Note: Descriptions are shown in the official language in which they were submitted.
10,6
ll~Z7ZS
This invention r~lates to a process for steril-
ization fermentation media. More particularly, this
invention relates to the use of glutaraldehyde as a
chemical sterilant for microbial media and equipment used
in fermentation processing.
Liquid media for the growth of microorganisms
in submerged culture fermentation are most commonly
sterilized with high-pressure steam prior to inoculation
wi~h the desired product-producing organism. Although
high-pressure steam is an effective sterilant for ferm-
entation media, there are, nevertheless, numerous dis-
advantages associated with its use. One of the most
obvious disadvantages is the economic penalty associated
with the energy requirement for heating and cooling large-
scale reactors and large-volume fermentation media which
are commonly used in present day fermentation processing.
In a period of high energy costs and limited energy
supplies, the economic disadvantage of steam sterilization
is significant.
Furthermore, the use of high-pressure steam pre-
cludes using low-cost materials of construction, such as,
fibergla~s, in the processing plant, and instead, nec-
essitates the use of relatively costly materials such
as stainless and high carbon steel. In addition, rela-
tively long periods of time are required to effectively
sterilize large volumes of microbial media thereby ad-
versely affecting process economics. Moreover, steam
sterilization often results in loss of nutrient values
10,611
~ ~ 27 ~ ~
of the microbial media by overheating, and may cause un-
desirable chemical and physical changes in the substrate
components resulting in decreased production of desirable
products and a concomitant reduction in overall process
efficiency.
Biocidal chemical treatment of fermentation
media, as an alternative to steam sterilization, is
well-known in the art. For example, beta-propiolactone
and ethylene oxide have been disclosed as effective
chemical sterilants; see "The Chemical Sterilization of
Liquid Media with Beta-Propiolactone and Ethylene Oxide",
Toplan et al,Journal of Biochemical and Microbiological
Technology and Engineering, Volume III, No.3, Pages
311-323 (1961). Other chemical anti-microbial agents
used in fermentation processing include formaldehyde
(U.S. Patent No. 2,991,231) furacilin (U.S Patent No.
3,202,587) and antibiotics such as pencillin and strepto-
mycin (U.S. Patent No. 3,494,832). However, apart from
ethylene oxide, all of the chemical agents which have
been used to prepare microbiological media exhibit only
limited biocidal activity. That is, they are specific
towards certain classes of microorganisms, or merely
inhibit the growth of specific contaminating organisms,
and are therefore of limited value. While ethylene oxide
~s a broad spectrum microbicide capable of achieving de-
sirable high levels of microbial purity, safety consider-
ations alone preclude its use in a commercial process.
The highly flammable nature of ethylene oxide is such
. . . , .- , .
10,611
~Z725
that it forms explosive mixtures with air at very low
concentrations. Consequently, its use in a fermentation
process is limited to specially designed autoclaves which
must be operated with stringent safety precautions, and
hence, as a practical matter, it has little commercial
utility.
The effectiveness of glutaraldehyde as a bio-
cidal agent is well documented in the patent literature.
For examp~e, U.S. Patent Nos. 3,016,328, 3,697,222 and
1~ 3,912,450 disclose methods of sterilizing contaminated
objects such as surgical and dental equipment with
glutaraldehyde solutions. However, it is the very
potency of glutaraldehyde as a biocidal agent which has
made it undesirable as a chemical sterilant for ferment-
ation media. That is, glutaraldehyde is biocidal toward
practically all species of microorganisms, including those
-~ which are innoculated into the microbiological media to
produce the desired products by culture fermentation.
Thus, unlike sterilization with high-pressure steam, the
2~ biocidal e~fects of glutaraldehyde are not dissipated
after the li~uid media and related fermentation equipment
have been sterilized. Concequently~ glutaraldehyde has
heretofore not been considered a vlable commercial al-
ternative to high-pressure steam as a sterilant for
fermentation systems.
