Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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25547
FERMENTATION WITH THERMOPHILIC MIXED_ CULTURES
Field of the Invention
The invention relates to the production of single cell pro-
teinO In another aspect, the invention relates to a novel thermo-
philic mixed culture.
3e_
Efforts to relieve the impending worldwide shortages of pro-
tein have included various biosynthesis processes wherein bio-
logically produced single cell protein (SCP) is obtained by the
growth of a variety of microorganisms on a variety of carbon-
containing substratesO
The carbon and energy sources used as substrates for such
processes should be available widely, relatively cheap, uniform
and safe in that they do not leave harmful residues in ~he pro-
teinaceous product ultimately obtained by the microbial conversions.
Petroleum hydrocarbons have been employed as the carbon and energy
source material, but have faced practical difficulties in the lack
of water solubility, in the high consumption of oxygerl to assist
in the microbial conversion, and allegedly in traces of potentially
carcinogenic agents from the petroleum feedstocks entering or ad~
hering to the protein product.
Other proces~es have used oxygenated hydrocarbon derivatives
as feedstocks due to the water solubility of such derivatives and
hence ease of handling since microbial conversion processes are
essentially conducted under aqueous conditions. Such feedstocks
are readily available either from petroleum sourcesl natural
yas sources, various waste/garbage processing and conversion of
methane~ and the like, from fermentation of variou grains and the
like, destructlve aiskilLation Qf wood, and so on. Such oxygenated
hydrocarbons, whatever their source, are widely available and
relatively cheap feedstocks ~or fermentation proce~ses. Advantages
accrue in that these feedstocks are partially oxygenated, so that
substantially reduced molecular oxygen requirements are involved
for the microbial conversion growth process itself.
However, another difficult and limiting factor in the commer-
cialization of single cell protein processes has been the necessity
to conduct the fermentation at relatively moderate temperatures
of about 20 to 50~ C., and preferably not over about 35 C.
Microbial conversions are exothermic oxidation reactions with
large quantities of heat being pxoduced. Heat must be removed
from the fermentation admixture continuously and consistently,
or risk the overheating of the system and either the death of the
microorganisms or at least severe limitations of growth encoun-
tered as temperatures rise, and hence loss in efficiency.
Many processes have concentrated on the employment Qf one
or other of the many available yeasts as the microorganismO
Yeast cells generally are slightly larger than a bacteria cell,
and sometimes provide easier separation from the fermentation
process media.
However, bacteria offer advantages, since higher crude
protein contents of the cell are obtained from bacteria as com-
pared to the product obtainable from yeasts in general, sincethe yeasts have higher proportions of nonprotein structural
material in their cells. Bacteria usually have a significantly
higher true protein content, frequently being higher in the
nu~ritionally important sulfur amino acids and lysine.
Discovery of bacteria with the capability o~ rapid growth
and high productivity rates at relatively high fermentation
process temperatures would be advantageou~. High temperature
growth operation means less heat to be removed, less cooling
apparatus involved, and ultimately relatively smaller amounts of
heat needed for sterilization, coagulation, and separation pro-
cesses. Danger of contamination with othex microorganisms is
greatly reduced when high temperature ~ermentation can be employed.
Thus, thermophilic or therrnotolerant bacteria are definitely
J ~
needed for commarcialization of the single cell protein process.
Summary of_the Invention
I have discovered a unique thexmophilic mixed culture of
bacteria, containing three separate species of bacteria. These
bacteria are individually classified as (1) a large gram-positive
curved rod, division bacteria, class ~ , ~rder
Eubacteriales, family Bacillaceae, genus ~acillusi (2) a large
gram-negative rod, division Bacteria, class ~ , order
Eubacteriales, family Bacillaceael genus ~acillus; (3) a short
.
gram-negative rod, division Bacteria, class ~ . The
mixed thermophilic culture exhibits highly desirable and useful
properties. My Mc mixed culture exhibits improved growth at
higher temperatures than at conventional temperatures, producing
higher cell yields, with reduced foaming tendencies under fer-
mentation conditions.
My mixed culture is thermophilic, grows effectively with
high productivity on oxygenated hydrocarbon feedstocks, particu-
larly lower alcohols, most preferably methanol or ethanol, at
temperatures wherein most other kno~m bacteria speci~s either
are relatively unproductive, or simply cannot survive/ or are
unproductive and intolexant of an oxygenated hydrocarbon feedstock.
This unique mixed culture which I have discovered, and employ
in my process, is designated as follows:
Culture Name My Strain Desi~nation
.
MC HTB-53 NRRL ~ 8158
The designation NRRL B-8158 reflects the fact that I have
deposited my thermophilic mixed cul~ure with the o-f~icial deposi-
tory United States Department o~ Agriculture, Agricultural Research
Service, North Central Region, Northern Regional Research
Center, 1815 North University Street, Peoria, Illinois 61604, by
depositing therewith thirty lyophilize~preparations of my mixed
culture, prior to filing of this application, and have received
rom the depository the NRRL designation B-8158 as indicated.
