Microbioogical

Process Discussion

Calculation of Heat Sterilization Times for Fermentation Media

FRED H. DEINDOERFER1
Merck Sharp & Dohme Research Laboratories, Merck & Co., Inc., Rahway,

New

Jersey

Received for publication September 14, 1956

Sterilization of nutrient media is an operation

essential to all industrial fermentation processes requiring pure culture maintenance. AMethods for sterilizing nutrient media include 1) heating, 2) filtration,
3) irradiations, 4) sonic vibration, and 5) exposure to
chemical agents. Because heating the medium, usually
by steam, is the most reliable and on a large scale the
easiest to control, it is the method of choice throughout
the fermentation industry. The increasing availability
of radioactive isotopes undoubtedly will stimulate
further study of the use of gamma radiation for sterilization. However, a great deal of work is needed before
commerically feasible gamma ray applications will be
used in the fermentation industry. The other methods,
although successful on a laboratory scale, present too
many operational drawbacks for large-scale use in
fermentation processes.
How long should a nutrient medium be exposed at
high temperatures to achieve sterile conditions? This
question arises often and usually is answered in benchscale or pilot plant tests. Improper translation of these
test results with large equipment may subject the
medium to unnecessary overheating. A method by
which minimum exposure time to achieve sterile conditions can be calculated from easily obtainable thermaldeath relationships and the temperature conditions in a
heat sterilization process is presented in this paper. The
method can be used to correlate sterilization conditions
among various sized fermentation vessels. It also permits evaluation of the temperature and retention time
relationship in continuous sterilizers. It need not be
restricted to fermentation processes, but should be
applicable to any process involving a heat sterilization
or pasteurization operation.

Bacterial spores are by far the most heat resistant forms

of microorganisms.
The thermal resistance of bacterial spores is inherently different among species and even among strains
of the same species. Besides thermal resistance inherent
to spores of a particular species, variations can be caused
by a number of environmental factors. These can be
generalized into two groups: 1) environmental factors
affecting sporulation prior to sterilization, and 2)
environmental factors affecting spores during the
sterilizing heat exposure. The most important factors
in nutrient media sterilization fall in the latter category
and include factors such as the pH during sterilization
and the osmotic nature of the media. The presence of
suspended solids also affects sterilization by physically
insulating spores from heat exposure.
Thermal-Death Relationships of Bacterial Spores
Because bacterial spores are the most heat resistant
forms of microorganisms, their germinated cells are the
most frequent contaminants encountered in industrial
fermentations due to improper sterilization. The ensuing
discussion will deal, therefore, with the thermal-death
relationships of these forms. Relationships between the
number of viable spores and exposure time to heat
demonstrate a logarithmic rate of spore viability destruction. Two typical survivor curves for spores of
Bacillus stearothermophilus strain 1518, an organism
often used for sterilization studies in the food industry,
are shown in figures 1A and lB. These curves were
obtained in buffer solutions and would differ from their
respective curves in other media. Spore destruction at
the higher temperature is more than 400 times faster
than at the lower temperature.

Thermal Resistance of Microorganisms

The ability of microorganisms to withstand heat is
much greater than that of other forms of life. Thermophiles capable of even tolerating common heat sterilization conditions, such as exposure to steam at 250 F
for 20 to 30 min, exist. Their occurrence in nutrient
media, however, is rare. Relative resistances of several
microorganisms to moist heat are shown in table 1.

TABLE 1. Relative resistances of microorganisms to sterilization

by moist heat*
Organism

Organism

Escherichia coli .............................

Bacterial spores .............................
Mold spores .................................
Viruses an(d bacteriophages .................

1 Present Address: E. R. Squibb & Sons, New Brunswick,

New Jersey.