~ 7;~ 10,611
SUMMARY OF THE INVENTION
This invention describes a process for steril-
izing a culture medium with glutaraldehyde wherein the
biocidal activity of glutaraldehyde is eliminated after
sterilization has been completed. The culture medium
is initially contacted with glutaraldehyde for a period
of time and in a concentration sufficient to achieve the
desired degree of microbial purity. Following steriliza-
tion, the pH of the medium is raised to a value above
about 6.5 and then contacted with one or more nitrogen-
containing compounds selected from the group consisting
of ammonia, primary amines, secondary amines, heterocyclic
amines, ureas, and amides so as to inactivate the glu-
taraldehyde and eliminate its biocidal effects. The pH
of the medium is maintained above 6.5 during this in-
activation step. The medium may then be inoculated with
the desired organism to initiate fermentation.
The term "sterilization" as used herein refers
to decreasing the level of microbial contaminants to very
low values and is not used in its strictest technical
sense as referring to the absolute elimination of all
contaminants.
The term '1nitrogen-containing compound" as used
throughout the specification and claims refers to a com-
pound contain~ng at least one "N-H'~ond reactive to
glutaraldehyde, i.e., it contains at least one nitrogen-
bonded hydrogen capable of forming an addition compound
with glutaraldehyde.
- 10,611
Z~ .
The term "amide" as used throughout the spec-
ification and claims refers to a compound having the
following structure:
O H
Il I
R - C - N - Rl
where R and Rl are hydrogen, alkyl or aryl. It is to be
understood that "alkyl" and "aryll' are intended to include
unsubstituted groups as well as those substituted with
substituents, such as halogen or nitrogen.
DETAILED DESCRIPTION OF THE INVENTION
The desired level of microbial purity for a
given fermentation process will determine the necessary
operating vàriables to effect sterilization, namely, the
concentration of glutaraldehyde in the culture medium,
the pH of the medium and the time of treatment. At a
pH of below about 6.5, a glutaraldehyde concentration of
less than about 0.5 percent, by weight, of the fermenta-
tion broth will generally suffice. Typically, for a
culture medium at ~mbient temperature and maintained at
a pH of about 5, a two hour treatment with glutaraldehyde
at a concentration of about 0.05 percent, by weight, of
the medium will produce the desired degree of microbial
purity; for a glutaraldehyde concentration of about 0.2
percent, by weight, the time of trea~ment will generally
10~611
~ 2 ~
be on the order of about 30 minutes at the same tempera-
ture and pH. The temperature of the liquid medium during
treatment may conveniently vary from about 15C to 75C;
a temperature of from about 25C to 30C beîng preferred.
The pH of the medium may be within the range of from about
2 to 10, but is preferably below a value of about 6.5,
and most preferably from about 5 to 6. At a pH above
6.5, ammonium salts, frequently present as substrate
components in fermentation media, chemically react with
glutaraldehyde thereby necessitating substantially higher
concentrations of glutaraldehyde to sterilize the medium.
This reaction is, of course, also undesirable insofar as
it depletes the medium of nutrients intended to promote
the fermentation reaction. Consequently, sterilization
of media containing ammonium salts such as, ammonium
sulfate and ammonium phosphate,is preferably carried out
at pH values below about 6.5 to suppress the reaction of
glutaraldehyde with the ammonium compounds.
Nitrogen-containing compounds which are useful
for chemically inactivating glutaraldehyde in accordance
with the present invention include~ for example, ammonia;
methylamine; ethylamine; propylamine; butylamine;
t-butylamine; nonylamine; octadecylamine; dimethylamine;
diethylamine; dipropylamine; diisopropylamine; cyclo-
hexylamine; piperazine; morpholine; urea; methylurea;
ethylurea; dimethylurea; formamide; methylformamide; ethyl-
formamide; N-methylformamide; N-ethylonmamide;
N-propylformamide; benzamide; N-phenylformamide and the
like. Ammonia is the preferred compound in terms of its
ava~lability and cost.