G2~
My unique mixed culture has been deposited in accordance with
the pxocedures of the Department of Agriculture such that progeny
of the mixed culture will be available during pendency of this
patent application to one determined by the Commissioner of
Patents and Trademarks ~o be entitled to access thereto in accord-
ance with the Rules of Practice in Patent Cases and 35 U.S.C. 122.
The deposit has been made in accordance with the practices and
requirements of the United States Patent and Trademark Office
such that all restrictions on availability to the public of pro-
geny of the unique mixed culture will be irrevocably removed upongranting of a patent of which this important mixed culture is
the subject, so that said culture will be available to provide
samples for utilization in accordance with my invention. Thus,
any culture samples from this deposit, or from cultures from which
the deposit was derived, thus provide mixed culture strains de-
rived from the thermophilic mixed culture of my discovery.
Detailed Desc ~
I have discovered a peculiarly and uniquely effective
thermophilic mixed culture of bacteria which I have designated
as mixe~ strain HTB-53 and which has received U.S.D.A. depository
designation NRRL B-8158. This mixed culture, for which I us~ the
shorthand designation Mc, is a culture which is highly productive
at relatively high fermentation temperatures, producing desirable
and valuable single cell protein products with a high protein
content of desirable amino acid ~ype and balance.
My unique thermophilic mixed culture is compositionally
stabla. The mixed culture employed for lyophilization was iso~
lated from a fermentation run and lyophilized under usual condi-
tions which comprise rapidly reezing the microbial cells at a
3~ very low temperaturP followed by rapid deh~dration under high
vacuum, and storage at room temperature. To determine viability,
some of the lyophilized samples were subsequently reactivated
2~2~
and sub~ected to g~owkh of the lyophilized mixed culture under
the same conditions utilized previously. The subsequent fermenta-
tions employing the reactivated Mc culture gave essentially the
same results in terms of high cell yield and the like, and appar-
ent composition of the fermentation culture, as did the earlier
runs. Microscopic examination of the reactivated Mc culture
indicated ~he same three organisms in the same form and relation-
ship existing in the reconstituted culture as in the source
fermentation.
This unique hi~h temperature preferring culture provides
improved rates of single cell protein production,with reduced
cooling requirements, when grown on a carbon and energy substrate
of an oxygenated hydrocarbon, preferably a lower alcohol, more
preferably methanol or ethanol, and presently preferred is methanol
or a substantially methanol-containing substrate.
There are distinct advantages in employing my unique thermo-
philic mixed culture Mc in comparison to the utilization of a
pure thermophile in the fermentation of methanol or of a similar
carbon and energy source material. Firstly, a higher cell yield
is obtained using the mixed culture. Cell yield as described
herein is defined as the grams of cells produced per 100 grams of
carbon and energy source material utili2ed, such as methanol. The
higher cell yield is an important practical commercial aconomic
advantage.
Another advantage fox my unique thermophilic mixed culture
in ~omparison to a pure thermophile, is the fact that foam gen~
eration is substantially less as compared to pure cultures which
I have studied. Pure cultures generally tend to produce large
amounts of foam during the fermentation process. While foam may
be desirable in certain apparatus as a means of assisting in the
fermentation process in heat transfer, and in assisting in provid--
ing the desired quantities of molecular oxygen for the aerobic
fermentation process, nevertheless, many types of fermentation
6~
apparatus means are not designed or equipped to handle excessive
or large amounts of foam produced ~y some pure-strain microorgan-
isms. Thus, the moderate amounts of foam produced by my unique
culture are certainly a distinct advantage.
Another advantaye for my unique mixed culture is the fact
that the mixed culture inherently produces a single cell protein
product which is a mixture of several varie~ies of cells, and
thus the balance of amino acids in the recovered microbial cells
from the Mc is expected to have a more desirable balance than
exhibited by the product of any single pure culture. A better
amino acid balance simply means that there is less likelihood of
a deficiency of a particular essential amino acid.
My unique thermophilic mixed culture was discovered by me
during work to discover and develop a variety of cultures suitable
for microbial conversion processes. A sample of soil was taken
two feet below the surface of earth covering a steam line at the
Bartlesville, Oklahoma, Research Center of Phillips Petroleum
Company. Conventional enrichment techniques in the presence of
methanol were employed to isolate several separate or distinct
cul~ures. During the course of the work in which several pure
thermophilic cultures were isolated, it became apparent that a
most unusual compositionally stable mixed culture of thermophilic
organisms al90 had been obtained.
The thermophilic mixed culture which I have discovered is
composed of three separate microorganisms. The three types of
bacteria in my compositionally stable thermophilic mixed culture
are described as (1~ a large gram~positive curved rod, (2~ a large
gram-negative rod, and (3) a ~hort gram-negative rodO
Repeated attempts at separation were made on this culture
3~ in order to isolate the pure microorganisms. For example, streak
plates gave isolated colonies o~ the three organisms on a mineral
agar media containing methanol. ~owever, when the isolaked
colonies were transfarred to aqueous media for further growth
.