221

Rahn (1945).

~~~~~~Relative
Resistance
1

3,000,000
2-10
1-5

F. H. DEINDOERFER

222

The curves in figures IA and lB represent first-order

reactions, where the rate of spore destruction is directly
proportional to the number of surviving spores. This
can be described mathematically as follows:
dN
(1)
dO
where N is the number of surviving spores in the volume
under consideration, 0 is the time of exposure, k is a
is the rate of spore destrucvelocity constant, and dN
dO
tion.
By integrating this equation, it can be modified to a
form that can be used to calculate the time for sterilization at a particular temperature where the velocity
constant, k, is known. Then,

N1
6=2.3l
k
N

spores of a particular species will be a function of

temperature only. It will behave similarly to other velocity constants in its relation with temperature, quantitatively expressed by the empirical Arrhenius equation
as follows:
k = Ae RT

(3)

where A is a proportionality constant, R is the gas

constant, T is the absolute temperature, and ,u is an
apparent activation energy for heat destruction of the
spores. No theoretical significance need be attached to
this equation for this discussion. The conformity of
many destruction rates to the Arrhenius equation
provides sufficient justification for its generalization
here. In logarithmic form, equation 3 may be written
as follows:

(2)

where N1 is the number of particular spores at the start

of the heat exposure and N2 is the number of the same
spores surviving at any time, 0, after exposure has
started. N2 in this relationship can never reach zero,
but practikally this does not matter. For destruction of
spores wAith only one chance in one hundred of failing to
destroy all spores, N2 should be set equal to 0.01.
Higher degrees of confidence result from smaller values
of N2.
In a given environment the velocity constant, k, for

[VOL. 5

logk= - - /+logA

(4)

For all practical purposes, log A may be treated as a

constant.2 Then equation 4 is of the form y = mx + b
2 The significance of the term, log A, is evident from examination of the Eyring rate equation. See Johnson et al. (1954).
It includes an entropy term,
which in cases of spore destruction is quite large. Occasionally, values of AS along with
values of /Aare listed for thermal spore destruction. This completely defines the velocity constant, k, as a function of temperature.

05\

'OS

A)

14

TEMPERATURE 220 F.
REDRAWN FROM DATA
OF BALL (1943)

B)

104

TEMPERATURE 268.7 F.
REDRAWN FROM DATA
\OF STERN AND PROCTOR (1954)

-I

-J
103

\ k .

102

00057

SEC.-'I

k - 0.25

,o

SEC.-'

z
z

TIME

MINUTES

-0-

TIME

MINUTES

FIG. 1. Survivor curves for spores of Bacillus stearothermophilus strain 1518 at two different temperatures

STERILIZATION OF NUTRIENT MEDIA

1957]

particular
species in a given medium should yield a straight line
having a slope equal to 23RT* A typical plot is shown
in figure 2. Such plots are constructed by determining
the velocity constant, k, at at least two temperatures.
If there is doubt that the kinetics of the spore destruction are such that the velocity constants are not related
to temperature as in the Arrhenius equation, more k
values should be determined. The subsequent treatment is not affected by another relationship, as long as
it is known. For spores of B. stearothermophilus strain
1518, characterized in figure 2, the activation energy
for destruction is 67.7 Kcal/mole. Activation energies
and entropies for thermal destruction of other bacterial
spores are listed in table 2.3

and

plot of log k

for

versus

spores

of

223

Injection of steam introduces some dilution to the

medium, but this is taken into account during batch
make-up prior to sterilization.
Batch sterilization conditions are often specified as a
holding period at a certain temperature. The heat
effects involved during the time required to reach the
desired sterilizing temperature and the time required in
cooling down from this temperature are usually neglected. In large-scale equipment, the rising and falling
temperature portions of the heating cycle are much
longer than the constant temperature portion.
Figure 4 compares actual fermentor heating cycles for
various sized vessels. Note that 110 F is the starting
temperature for the heating cycle. Batch make-up using
warm water can save considerable time in the solution

Batch Sterilization

The most common method of heat sterilizing nutrient

media is the batch method. The medium ingredients are
charged directly into the fermentor. The fermentor and
medium are sterilized by heat transferred across the
jacket and/or coil surfaces from condensing steam.
Fermentors are usually of the geometric design shown
in figure 3. The heat transfer surfaces are marked in
figure 3 by heavy lines. The medium is agitated and
often steam is injected directly into the medium
through the air sparger to speed up the sterilization.
3Various other terms commonly are used to describe thermal behavior of bacterial spores during heat sterilization,
especially in the food industry. The terms used in this article
are consistent with those used in chemical reaction kinetics.
Some other terms and their relationship to the velocity constant, k, are tabulated for reference purposes.

-1

I.z
4n

z
0

Fa
3-0

-J

>

1i0

0.00135

Term

Relation

Definition

kT

The ratio of the velocity constant at a

particular temperature to the velocity constant at a temperature ten
degrees lower. This ratio is often
falsely assumed constant over the
entire temperature range. It diminishes as temperature is raised

Qio

klTjo

TDT

Two synonymous terms representing

the time required for 90 per cent destruction of a spore population at a
particular temperature, often called
the decimal reduction time

2.3
k
2.3
k

2 3 log N,

N2

as above

Thermal death time, a term attached

required for "complete"

destruction of spores in a particular
environment. Algebraically, this
term is meaningless unless N2 has
some finite value
to the time

The thermal death time at 250 F

000137

0.00139

0.00141

0.00143

0.00145

RECIPROCAL TEMPERATURE
T

0.00147

0.00149