~ 10,611
7~
The nitrogen-containing compounds of the i~ven-
t}on may be introduced directly into the medium to react
with free glutaraldehyde, or optionally, may be formed
in-situ, at a pH above about 6.5, from a coordinate
covalent ammonium compound. Thus, for example, ammonium
hydroxide and/or ethylammonium hydroxide may be introduced
into the medium wherein they will be converted, at a pH
above 6.5, to reactive nitrogen-containing compounds,
n~mely, ammonia and ethylamine, respectively. Coordinate
covalent ammonium compounds useful for the present inven-
tion include, ammonium hydroxide; ammonium chloride;
ammonium sulfate, ammonium phosphate; ammonium acetate;
ammonium propionate; methylammonium hydroxide; ethyl-
ammonium hydroxide; methylammonium acetate; methylammonium
sulfate; dimethylammonium hydroxide; diethylammonium
acetate; dipropylammonium hydroxide and the like.
Introduction of nitrogen-containing compounds,
or optionally, coordinate covalent ammonium compounds,
into the medium may be effected by any convenient means.
If the compound is a gas, such as, ammonia, methylamine
or d~methylamine, it may be bubbled directly into the
liquid medium. Liquids, such as ammonium hydroxide,
ethylamine, diethylamine and the like may be added
directly to the medium volumetrically, or by weight.
Solid compounds, such as, urea, ammonium sulfate and
ammonium phosphate, are d~sirably diluted with a solvent,
such as, water, and the resulting solution sterilized
with steam or a chemical sterilant, or by filtration,
prior to introduction into the medium.
72~ lo, 611
As previously noted, ammonium compounds, such
as, ammonium phosphate and ammonium sulfate,are commonly
used as nutrients in certain fermentation media. When
the pH of the medium is above about 6.5, these compounds
are converted to nitrogen-containing compounds which
chemically react with glutaraldehyde. In accordance
with a preferred embodiment of the invention, an excess
of a coordinate covalent ammonium c~pound, above that
required as a substrate component of the fermentation
broth, is introduced into the medium during its initial
formulation. The amount in excess is conveniently the
approximate stoichiometric amount of ammonium compound
which will be required to chemically react with gluta-
raldehyde following sterilization of the medium. In-
activation of glutaraldehyde may then be effected by
simply adjusting the pH of the medium above about
6.5 to favor the formation of the nitrogen-containing
compound from the coordinate covalent ammonium compound.
The pH of the medium is thereafter maintained above 6.5
until the reaction with glutaraldehyde is completed.
Inactivation of glutaraldehyde may be carried
out at any convenient temperature, although a temperature
of from about 20C to 30C is preferred. The pH of the
medium may vary from about 6.6 to 14, but is preferably
from about 7 to 9. The reaction time will generally vary
within the range of from about 0.5 to 4 hours depending
upon the pH of the medium and the activity of the nitrogen-
containing compound or coordinate covalent ammonium
10,611
~2~725
compound employed. Ammonium salts containing very weak
acid moieties, such as, ammonium carbonate, generally
require less time to inactivate glutaraldehyde than
ammonium salts containing strong acid moieties, such as,
ammonium sulate. Accordingly, the reactivity of some
typical a~monium compounds, in decreasing order, is as
follows: ammonium hydroxide, ammonium carbonate, ammonium
acetate, ammonium phosphate, ammonium chloride and
ammonium sulfate. Typically, for a fermentation medium
at 25C, maintained at a pH of about 7.5, a reaction time
of from about 1 to 1.5 hours is necessary for inactivation
when using a coordinate covalent ammonium compound such as,
ammonium hydroxide.
To effect complete inactivation of glutaralde-
hyde, the amount of nitrogen-containing compounds in the
medium should be approximately stoichiometrically equiv-
alent to that of glutaraldehyde in the medium.
Specifically, the number of reactive "N-H" bonds should
equal the number of available aldehyde groups of gluta-
raldehyde; there being present two aldehyde groups per
mole of glutaraldehyde. Thus, for example, 2/3rds of a
mole of ammonia, having three reactive "N-H" bonds per
molej is required to stoichiometrically react with one
mole of glutaraldehyde. Similarly, one mole of ethyl-
amine (having two reactive "N-H" bonds per mole), or two
moles of diethylamine (having one reactive "N-H't bond
per mole) are required per mole of glutaraldehyde.