- ~ - '
with methanol as the carbon and energy source material, surpris-
ingly and unexpectedly no growth took place. Repeated efforts
of this type were carried out, but without success.
The cooperative growth by this mixed culture indicates to
me a symbiotic relationship between the three microorganisms.
Theorizing, and without intending to be bound by such theorizing,
but rather in an effort to help explain the relationship observed,
it appears possible ~hat a metabolic product or products of at
least one of the microorganisms serves as a necessary substrate
for the growth of one or other of the other microorganisms. While
the exact nature of such a metabolite is not known at present, it
appears possible that such metabolite may be toxic in nature to
the microorganism which produces it, thereby explaining the need
for ~he presence of the additional microorganism to consume this
toxic metabolite in order for the first microorganism ~o continue
to grow ade~uately.
A further indication of this symbiotic r&lationship that
appears to exist between the three mi~roorgani~ms making up my
unique compositionally stable thermophilic mixed culture is the
fact that fermentations employing my Mc produce significantly
less ~l~antities of foam under typical aerobic fermentation condi-
tions as compared to the quantities of foam normally observed
under equivalent typical aerobic fermentation conditions but
employing pure thermophilic bacteria Bacillu_ ge~us~ This sug-
gests to me, though again I do not wish to be bound hy theorizing
when I have discovered a unique mixed culture and have demonstrated
how to employ it to obtain improved cell yields o~er that obtainable
by pure strains, that my Mc does not produce what would otherwise
be expected in the way of very large amounts of foam during fermenta~-
tion because in an Mc Eermentation an extracellular product,probably of proteinaceous nature~ i5 being consumed by at least
one of the symbiotic microorganismæ, thereby continuously deplet-
ing such product as a foam-generating material i.n the fermentation
- . ~
admixture.
Carbon and Energy Source
~ he carbon and energy source material or substrate for the
fermentation process of my inven~ion employing my novel and unique
mixed culture is an oxygenated hydrocarbon. The term oxygenated
hydrocarbon is a generic term descriptive of ~he compounds employ-
able, and not necessarily a limiting term referring to the source
of the substrate. The oxygenated hydrocarbons include alcohols,
ketones, esters, ethers, acids, and aldehydes, which are carbon-
oxygen-hydrogen~containing water-soluble compounds and are sub-
stantially water-soluble in character. The oxygenated hydrocar-
bons preferably should be of up ~o about 10 carbon atoms per mole-
cule for better water solubility, since higher molecular weights
tend to reduce water solubility level.
Illustrative examples include: methanol, ethanol, propanol,
butanol, pentanol, hexanol, 1,7-heptanediol, 2-heptanol, 2-methyl-
4-pentanol, pentanoic acid, 2-methylbutanoic acid, 2-pentanolr
2-methyl-4 butanol, 2-methyl-3 butanol, 2-butanol, 2--methyl-1-
propanol, 2-methyl-2-propanol, 2-propanol, formic acid, acetic
acid, propanoic acid, formaldehyde, acetaldehyde, propanal~ butanal,
2~methylpropanal, butanoic acid, 2-methylpropanoic acid r pentanoic
acid, glutaric aaid, hexanoic acid, 2-methylpentanoic acid, hep-
tanedioic acid, heptanoic acid, 4-heptanone, 2-heptanone, octanoic
acid, 2-ethylhexanoic acid, glycerol, ethylene glycol, propylene
glycol, 2-propanone, 2-butanone, diethyl ether, methyl ethyl ethe~r
dimethyl ether, di-n-propyl ether, n-propyl isopropyl ether, and
the like, including mixtures of any two or more.
Petroleum gases, such as natural gas, such as methane, or
other low carbon gases such as ethane and the lik~, can be oxidized
~0 to provide mixtures of predominantly the corresponding alcohols,
as well as miscellaneous minor amounts o~ misc~llaneous ketonas,
aldehydes, ethers, acids/ and the likeO
~6~
Among the oxygenated hydrocarbons, a presently preferred
are the water-soluble alcohols as being suitable carbon and
energy source materials for utilization by the thermophilic mixed
culture of my discovery. Generally, these will be alcohols of 1
to 7 carbon atoms per molecule. Such alcohols include both linear
and branched alcohols, primary, secondary, as well as tertiary.
Such alcohols can be monohydroxy, as well as polyhydroxy.
Exemplary alcohol species include such as methanol, ethanol,
propanol, butanol, pentanol, hexanol, 1,7-heptanediol, 2-heptanol,
2-methyl-4-pentanol, 2-p~ntanol, 2-methanol-4-butanol, 2-methyl-3-
butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol, 2-
propanol, glyceral, ethylene glycol, propylene glycol, and the
like, including mixtures of any two or more.
