO8
4.9
5.2
3.6
4.8
5.9
5.3
4.7

8.5
6.8







2.7


fragllls
s Resid
CHO


.29
.44
3.23
.66

.36
1.58
0.39

0.36
0.51



8.3
9.6
9.25
W*
Pack*«
ml T
0.5



.50

.70
.60
.775

.85
.70



0.6
0.5
0.5

-------
7 Dilution
of
various CHO
CHO 10
10
7
7
6
6
5
5
.0

.5
.5
.0
.0
.0
.0
whey to
levels
M 0.5

0.5

0.5

0.5
M

Y 0

0

0

0


.1

.1
_
.1
_
.1
-

7

6

8

7


.05

.45

.75

.55


6.1
5.8
3.1
2.1
.46
.k8
_
.05

.65
.65
.65
.60
.625
.60
.60
.60

3.1
3.4
4.1
2.8
3.3
3.6
4.9
5.8

6.1
5.9
2.4
2.2
-
•
-
•

.6
.68
.6
.6
.5
.5
.55
.55
          * Minimally  duplicate   standard  triplicate flasks were set for most experimental  conditions,
                 obtained with three  other yeast  cultures in the first five experiments are  not
            included above (see  text).
vn
          ** ml wet  cells/10 ml broth

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Live culture of yeast asceptically transferred from
yeast agar slant to shake flask.
                         76

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Growth of yeast on whey media in shake flasks.
 Growth  of  yeast  on whey media  in New Brunswick fermentors,
                          77

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Top view of 600 gallon fertnentor showing mechanical
foam breaker and yeast/whey fermentation broth.
Broth  from  continuous  yeast  fermentation of  whey.
                          78

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Growth of yeast on whey media in 500 gallon deep tank
fermentor.
Progress of yeast fermentation followed by actual cell
counts.
                         79

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Pilot spray dryer for production of experimental lots
of dried yeast.
 Spray  dried  yeast  from  continuous  growth of yeast  on whey.
                          80

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SELECTED WATER
RESOURCES ABSTRACTS

    ' TRANSACTION FORM
                                                  2.
                                                           3. Accession No.
                                                           w
                                                            at
PROTEIN PRODUCTION PROM ACID WHEY VIA FERMENTATION
 7. Author(s)                                J
 Sheldon Beenstein, Ph.D.; and Thomas  p.  Everson,  Ph.D
   Milbrew Incorporated
   Amber Laboratories Division
   Juneau, WI  53039
                                                           10. Project No.

                                                              S-800747
                                                           11. Contract/Grant No.
                            .tAl, Pro tec tiojo _ Agency;
15. Supplementary3 Notes                         *

   Environmental Protection Agency report number, EPA-660/2-7U-025, May
16  Abstract                                 '
   Prom the operation of a  demonstration pilot plant over extended periods
   of time, it has been shown that yeast may be grown on an acid whey or
   sweet whey medium in a continuous, deep tank aerated fermentor.  Varia-
   tions in fermentation conditions,  strain selection, and medium cpmposi-
   tion produced  cell concentrations  of  several billion cells per milli-
   liter.  Up a process of  evaporation and spray drying the whole fermented
   whey mass and  the Utilization of the  evaporator  condensate to dilute
   Incoming condensed whey,  a high grade, non-toxic,  protein feed material
       be produced without  any effluent  streams.  Amlno acid analyses and
           efficiency wetioff -are pre&j^ed -for thfc* feedr materialv

  Economic estimates show that while": a large capital investment  and low
  cost raw material are required for the commercial,feasibility  of this
  fermentation process, it will be competitive with  other methods for the
  manufacture of singl« cell proteini  This whey fermentation Is one
  means of convertingllarge quantities of a potential environmental
  pollutant Into a useful and  needed! product.
 l?a. Descriptors ^Illdua(:rl£|1 WaataSj *Fennentati
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