Amounts in excess of stoichiometric may be used, if
desired.
.
. 10,611
~19272~
EXAMPLE 1
The following experiment was conducted to demon-
strate glutaraldehyde sterilization of production media
for Penicillin-G submerged culture fermentation and to
provide a comparison with steam sterilization.
Three~ fourteen liter fermenters equipped with
an automatic pH control unit, a dissolved oxygen probe,
an automatic glucose feed system~ and an automatic
demand-feed foam control system, were each charged with
the following g_owth medium:
Comp nentAmount (Grams~
Glucose 20.00
(NH4)2S04 120.00
KH2P04 24.00
CaS4 2H2 66.00
Na2S4 4.00
: MgSO4.7H2o 5.60
~eS04
MnC12 0.40
ZnS04 0.40
CaC12 0.40
CuS04 0.06
Water 8000. 00
11
` 10,611
~ 7 ~ ~
The automatic glucose feed system for each unit
was charged with an aaueous solution containing twenty-five
percent, by weight, of glucose and 2.08 percent, by weight,
of phenyl acetic acid. To assure uniformity, these solu-
tions were sterilized for thirty minutes with steam at
121C and 29.7 psia steam pressure.
The automatic foam control addition units were
charged with one hundred grams each of UCON TM Fluid
LB-625 fermentation foam control agent, which was used
to control foaming throughout the runs.
Fermenter No. 1 and its contents were steam
sterilized for forty-five minutes at 121C and 29.7 psia
pressure. The pH after sterilization was 5.2, and was
adjusted to 6.8 by the addition of sodium hydroxide solu-
tion (5.0 N). Approximately five hours were required to
heat the fermenter and contents to 121C, maintain the
temperature for the prescribed time, and cool the unit
to 24C for inoculation.
Fermenter No. 2 was not sterilized, and the pH
of the contained medium was 5.6. This pH was maintained
for one hour, and then adjusted to a pH of 6.8 using sodium
hydroxide solution (5.0 N). The temperature of the medium
was 24C.
Thirty-two grams of 25.2 percent, by weight,
aqueous glutaraldehyde solution was added to the contents
of fermenter No. 3, resulting in a glutaraldehyde content
of approximately 0.1 percent, by weight, based upon the
total charge. The temperature of the medium was 24C.
12
10,611
272~;i
The pH of the medium was 5.5 immediately after the addi-
tion, and this condition was maintained for one hour.
The pH of the medium was then adjust~d to 7.0 using
sodium hydroxide solution (5.0 N). During the next hour
and twenty minutes, the pH of the medium was automatic-
ally adjusted by pH controller, and maintained at a
value of 6.8. It should be noted, that as a portion of
the (NH4)2S04 in solution is converted to ammonia which,
in turn, reacts with glutaraldehyde, the pH of the medium
tends to drop due to the presence of sulfate ion in solu-
tion. Periodic addition of an alkaline material, such
as, sodium hydroxide is therefore required to maintain
the medium at constant pH. When the inactivation of
glutaraldehyde is completed, the pH of the medium remains
stable.
Fermenters No. 1, No. 2, and No. 3 were ~ach
inoculated with 200 cc of seed medium cultured using the
organism Penicillium chrysogenum (A.T.C.C. 12690), and
incubated for forty-eight hours in 500 cc shaker flasks
at 25C. The wet volume of mycelium in the inoculum was
ound to be sixteen percent after centrifugation at 5000
rpm for fifteen minutes.
The agitation rate for each inoculated fer-
menter was adjusted to 360 rpm, and air flow rates were
adjusted to 4000 cc/minute. The temperature of each
fermenter was maintained at 24.0 + 0.5C throughout
the fermentation. The saturation dissolved oxygen con-
tent for each unit was found to be, at one h~l.ndred
percent meter reading, 8.8 mg 02/liter.