The preferred alcohols are those of 1 to 4 carbon atoms per
molecule, and most preferred are the monohydroxy alcohols,
because of availability, water solubility, and economics. Methanol
presently is an especially preferred alcohol because of its rela-
tively low cost, wide availability, wa~er solubili~y, and since
governmental regulations on use are less cumbersome than with
ethanol. Methanol also is readily obtainable by simplP oxidative
conversion of natural gas, which is primarily methane, and thus
is or can be made readily available in many areas of the world
which presently have surplus stocks of methane and little market
or Sanle~ and correspondingly also sometimes have large populations
of hungry people in this needlessly protein-deficit world.
~ presently commercially available material sometimes termed
"Methyl Fuel'l (C&EN, September 17, 1973, page 23) is exemplary of
a commercially available mixture of methanol and controlled per-
centages of higher alcohols containing up to about 4 carbon atoms
0 per molecule, and could be employed as a suitable substrate.
Fermentation Condition~
Since the fermentation process employing the thermophilic
~G22~
mixed culture in accordance with my invention is a process of
aerobic fermentation, there must also be supplied adequate oxygen
for the fermentation admixture. Aerobic fermen~ation processes
basically are well known in the art, and means of supplying oxygen
to fermentation admixtures are also well known. Generally, the
supply of molecular oxygen to the aqueous fermentation reaction
admixture can be provi~ed by passing adequate volumes of air of
ordinary oxygen content, or oxygen-enriched air if desired, or
air and an ~ugmented supply of such as pure oxygen separately,
through the fermentation vessel. Offgases can be recovered,
recycled if desired, for maximum utiliæation of oxygen, such as
by stripping carbon dioxide ~rom the offgases and recycling. In
effect, using the oxygenated hydrocarbon as carbon and energy
source substrate, a part of the oxygen demand needed for growth of
the micro organism is supplied by the oxygen content of the
substrate. Nevertheless, additional quantities of molecular oxygen
must be supplied for suitable growth, since the assimilation of
the substrate and corresponding growth of the microorganism is,
in a sense, a combustion process. In general, exemplary is a
range o~ about 0.1 and 10, more usually about 0.7 and 2.5, volumes
per minute of air of normal oxygen content are supplied to the
ermentation admixture per volume of aqueous liquid in the
fermentor, ox in terms of oxygen, the respec~ive ranges would be
about 0.02 to 2.1, and 0.14 to 0.55.
Pressure employed for my aerobic fermentation process can
vary over a wide range. Exemplary would be considered a range of
about 0.1 to 100 atmospheres (10.13-10,132 kPa), more usually from
about 1 to 30 atmospheres (101.3-3,039 kPa), presently prèferably
about 1 to 5 atmospheres (101.3-506.5 kPa), as being suitable and
convenient. Pressures greater than atmospheric pressure are
advantageous in ~he proces~ since such higher pressures tend to
increase the dissolved oxygen content in the aqueous fermen~ation
medium, which in turn tends to promote more rapid microbial growth.
.
.
Pressures greater than atmospheric are especially useful employing
my thermophilic }nixed culture, since the higher temperatures em-
ployed in my thermophilic fermentation tend to decrease oxygen
solubility in the aqueous fermentation medium admixture.
The culturing of my unique and novel Mc mixed species of
bacteria with the oxygenated hydrocarbon feedstock can be advan-
tageously carried out at a temperature in the range of about 45
to 65 C., more preferably for optimum growth rates in accordance
with my invention in the range of about 50 to 60 C. It should be
noted that the temperature ranges given clearly indicate that the
microorganisms utilized in my process are thermophiles in the
accepted usage of the term, i.e., ~he microorganisms require such
relatively high temperatures for suita~le growth. Lower tempera-
tures tend to inhibit (retard) growth rate~.
High concentrations of some of the described carbon and
enexgy source substrates, such as methanol, may be inhibitory
to satisfactory microbial growth or even toxic to ~he micro-
organisms in the fermentati~ns employing the mixed culture, and
should be avoided.
The fermentation process can be carried out in a batch or in
a continuous fashion t the presently preerred for economical SCP
production is the growing of microbial cells in large quantities
in a continuous process. A continuous process is particularly
suitable when a carhon and energy source material is a low~r
alcohol, such as methanol or ethanol, which lends itself readily to
controllable feed. ~he use o such microorganisms and the use of
such feedstocks in a batch fermentation process is considered to
be generally uneconomical on a practical basis. However, such
substrates are conveniently utilized in a continuous fermentation
process with overall high efficiencies and ~conomies. In the
fermentation, a~ter the fermentor has been properly inoculated
with the mixed culture species, the oxygenat0d hydrocarbon can be
Z~
added as a separate stream, or admixed with water as an a~ueous
stream to sterilize same, or with mineral media to sterilize same,
or any or all of these~ Usually, it is fed separately for ease of
control in a concentration in ~he feed stream to the fermentor
broadly in the range of about 2.5 to 35 weight percent, more
usually and conveniently ahout 10 ~o 15 weight percent~
Culturing is accomplished in an aqueous growth medium compris-
ing an aqueous mineral salt medium, the carbon and energy source
material, molecular oxygen, and, of course, a starting inoculum
o~ the Mc mixed culture.