~~~~~~~R

FIG. 2. Effect of temperature on the velocity constant for

destruction of spores of Bacillus stearothermophiluis strain
1518.
TABLE 2. Activation energies and entropies for first order
degradation of B-complex vitamins and death of bacterial
spores
Energy

Vitamin or Bacterial Spore

Entropy
AS

cal/mole

cal/mole K

Folic acid ............................... 16,800*

........... 21,000*
d-Panthothenyl alcohol .......
Cyanocobalamin ............. ............ 23,100*
Thiamine hydrochloride .................. 26,000*
Bacillus stearothermophilus strain 1518.... 67,700
Putrefactive anaerobe NCA 3679 ..... .... 72,400t
........... 82,l00t
Clostridium botulinum .........

-14.1 t
5. it
2.2t

Garrett (1956).

t Calculated from data of Garrett (1956).

t Levine (1956).

11.4t
105

1234
160t

F. H. DEINDOERFER

224

of batch ingredients and in heating time. If water at

50 F were used in batch make-up it would have taken
an additional 30 min to heat the medium in the 15,000gal fermentor to 110 F. In order to avoid extending the
graphs unduly, 110 F was used as the finishing temperature. Actually, temperatures below 200 F have little
effect on the sterilization.
All the vessels characterized in figure 4 are reasonably
geometrically similar. Because of this congruency, the
heat transfer area per unit volume decreases as vessel
size increases. Also, since the vessels operate at similar
power input per unit volume levels, the heat transfer
coefficient also decreases as vessel size increases. This

[VOL. 5

explains the different shapes of the heating cycles in

figure 4. Coils are added to larger fermentors to provide
additional heat transfer surface. Very often, too, the
amount of steam directly injected into the medium
per unit volume is increased for larger fermentors. Despite this, however, the disadvantages of increased size
are not fully compensated.
Obviously, the rising and falling portions of the
sterilization cycle contribute significantly to fermentor
and medium sterilization, especially in larger vessels. A
method for determining the contribution of these
portions to the sterilization is suggested through the
use of the thermal-death relationships discussed earlier.

RAW INGREDIENTS
WATER
ACID OR BASE

STEAM INILETS

{=

AND
COOLING WATER

EXHAUST AIR OUTLET

(STEAM VENT DURING STERILIZATION)

OUTLEETS

3 STEAM CONDENSATE OUTLETS

AND

3 COOLING WATER INLETS

C
STERILE AIR INLET
($TEAM DURNIN STERILIZATION)

FIG. 3. Geometric design of large-scale fermentors

TimE

MINUTES

TIME

MINUTES

FIG. 4. Temperature rising and falling curves during sterilization of medium in various sized fermentors

225

STERILIZATION OF NUTRIENT MEDIA

The method involves a graphical integration of the
velocity constant over these portions of the cycle. k is a
function of T as shown in figure 2 and described in
equation 3. T is a function of time, 0, as described by the
heating cycle. Therefore, k is also a function of 0. An
average value of k for each portion of the cycle can be
obtained by graphically integrating k over the time
period involved and dividing the integral by the time
represented by the portion, as in equation 5 below.
2

lCv

kavg

k dO

presterilized medium. For how long must the medium

be sterilized at 250 F to be sure of sterilization in 999
out of 1000 batches?
Solution. For sake of illustration, assume the curve
in figure 2 characterizes the most heat resistant bacterial
spores in the penicillin medium. Also, let the heating
cycle of the 15,000-gal fermentor shown in figure 4
represent the heating cycle of the fermentor under
consideration. 220 F is chosen as the minimum lethal
temperature. This is an arbitrary choice, of course.
Actually lower temperatures are lethal but their
relative lethality is small and neglecting them does not
introduce any significant error.
Since the percentage of the laboratory bacterial
count that is contributed by the most heat resistant
spores was not determined, assume that the entire
count was contributed by these spores. This is equivalent to adding a safety factor in the calculation.
Then
N1 = (20 X 106 spores/ml)
(3.78 X 103 ml/gal)(12 X 103 gal)
= 9.07 X 1014 spores
The chance for failure has been set at one in a thousand so that N2 = 0.001 spore.
The velocity constants at the temperatures along the
rising portion of the sterilization cycle in figure 4 are
determined from figure 2. These values are plotted
versus the time corresponding to the temperatures
along the cycle as in figure 5A. The procedure is repeated for the falling portion of the cycle in figure 5B.
Note that the time of each of these portions below 220 F
is not shown on the figures. Graphical integration of the
area under the curves in figures 5A and 5B, and solu-