13
10,611
Twenty-four hours after inoculation, the dis-
solved oxygen content of the broth in each fermenter was
as follows: No. 1 - ninety-seven percent, No. 2 - ten
percent, and No. 3 - ninety-five percent. Microscopic
examination using a simple stain technique indicated
that ferments No. 1 and No. 3 contained predominantly
the organism Penicillium chryso~enum, with essentially
no trace of other contamination organisms. By contrast,
the ferment in fermenter No. 2 contained only traces of
Penicillium chrysogenum, and considerable amounts of
contamination consisting of bacilli and cocci bacteria.
Due to the large population of contaminating organisms
in fermenter No. 2, it became necessary to discontinue
this fermentation after thirty hours.
The fermentations in fermenters No. 1 and No. 3
were allowed to continue for one hundred ninety-six hours,
and were facilitated by the addition of sterile glucose/
phenyl-acetic acid solution to each unit. At the end of
one hundred ninety-six hours, microscopic examination of
the broths indicated that both cultures were essentiaily
Penicilliwm chrysogenum, and contained no visible signs
of contamination.
The Penicillin-G content of the broths was
measured by the method of Bethel and Bond (J. Anal.
Chem., Volume 86, pages 448-457, 1961), and found to
be as follows: 865 units/ml in fermenter No. 1 and
858 units/ml in fermenter No. 3.
.
10,611
7 ~ ~
EXAMPLE 2
The following experiment was conducted to
demonstrate the sterilization of a Cephalosporin "C"
fermentation medium with glutaraldehyde and to provide
a comparison with steam sterilization.
Three, fourteen liter fermenters were each
charged with th following Cephalosporin fermentation
medium:
Component Grams of Component
Beet Molasses 240.0
Vita Pro 70 320.0
(Rath Chemical Company)
Cornsteep Liquor 40.0
(C.P.C. Int.)
D L-Methionine 40.0
Methyl Oleate 160.0
CaC03 12.0
NH4C03 7.0
SAG TM 4130 Silicone Antifoam 0.160
Water 8000
Fermenter No. 1 and its contents was sterilized
at 121C with steam at fifteen psig pressure for forty-
five minutes. Approximately six hours were required to
sterilize the system and reduce the temperature to 29C
for inoculation.
The pH of the medium in fermenter No. 2 was
adjusted to 5.5 and the temperature maintained at 29C
for one hour. The pH was then adjusted to 6.8 using
ammonium hydroxide solution (30V/o~ by weight).
:
10,611
72~
Sixty-four grams of 25%, by weight, aqueous
glutaraldehyde solution were added to the cont~nts of
fermenter No. 3 and the temperature was maintained a~
29C. The pH of the medium after addition of glùtaralde-
hyde was 5.5, and this condition was maintained for one
hour. The pH was then adjusted to 6.8 by the addition of
~mmonium hydroxide solution and maintained at constant
pH for one hour.
Fermenters No. 1, No. 2, and No. 3 were then
inoculated with 500 cc each of seed medium cultured for
48 hours using the organism CePhalosporium acremonium
(A.T.C.C. No. 11550) and fermentations were conducted
at 29C using an agitation rate of 300 rpm and an air
flow rate of 4000 cc/minute.
After ninety-six hours of fermentation,
fermenters No. 1 (steam sterilized unit) and No. 3
(glutaraldehyde treated unit) were each found to contain
in excess of five hundred units/ml of Cephalosporin "C".
Fermenter No. 2 (no sterilization treatment) contained
no trace of Cephalosporin "C" and was overgrown with at
least 5 different types of contaminating organisms as
determined by microscopic examination.
EXAMPLE 3
The following experiment was conducted to demon-
strate glutaraldehyde sterilization of production media
employed in the manufacture of ethyl alcohol by anaerobic
fermentation and to provide a comparison with steam
sterilization.
16
10,611
~ ~ 27 ~ ~
Two, four liter anaerobic fermenters were each
charged with 2000 cc of distilled water and 500 grams of
Minnesota Beet Molasses.
Fermenter No. 1 was steam autoclaved at 121C
and fifteen psig steam pressure for fifteen minutes. The
pH of the media in the fermenter was then adjusted to
pH = 4.9 using lactic acid.