The fermentation growth rates can be adjusted by controlling
the feed of oxygenated hydrocarbon. The feed rate of the carbon
and enexgy source material should be adjusted so that the amounts
being fed to the fermentor substantially are the same as the rate
of consumption by the organism to avoid a significant buildup in
the fermentor, particularly of any toxic materials which might
inhibit the growth or even kill the microorganisms. A satisfactory
condition usually can be exhibited by observation of little or no
carbon and every feed material in the effluent being withdrawn
from the ferme~tor, though a satisfactory check also can he obtained
by watching the feed source material in the fermentor e~luent
so as to maintain at a desirable low level of about O to 0.2 weight
percent.
Generally the retention time of microbial cells in the fer-
mentor means in a continuous process is of the order on the average
o~ about ~ to 4 hours under such conditions, though this is not
critical and can vary widely.
The unique mixed culture Mc of my discovery requires mineral
n~trients and a source of asæimilable nitrogen~ in addition to
the molecular oxygen, and the carbon and energy sources as des-
cribed. The source of nitrogen can be any nitrogen-containing
compound capable of releasing nitrogen in a form suitable for
12
metabolic utilization by the organism. While a variety of organic
nitrogen source compounds such as other proteins, urea, or the
like, can be employed, usually inorganic nitrogen source materials
are more economical and practical. Typicall~, such inorganic
nitrogen-containing compounds include the presently preferred
ammonia or ammonium hydroxide, as well as various other ammonium
salts such as ammonium carbonate, ammonium citrate, ammonium
phosphate, ammonium sulfate, ammonium pyrophosphate, and the likeO
Ammonia gas is convenient and can be employed by simply bubbling
such through the aqueous fermentation media in suitable amounts.
The pH of the aqueous microbial fermentation admixture should
he in the range, in accordance with my investigations, broadly
from about 5.5 to 7.5, with a presently preferred range of about
6 to 7. Feed of the ammonia assists in controlling the pH desired,
since otherwise the aqueous media tends to be slightly acidic. Of
course, pH range preferences for microorganisms generally are
dependent to some extent on the media employed, and thus the pH
preference may change at least slightly with a change in mineral
media, for example.
In addition to the oxygen, nitrogen, and carbon and energy
source, it i5 necessary to supply selected mineral nutrients in
necessary amounts and proportions to the feed media in order to
assure proper microorganism growth, and to maximize the assimila-
tion of the oxygenated hydrocarbon by the cells in th~ microbial
conversion processO
A source of phosphate or other phosphorus, magnesium, calcium,
sodium, manganese, molybdenum, and copper ion~ appear to provide
the essential minerals. The recipe shown below can be used to
culture my novel Mc culture of my discovery. A mineral nutrient
medium designated by me as FM-12 is useful in the fermentation pro~
cess and is listed below along with the trace mineral solution
which forms a part of the FM-12 nutrient medium:
FM-12 Medium
Component _ ~mount
.
H3PO4 (85%) 2 ml
KCl 1 g
MgSO4-7H2O 1.5 g
CaC12~H2O 0~2 g
NaCl 0.1 g
Trace Mineral Solution 5 ml
Distilled Water To make 1 liter
Trace Mineral Solution
-
CUSO4 ~ SH2O O . 06 g
KI 0.08 g
Fecl3 6~2o 4.80 g
MnSO4 H2O
Na2MOO4 2H2o 0.20 g
ZnS04 7H20 2.00 g
H3BO3 0.02 g
H2SO4 (conc.) 3 ml
Distilled Water To make 1 liter
Other mineral medium concentrations al~o can be employed,
examples of which are provided in this disclosure in the Examples
sec~ion.
In either a batch, or in the preferxed continuous operation,
all equip~ent, reactor or fermentation means, vessel or other
container, piping, attendant circulating or cooling devices, and
the like, should be sterilized, such as by the employment o~
steam such as about at least 250 F. ~or at least sevexal minutes,
such as about 15 minutes, prior to actual staxtup. The sterilized
reactor then IS inoculated with a culture from my mixed culture
in the presence o~ all the required nutrients, and including the
molecular oxygen and the oxygenated hydrocarbon feed.
In the f0rmentation process, as the culture begins to grow,
the introduction of air or other molecular oxygen, nutrient medium,
14
nitrogen source if added separately, and the alcohol or other
oxygenated hydrocarbon, are maintained. The addition rate of the
feed stream or streams can be varied so as to obtain as rapid a
cell growth as possible consistent with the utilization of the
carbon and energy source input, so that the objective of a maxi-
mized high yield cell weight per weight of feed charged is obtained.