(5)

02

01

It should be kept in mind that the heating cycle

rising and falling curves for a vessel will vary with a
number of factors. These include the liquid physical
properties of the nutrient medium such as density,
viscosity, thermal conductivity, and specific heat;
extent of fouling of the heat transfer surfaces; amount
and enthalpy of steam sparged directly into the medium; medium charge volume; temperature and
enthalpy of the heating steam and temperature of
the cooling water in the vessel coils and jacket. For a
given process and given vessel these variables are
maintained reasonably constant, and good replication
in heating cycles occurs.
The method is illustrated by way of the following
hypothetical example.
Example 1. A 15,000-gal fermentor containing 12,000
gal of a penicillin production medium is to be sterilized.
The medium contains 4 per cent corn steep liquor. Corn
steep provides an excellent source of contaminating
organisms. Laboratory checks have shown bacterial
counts not exceeding 20 X 106 cells per ml in the

.024

A)

8) FALLING PORTION
OF CYCLE

RISING PORTION OF CYCLE

-10
I- .020

0
.016

0
-I

w .012

.008o
f

.004

kd

fkde

o. 331

2kav0.335
9

0.0112

SEC.-l

~~~~I
0

10

15

25

20
0

FIG. 5. Velocity constant variation

over

TIME

kav

i-31 0

0.087
0.0870,0066

I
______

I.

SEC. 1
I

10

MINUTES

rising and falling temperature portions

of sterilization

cycle

226

F. H. DEINDOERFER

tion of equation 5, yields values of kavg for each of these

portions of the cycle. For 29.5 min of the rising portion,
kavg is equal to 0.0112 sec-1 and for 13.2 min of the
falling portion, kavg is equal to 0.0066 sec-'.
Therefore, for portions of the heating cycle totalling
42.7 min, a kavg of 0.0098 sec-' can be used to calculate
the contribution of these portions to the sterilization.
Using equation 2, the population remaining if these
two periods followed each other can be calculated.
X 1014
log-9.07 N2

(0-0098 sec') (60 sec/min) (42.7 min)

2.3
N2 = 1.14 X 10' spores
The N2 just calculated becomes the new N1 to be used
in equation 2, this time to calculate the time the
fermentor must be held at 250 F to reduce the population to the desired N2 of 0.001.
2.3
1.14 X 103
0 =
0
(0.0265 sec-) (60 sec/min) log 1 X 10-3
0 = 8.8 min
TABLE 3. Time at sterilization temperature to achieve the same
degree of sterilization in different sized fermentors
Fermentor Size

Total
of Heating
220 F
Above
CycleTime

gal
50
150

1,500
15,000

Time at 250 F

m#

mn

28.0
33.7
41.3
51.5

17.5
12.6
11.3
8.8

[VOL. 5

Thus, only 8.8 min at 250 F are required for the

sterilization of the fermentor.
For comparison, similar calculations have been
made for the other sterilization cycles depicted in
figure 4. The results are listed in table 3. As vessel size
increases, the respective contributions of the rising and
falling portions of the cycle increase, and consequently
the time required at so-called sterilization temperature
decreases.
Heat Effects on Nutrients
The time required for batch sterilization is not
usually the optimum time of heating when product
yield is considered. The thermal effects on nutrient
quality of the medium must also be taken into account.
Sometimes, productivity is increased by prolonged
heating of the medium. In most media, however, the
deleterious effects of extensive heating are more apparent than any beneficial effects, and overheating of the
medium must be minimized.
An easily destroyed nutrient quality might be any one
of the B-complex vitamins. Vitamin degradation is
known to occur rapidly at high temperatures. Although figures for vitamin destruction in fermentation
media are not published, Garrett (1956) has studied
vitamin stability at elevated temperatures in liquid
preparations. He found activation energies of from
16,800 to 26,000 calories/mole for destruction of several
B vitamins. The vitamins investigated by Garrett are
listed with their activation energies and entropies in
table 2. The activation energies and entropies for the
vitamins are much lower than for the three bacterial
spores also listed.
STERILE

STEAM

RAW INGREDIENTS
WATER
ACID OR BASE

MEDIUM

MAKE -UP
TANK

UNSTERILE

MEDIUM

FIG. 6. Steam-injection type of continuous sterilizer

WATER

227

STERILIZATION OF NUTRIENT MEDIA

1957]

The conditions in a batch sterilization carried out at