Five grams of 25 percent (by weight) aqueous
glutaraldehyde solution were added to fermenter No. 2 at
pH = 5.6 and at a temperature of 25C. After twenty
minutes, the pH was adjusted to 7.0 using about 8 cc of
30%, by weight, ammonium hydroxide solution, and this pH
was maintained for one hour. The pH was then adjusted to
4.9 using lactic acid.
Fermenters No. 1 and No. 2 were inoculated with
2 grams of dry Sacchar~myces cerevisiae (Wine Arts Company)
and the fermentations were conducted at 25C.
The ethyl alcohol content of the broths was
determined by gas chromatography with the following
results:
PERCENT BY VOLUME ETHANOL (~ 0.1%)
Time Fermenter Fermenter
Hours No. 1 No. 2
O O O
12 1.1 1.4
24 4.6 4.8
48 6.0 5.8
17
10,611
72~i
EXAMPLE 4
This example is intended to demonstrate gluta-
raldehyde sterilization of the production media employed
in the manufacture of Xanthan Gum and provide a comparison
with steam sterilization. Further, this example illus-
trates the effect of simply raising the pH of the culture
medium to above 6.5 following sterilization but failing to
introduce a nitrogen-containing compound into the medium
to inactivate the glutaraldehyde (fermenter No. 2).
Three, fourteen liter fermenters were each
charged with the following Xanthan Gum production medium:
Component Amount ~rams)
Yeast Extract 24
Malt Extract 24
Bacto Peptone 40
Glucose 160
Distilled Water 8000
Fermenter No. 1 was steam sterilized at 121C
and fifteen psig pressure for twenty minutes. The pH
of the medium was adjusted to pH = 7.0 by the addition
of sodium hydroxide.
Fermenter No. 2 was sterilized at 29C by the
addition of thirty-grams of 25 percent (by weight)
aqueous glutaraldehyde, with the pH adjusted to 5.5.
The pH was maintained at 5.5 for one hour. The pH was
then adiusted to 7.2 using 5 N sodium hydroxide solution,
and maintained at constant pH for 2 hours.
~ .
~ 18
.
10,611
~ 7 ~
Fermenter No. 3 was sterilized at 29C by the
addition of thirty-two grams of 25 percent (by weight)
glutaraldehyde solution, with the pH adjusted to 5.5.
The pH was maintained at 5.5 for one hour. The pH was
then adjusted to 7.0 using about 8 cc of 30%, by weight,
ammonium hydroxide solution, and maintained at constant
pH for 2 hours.
All three fermenters were then inoculated with
1000 cc of seed medium containing pure cultures of
Xanth$monas campestris (N.R.~.L. 1492A), and the fer-
mentations were performed at 29C with air flow rates
equal to 4000 cc/minute and agitation rates of 300 rpm.
Immediately after inoculation, the dissolved
oxygen content of the broth in each fermenter dropped to
about 92 percent of saturation, indicating the growth
of _anthamonas campestris. Within fifteen minutes, the
dissolved oxygen content of fermenter No. 2 was one
hundred percent; an indication that the biocidal prop-
erties of glutaraldehyde were still present due to the
~0 fact that no ammonium compound or nitrogen-containing
compound was added to the medium to effect inactivation.
The dissolved oxygen content of fermenter No. 2 remained
at one hundred percent ~hroughout the forty-eight hour
fermentation, and no change in viscosity was observed.
The dissolved oxygen contents of fermenters
No. 1 and No. 3 continued to drop, and approached zero
percent saturation in twelve hours. The agitation rates
of these fermenters were adjusted to 500 rpm to maintain
a dissolved oxygen content of from about twenty to
10,611
~L~Z72S
thirty percent. The viscosities of the broths in
fermenters No. 1 and No. 3 continued to increase through-
out the following forty-eight hours, and at the conclu-
sion of the fermentations, the broths were essentially
too viscous to flow.
Microscopic examination of the extremely viscous
fluids in fermenters No. 1 and No. 3 indicated that the
cultures contained predominately the organism Xanthamonas
campestris. Microscopic examination of the low viscosity
liquid in fermenter No. 2 indicated that a very low
population of this organism was present.
-
,',