Of course, any of the feed streams can be added either incrementally
or continuously as desired ox convenient.
Instrumentation can be maintained to measure cell density,
pH, dissolved oxygen content, oxygenated hydrocarbon concentration
in the fermentor admixture, temperature, feed rates of input and
output streams, and the likeO It presen~ly is preferred ~hat
materials fed to the fermen~or be sterilized prior to introduction
into the fermentor. When the oxygena~ed hydrocarbon is a material
capable of sterilizing other makerials, such as ethanol, or
methanol, in some instances it may be convenient to add this
component to other streams, such as the mineral media, in steril-
izing amounts, and thus accomplish several purposes without the
necessity for separate heat sterilization of the min~ral media,
for example, thus providing as maximum and economical an operation
as possible. Heat added to any stream ultimately generally must
be taken out by cooling means in the fermentor, since the aerobic
fermentation is one in which heat is being produced.
The type of fermentor employed appears not to be critical
in the practice of the fermentation process employing the mixed
culture in accordance with my discovery. High productivity of
the mixed culture with alcohol appears to be best achieved in a
continuous process. Of course, watch must be maintained to con-
trol growth rates to avoid foam-out o~ the fermentor which could
lower the effective liquid volume and cause some loss of fermentor
contents. My unique mixed culture, has a distinct advantage, since
while it is a "foamer", it is not an excessive foamer, and liquid
levels can be more readily maintained in the fermentor without
.
~ 2~
dang~r of foam-outO Particularly, addition of antifoam to the
fermentation admixture is to be avoided, if at all possible, since
antifoams such as the silicones may be detrimental to the dissolved
oxygen content at the reco~nended high fermentation temperatures,
and may cause organisms to grow at a slower rate, or even to die.
The foam produced with my Mc mixed culture is not harmful to
growth, and definitely is beneficial in maintaining the organisms
in a system of high dissolved oxygen content. Foam helps provide
relatively large areas of gas/liquid contacting interfaces. ~hus,
fermentation can be improved and heat transfer improved as to
control, uniformity, and avoidance of hot spots.
Of course, a foam-inducing substance such as some of the
detergents, preferably nonionic, could be employed, if desired,
to assist or induce additional foaming, though normally this is
not necessary, or even desirable, with ~he Mc mixed culture of my
discovery.
Product Recovery
The recovery of microbial cells from my fermentation process
can be accomplished by the usual techniques, such as acidification
of fermentation effluent to a pH of such as about 4, and heating
the acidified effluent to a temperature suitable to kill the
microorganisms, such as about 80 C., though low enough not to
damage the protein product. The effluent then can be centrifuged,
washed, and recentrifuged to recover the microbial cells from the
ermentor effluent. The cells can be treated to cause lysis to
expedite recovery of protein and other materials from the cell.
The fermentation also produces a number o~ desirable by-pxoducts
which can be recovered from th~ fermentor effluent. These extra-
cellular produats can be economically helpful in the overall
process, since they include valuable products such as poly-
saccharides, amino acids, such as glutamic acid, enzymes, vitamins,
and the like.
The single cell protein product in accordance with my process,
16
is a valuable source of protein for humans as well as for animals.
For human consumption, the cells can be trea~ed to reduce the
nucleic acid content, if desired, though for animal feed purposes
such ~reatment does not appear necessary.
EXAMPLES
Examples following are intended to be descrip~ive of runs
employing ~he novel mixed cul-ture of my discovery. Particular
amounts and materials, or alcohols employed, should be considered
as illustrative, not as limitative of my invention.
XAMPLE I
A continuous fermentation run utilizing the thermophilic
mixed culture of the instant invention was carried out. ~ 7 liter
fermentor equipped with an aerator, stirrer, dissolved oxygen
monitor, and means for measuring and controlling temperature and
pH of the fermentation mixture, was charged with about 500 ml of
~ermentation reaction mixture from a previous fermentation run
utilizing the thermophilic mixed culture, and 10 ml of methanol
as the inoculant. The reactor also was charged with 2 liters of
fermentation mineral medium FM-12.
~he stirring rate was maintained at 1,000 rpm throughout
the course or the run and the p~ was controlled at from 6.2 to
6.35 by addition as necessary of ammonium hydroxide solution.
~ir wa~ introduced into the fermentor at a rate o~ 2 liters per
minute throughout the course o~ the run. At 6 hours into the run
essentially pure oxygen also was introduced to the fermentor at a
rate of 0.5 liters per minute, then was increased to 0 75 liters
per minute at 118 hours, to 1.5 liters per minute at 174 hours
and to 2 liters per minute at 190 hour~ duriny the run.