250 F cannot always achieve sterilization without
impairing nutrient quality of the medium. Higher temperature-shorter time batch sterilizations can be carried
out, but fermentation vessels are usually limited to
operation at not more than 30 psig (274 F) by design
restrictions.
Continuous Sterilization
A sterilization method becoming increasingly popular
and having several advantages over the conventional
batch sterilization is continuous sterilization. Continuous sterilization of media for riboflavin fermentations
has been reported by Pfeifer et al. (1950) and for
penicillin fermentations by Whitmarsh (1954).
In one type of continuous sterilization, preheated
unsterile medium passes through an injection heater in
which steam is introduced. The vigorous and almost
instantaneous mixing obtained raises the medium to
sterilization temperature immediately. This temperature is maintained for the required amount of time in an
insulated retention tube through which the hot medium
flows. A diagram of a typical continuous sterilizer is
shown in figure 6. The hot medium passes through a
heat exchanger where it is cooled to below its flash point
as it preheats unsterile medium. Final cooling to process
temperature is accomplished in the fermentor.
The activation energy of bacterial spore destruction
is much higher than the activation energy of simpler
chemical reactions. It is, therefore, an advantage to use
a high temperature-short exposure time sterilization
operation whenever nutrient degradation occurs to the
extent that it lowers the process yield. Continuous
sterilization not only overcomes unfavorable nutrient
destruction, but has a number of operational advantages. Pfeifer and Vojnovich (1952) point out these
advantages in an excellent paper on continuous sterilization. Operating conditions employed by these authors
for continuous sterilization of several types of media
are shown in table 4. In a batch sterilization, probably
TABLE 4. Operating conditions employed for
continuous sterilization of mnedia*
Media Used

Suspended Solids

%t70
Riboflavin ........... 1.8 corn steep liquor
Cyanocobalamin ..... 4.0 soybean meal
and distillers'
solubles
Acetone, butanol ...1 1.8 ground corn
Sodium gluconate 0.4 corn steep liquor
Itaconic acid ........0 .2 corn steep liquor
Fungal amylase...... 4.0 distillers' solubles and
3.0 ground corn

* Pfeifer and

Vojnovich (1952).

pH

Temra- Time
F

min

4.5
4.5

275
325

4
13

6.5
4.5
6.1
5.0

275
275
300
325

3
5
5
13

only those sterilizations at 275 F could be approached,

if at all, in conventional fermentors.
The calculation of sterilization conditions for continuous operation is simplified by almost instantaneous
rising time to sterilization temperature and a rapid
cooling period thereafter. For this type of sterilization,
only the velocity constants of the most resistant spores
present in the medium and the total initial bacterial
concentration need be known to perform the calculation. Usually, in production operations the sterilizer is
of a fixed length. Different retention times are achieved
by controlled pumping. If temperature conditions are
specified, the retention time can be calculated easily
from equation 2. If process conditions dictate a certain
flow rate, as may be the case in continuous fermentations, any modification in sterilizer operation will have
to be made temperature-wise. Consider the following
example.
Example 2. Process conditions are such that a retention time of 5 min is required in the sterilization of a
sodium gluconate production medium containing 0.4
per cent corn steep liquor. Plate counts of 3 X 106
bacteria per ml in the presterilized medium have been
determined in the laboratory. At what temperature
should the sterilizer be operated to achieve a sterilization having 99 per cent certainty of being successful?
One hundred thousand gallons of medium are to be
sterilized in this manner.
Solution. For sake of illustration, the curve in figure
2 again will be assumed as characterizing the most heat
resistant spores in the medium. The initial population
again will be assumed as entirely consisting of this
species.
Then
N1 = (3 X 106 spores/ml)
(3.78 X 103 ml/gal)(1 X 101 gal)
= 1.13 X 1015 spores

Since one chance in one hundred was chosen as the