The medium continuously charged initially was the FM-12
medium previously descri~ed. At 22 hours, the mineral medium
was changed to an aqueous composition of 7~5% by volume methanol
in addition to the FM-12 medium plus 0.75 grams of potassium
chloride~ twice the normal trace mineral content, three times
the normal manganese component content (all on a per liter basis),
and with dele~ion of sodium chloride. A~ 166 hours, tha feed was
changed to an aqueous composition of 10% by volume methanol, 2.5
ml phosphoric acid (85%) per liter, 2 grams per liter potassium
chloride, 1.75 grams per liter magnesium sulfate 7H20, 0.25
grams per liter of calcium chloride .2H20, 20 ml per liter of a
manganese sulfate H2O aqueous solution (0.3 g/l), and 35 ml per
liter of the trace mineral solution previously described.
The media feed rate during the run ranged from 700 ml per
hour at 22 hours to 817 ml per hour at 118 hours, 781 ml per hour
at 166 hours, and 763 ml per hour at 382 hours.
Samples of the fermentation efluent were with~rawn from
time to ~ime to recover the cells therefrom. Values obtained
for cell content in terms of dry weight of cells in grams per
liter and ~he yields calculated are presented below in Table I.
Also shown in the table are values calculated for productivity
in terms of grams of cells per liter per hour for the fermentation
process.
At about 126 hours into the run, samples of the fermentation
admixture were withdrawn and prepared for lyophilization o~ the
microbial cells according to procedures known in the art. These
lyophilized samples then were stored for subsequent use in a
later fermentation run and for supplying samples of HTB-53 to a
deposi~ory for microorganims operated by the United States
Department of Agriculture, Northern ~egional Research Laboratory
at Peoria, Illinois.
Periodic samples also were tak.en from the fermentation reac-
tion mixture for microscopic examination o~ the microbial cells
in terms of their gross morphology. Such microscopic examination
showed that the Mc culture was composed of a large Gram positive
curved rod, a large Gram negative rod, and a small Gram negative
rod. Occasionally, a large Gram positive rod (not curved) also
was observed, but this was believed to be a transitory variant of
18
the large curved Gram positive rod.
Table I
Time, Hours46 118 166 190 Z86 358
Retention
Time, Hours2.36 2.27 2.38 2.83 2.33 2.43
Cells(a)
g/l 2~.86 27.13 27.04 35.21 35.53 35.57
Solids(b)
g/l 26.82 27.66 27.87 35.5 36.66 36.76
10 Cell Yieldtc)
~ 44.7 46.1 46.47 44.4 45.8 45.9
Productivity(d)
g/l/hr 1104 12.2 11.7 12.5 15.7 15.1
(a) Value obtained by evaporating a 10 ml sample of
fermentex effluent overnight at 100 C and subtractlng
weight of mineral solids contained in 10 ml of medium.
(b) Value obtained by centrifuging 100 ml sample of fermentor
effluent, resuspending solids in distilled water and
centrifuging again to recover solids which are dried
overnlght at 100 C.
(c) Value obtained by dividing recovered solids (g/l) by
methanol charged (g/l)x 100.
(d) Value obtained by dividing recovered solids (g/l) by
retention time (hr).
After about one month, a single tube of the lyophilized HTB-53
microbial culture from the fermentation run of Example I a~ove was
opened aseptically by conventional procedures and added to 100
ml of a fermentation medium designated IM2 which also contained
0.5~ methanol. The composition of medium IM2 is ~hown below.
IM2 ~edium
C9 ~S~Y~ Amou_t, g
XH2PO4 2.0
K2HPO4 3.0
MgS4 7H20 0.4
CaC12 2H20
Narl 0. 1
(NH4)2so4 2.0
Trace Mineral Solution 0.5 ml
Distilled Water To make 1 liter
19
. . ..
- ... . : . : .
; .. :" ' '
~6~
The flask charged with the revived lyophilized culture was incu-
bated with shaking a~ 55 C. After 24 hours, the shake flask
showed good growth of the culture and 5 ml of the mixture was
transferred to 100 ml of medium IM2 also containing 1.5% by volume
methanol. Good growth developed within 24 hours and a third tran~-
fer was made to the same medium in two flasks, each containing
500 ml of IM2 medium plus 1.5% by volume methanol. This third
transfer involved 100 ml of the culture being added ~o each of
the two flasks. The culture was allowed to grow for 32 hours and
then was utilized as the inoculant for a continuous fermentation
run in the apparatus described above in Example I. The fermentor
was charged with 1,000 ml of FM-12 mediuml and 1,000 ml of the
inoculant to which was added 10 ml of methanol. The temperature
was maintained a~ 55 C, and the pH was controlled at from 6.25
to 6.4 by the continuous addition of ammonium hydroxide solution
as before. Initially the stirrer was operated at 300 rpm, and
air was introduced at a rate of 0.5 liters per minutel while the
culture was permitted to establish itsel in the fermentor. After
7 hours, a continuous introduction of feed media having the same
composition as the last named feed media shown in Example I above
was introduced. In addition, the air rate was increased to 2
liters per minute while the rpm was set at 1,000 for the stirrex~
After 30 hours, the air rate was reduced to 1.75 liters per
minute, while oxygen was introduced at 0.75 liters per minute,
later increased to 1 liter per minute at 54 hours, and to 1.5
liters per minute at 198 hours. Again the fermentation effluent
was sampled from time to time to provide data on the cell content
in terms of grams per liter based on a dry weight and in terms
of the yield and productivity of the fermentation. The data
obtained during the run are presented below in Table II.
.
Table II(a)
Time, Hours 70 166 190 214
Retention
Time, Hours 2.60 2.70 2~63 2.64
Cells
g/l 3~.33 33.56 33.87 34.8
Solids
g/l 34.44 35.4 34.55 35~52
Cell Yield
~ 43.6 44.2 43.2 44.2
Productivity
g/l/hr 13.2 13.1 13.1 13.5
(a) See footnotes for Table I above.
Samples of the mi.crobial culture were also obtained during
the fermentation run for microscopic examina~ion as described in
Example I. In this instance, there was also obser~ed the large
Gram positive curved rods and the large and small Gram negative
staining rods. At 238 hours into the run, the culture was lost
while attempting to change ~he feed to a higher alcohol concentra~
tion.
Example III
Presented below in Table III are analytical data character~
izing ~ha microbial ~ulture obtained in the run of Example I in
terms o a chemical analysis of the microbial cells recovered. In
Table IV there is also presented an amino acid content analysis
of microbial cells recovered from another fermentation run util-
izing the mixed thermophilic culture of my invention. For purposes
of comparison, an amino acid content analysis of a pure thermo-
philic microorganism obtained during the course o the isolation
; 30 of the thermophiles from the initial soil sample previously
described is also presented in Tahle IV.
. .
6~
Table III
Chemical Analysis of Microbial
Cells Obtained in A Run With HTB-53
Crude Protein(a), wt. % 85.63
Ash, wt. % 9.19
Amino nitrogen, wt. % 13.4
Carbon, wt. ~ 44.7
Hydxogen, wt. % 6.79
Nitrogen, wk. % 13.7
Phosphorous, wt. % 1.71
Potassium, wt. % 0.91
Magnesium, wt. ~ O.26
Calcium, wt. % 0.1
Sodium, wt. ~ < 0.01
Iron, ppm 1300
Zinc, ppm 55.8
Manganese, ppm 126
Coppex, ppm 20
(a) Nitrogen content (13.7) x 6.25.
TabIe IY
Amino Acid Content of Thermo~hilic
Cultures Grown on MethanoI- Grams
.
Per 100 Grams Product
_ _ _ .
Chem ~1)( Score(l)
Essential Amino Acids Pure ValuesMixed Values
(HTB-7)
leucine 5.37 75 5.76 104
isoleucine 4.56 83 4.90 118
30 lysine 5.54 96 5.67 141
methionine 1.50 f 1.22
34 ~36
cystine * * t
~hreonine 2.95 90 2.79 88
phenylal~nine 2.68 ( 2.78
: 80 88
tyrosine 2.55 2.72
tryptophan 0.60 43 0.89 90
valine 5.13 91 5.46 121
(*~= not detected)
`
22
,, :- , ~
,
&~
Table IV (continued)
Amino Acld Content of Thermophilic
Cultures Grown on Methanol _ Grams
Per lO0 Grams Product
. _
Chem.Chem.
Score(1)(2) score(l)
Essential Amlno Acids Pure _ Values~-HTB-~2) -
alanine 5.65 6.19
lO arginine 3.39 3.01
aspartic acids 6.50 6.26
glycine 3.71 4.19
glutamic acid 10.47 10.69
histidine 1.26 1.22
proline 2.32 2.38
serine 2.09 1.75
Total Essential
amino acids 30.88 32.19
Total ~mino Acids66.27 67.88
Crude Protein 85.63 84.4
(l) Chemical Score Values: based on essential amino acid
content of egg as 100 for same weight of protein.
(2) Based on averages from five pure thermophiLe runs.
It can be noted that the percentage of total amino acids
which are essential amino acids is slightly higher for the
mixed culture product (47%) than for the pure culture product
(46%). Furthermore, if the essential sulfur-containing amino
acids are supplied by addition of synthetic methionine, which
is very likely since essentially all SCP's have been ~ound to
30 ba low in these amino acids, the Chemical Score values show that
the mixed culture product is twice as good from a nutritional
standpoint as the pure culture product based on the next lowest
essential amino acid Chemical Score value.
The disclosure, including data, illustrate the value and
effectiveness of my invention. The examples, knowledge and
~3
background of the field of the invention, general principles
of microbiology, chemistry, and other applicable sciences, have
formed the bases from which the broad descriptions of my inven-
tion, including the ranges of conditions and generic groups of
operant componen~s have been developed, which have formed the
bases for my claims here appended.
24
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