margin of failure, N2 is equal to 0.01. Knowing the required retention time, equation 2 can be used to solve
for the necessary velocity constant.
2.3
300
= 0.131

1.13 X 1015
1 X 10-2

sec-1

The calculated value of k is used to read the required

sterilization temperature from figure 2. For k = 0.131
sec- 1 T = 0.1382. Thus, T = 724 R and a sterilization
temperature of 264 F is required.
To achieve the same sterilization at 250 F, a retention
time of 24.8 minutes would be required. Figure 7
illustrates the relationship between time and tempera-

F. H. DEINDOERFER

228

ture to achieve the above sterilization. Also listed in

table 5 are the relative effects of an adverse chemical
reaction destroying a hypothetical vitamin during the
sterilization, the degradation of which is characterized
by an activation energy of 22.6 Kcal/mole, one-third
that of the activation energy for bacterial spore destruction. Even for the retention time and temperature used
in example 2, the vitamin was almost completely
destroyed. Much of the vitamin quality can be retained,
however, by higher temperature-shorter retention time
sterilizations.

10.2

40

2
w

go-2 Il

o2~
0~~~~~~~~~~~~~

0.00129

0.00131

0.00133

0.00135

0.0057

0.00139

RECIPROCAL TEMPERATURE

000141

0.00143

FIG. 7. Time-temperature relationship to achieve the same

degree of sterilization in a continuous sterilizer.
TABLE, 5. Time-temperature relationship and its effect on vitamin
content in a continuous steri,lization
Temperature

Time

Relative Retention of
Original Vitamin
Content*

min

250
265
280
295
310
325

24.8
4.1
0.72
0.14
0.029
0.0061

0.0
0.0
2.3
28
64
89

* k for vitamin destruction assumed equal to k for spore

destruction at 250 F.

[VOL. .5

Design Method
The use of well-known thermal behavior characteristics of bacterial spores and the temperature characteristics of the sterilization cycle in calculating the time
for sterilization of fermentation medium has been
illustrated. This method offers an approach to the
correlation of sterilization conditions among various
sized fermentation vessels and between temperature
and retention time in continuous sterilizers. The advantages of continuous sterilization have been pointed out
in a sterilization where nutrient damage occurs.
The steps involved in calculating process conditions
for sterilization operations can be summarized as
follows:
(1) Periodically determine the thermal-death relationship of the most heat resistant bacterial spore in the
medium to be sterilized.
(2) Determine the initial population concentration of
the medium and choose a confidence level for the
sterilization. An added safety factor is introduced by
assuming that the total population consists entirely
of the most heat resistant spores.
(3) Calculate the contribution to the sterilization of
the rising and falling portions of the sterilization cycle;
this step is unnecessary for steam injection type continuous sterilizations.
(4) Calculate the time required to hold the medium
isothermally at the highest temperature chosen for the
sterilization. For continuous sterilizations, the time of
exposure is often chosen, and calculations are carried
out to find the required temperature.
REFERENCES
BALL, C. 0. 1943 Short-time pasteurization of milk. Ind.
Eng. Chem., 35, 71-84.
GARRETT, E. R. 1956 Prediction of stability in pharmaceutical preparations. II. Vitamin stability in liquid multivitamin preparations. J. Am. Pharm. Assn., 45, 171-178.
JOHNSON, F. H., EYRING, H., AND PILISSAR, M. J. 1954 The
Kinetic Basis of Molecular Biology, p. 220. John Wiley
& Sons, New York, N. Y.
LEVINE, S. 1956 Determination of the thermal death rate
of bacteria. Food Research, 21, 295-301.
PFEIFER, V. F., TANNER, F. W., VOJNOVICH, C., AND TRAUFLER, D. H. 1950 Riboflavin production by fermentation
with Ashbya gossypii. Ind. Eng. Chem., 42, 1776-1781.
PFEIFER, V. F. AND VOJNOVICH, C. 1952 Continuous sterilization of media in biochemical processes. Ind. Eng.
Chem., 44, 1940-1946.
RAHN, 0. 1945 Physical methods of sterilization of microorganisms. Bacteriol. Reviews, 9, 1-47.
STERN, J. A. AND PROCTOR, B. E. 1954 A micro-method and
apparatus for the multiple determination of rates of destruction of bacteria and bacterial spores subjected to
heat. Food Technol., 8, 139-143.
WHITMARSH, J. M. 1954 Continuous sterilization of fermentation media. J. Appl. Bacteriol., 17, 27.