Note: Descriptions are shown in the official language in which they were submitted.
37
HIGH ETHANOL PRODUCING DERIVATIVES OF THERMOANAEROBACTER ETHANOLICUS
Technical Field
This invention relates to derivative stralns of a ne~ly discovered
microorganism Thermoanaerobacter ethanolicus, a thermophilic anaerobe.
More speciflcally, this invention relates to producing ethanol using
derivatives of the microorganism Thermoanaerobacter ethanolicus.
Background Art
The microorganism Thermoanaerobacter ethanolicus has been described
as an extreme thermophilic, non-spore forming anaerobic bacterium which
ferments a variety of carbohydrates to ethanol as the main product
(Wiegel, J. and LJungdahl, L.G., Arch. Microbiol. 128, 343 - 348, 1981).
Relatively few thermophilic anaerobic bacteria, which ferment substrates
such as starch, cellobiose, glucose, xylose, and other sugars to ethanol
as the main product, have been reported. The microorganism
Thermoanaerobacter ethanolicus is described in U.S. patent No. 492929407,
September 29, 1981 of Ljungdahl et al. A related patent is U.S. patent
No. 4,292,406, September 29, 1981 of LJungdahl et al.
--2--
Disclosure of Invention
The present invention overcomes problems of fermentations
yielding low concentrations o-F ethanol using the microorganism
Thermoana robacter ethanolicus.
The present invention produces ethanol at a substrate concen-
tration in a fermentation medium greater than 1~ (w/v) using bio-
loyically pure cultures of derivatives of the microorganism Ther-
moanaerobacter ethanolicus. The present invention provides deriv-
atives having the ability to ferment substrates such as starch,
cellobiose, glucose, xylose and other sugars to ethanol under
anaerobic and thermophilic conditions in an aqueous~ nutrient
medium obtaining ethanol concentrations high enough to make contin-
uous fermentations possible. Specifically, substrate can be added
to a fermentation medium and ethanol can be removed therefrom
during the same continuous fermentation.
Microorganism strains of Thermoanaerobacter ethanolicus desig-
nated by the following accession numbers: ATCC 31550, ATCC 31936,
ATCC 31g37, and ATCC 31938, respectively, are on deposit with the
American Type Culture Collection, 12301 Parklawn Drive, Rockville,
Maryland 20852, U.S.A. The strain designated ATCC 31550 was
deposited with the American Type Culture Collection on August 9,
1979. Strains designated ATCC 31936, ATCC 31937, and ATCC 31938,
respectively, were all deposited with the American Type Culture
Collection on August 11, 1981. All of the above-referenced
deposits were made pursuant to the Budapest Treaty.
The derivative strains, produced both through induced muta-
yenesis and multiple selections for spontaneous mutations (de-
scribed in Examples I, II, and III below) differ from the parent
strain, strain JW200 of the microorganism Thermoanaerobacter etha-
nolicus (ATCC 31550), in that the production of ethanol continues
at a high rate although the ethanol concentration in the fermenta-
tion medium is greater than 1~ (w/v) and substrate concentrations
in the fermentation medium are greater than 1% (w/v). The follow-
ing derivative strains are representatives of a large number of
37
derivative strains obtained from the parent strain, strain JW200 of the
microorganism Thermoanaerobacter ethanolicus tATCC 31550). These
biologically pure cultures of the representative derivative strains,
speciEically, strain JW200L-Large of the microorganism Thermoanaerobacter
_hanolicus (ATCC 31936) representative of ultraviolet light mutagenesis
and pyruvate selection; strain JW200Fe(3) of the microorganism
Thermoanaerobacter ethanolicus (ATCC 31937) representative of iron
deprivation selection and low growth on pyruvate; and strain JW200L-Fe(7)
of the microorganism Thermoanaerobacter ethanolicus (ATCC 31938)
representative of ultraviolet light mutagenesis, iron deprivation and low
growth on pyruvate, are ethanol tolerant up to 10% (v/v) ethanol in the
fermentation medium. The foregoing representative derivative strains
produce ethanol at ethanol concentrations in a fermentation medium greater
than 1% (w/v) whereas the parent strain does not produce ethanol at ethanol
concentrations in a fermentation medium greater than 1% (w/v). The parent
strain, strain JW200 of the microorganism Thermoanaerobacter ethanolicus
(ATCC 31550), tolerates ethanol concentrations of up to 10~ (v/v) ethanol
in the fermentation medium only if given time to grow in low concentrations
first. The above-referenced derivative strains do not show this
characteristic of the parent strain, strain JW200 of the microorganism
Thermoanaerobacter ethanolicus (ATCC 31550).
Accordingly, the present invention seeks to produce ethanol at
substrate concentrations in a fermentation medium above 1% (w/v) using
biologically pure cultures of derivatives of the microorganism
Thermoanaerobacter ethanolicus.
Further, the invention seeks to continuously produce recoverable
amounts of ethanol from substrates such as starch, cellobiose, glucose,
xylose, and other sugars.
The invention in one aspect pertains to a biologically pure culture
of a derivative of the microorganism Thermoanaerobacter ethanolicus having
the ability to continuously produce recoverable amounts of ethanol such
that a substrate can be added to a fermentation and the ethanol can be
removed therefrom during a fermentation under anaerobic and thermophilic
conditions in an aqueous nutrient medium containing the substrate at a
3~
substrate concentration in the fermentation medium greater than 1% (w/v)
wherein the substrate is selected from the group consisting of starch,
pectin, glycerol, pyruvate, monosaccharides, and disaccharides.
Another aspect of the invention comprehends a process for continuously
producing recoverable amounts oE ethanol such that a substrate can be added
to a fermentation and the ethanol can be removed therefrom during a
fermentation which comprises subjecting an aqueous nutrient medium
containing the substrate at a substrate concentration in the fermentation
medium greater than 1% (w/v) wherein the substrate is selected from the
group consisting of starch, pectin, glycerol, pyruvate, monosaccharides,
and disaccharides, under anaerobic and thermophilic conditions to the
fermentation action of a derivative of the microorganism Thermoanaerobacter
ethanolicus.
These and other aspects and advantages of this invention will become
apparent from a consideration of the accompanying specification and claims.
Modes for Carrying out the Invention
The parent strain, strain JW200 of the microorganism Thermoanaerobacter
ethanolicus ~ATCC 31550), described in U.S. patent No. 4,292,40~ referred
to above is an extreme thermophilic, non-spore-forming anaerobic bacterium
which ferments a variety of substrates such as starch, cellobiose, glucose,
xylose, and other sugars to ethanol, carbon dioxide, lactate, acetate and
hydrogen gas. When growing in an aqueous nutrient medium containing 10
g per liter (1% w/v) or less of substrate the major products are ethanol
and carbon dioxide. If higher substrate concentrations are used, there
is a shift away from ethanol production to production of the other products.
Strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC
31550) under optimal conditions, produces 1.8 mol of ethanol per mol of
glucose. ~or example, at a concentration of 10 g per liter of glucose,
one approximately obtains 0.5% (w/v) ethanol. This level of ethanol is
not easily removed by known means of distillation; therefore, derivatives
of the parent strain, strain JW200 of the microorganism Ther _anaerobacter
ethanolicus (ATCC 31550), altered to grow at high substrate concentrations
to yield ethanol above 1% (w/v) are useful. The representative derivatives
.~
37
--5--
mentioned above, strain JW200L-I.arge of thc microorganism li rmoan_erobclc~cr
ethanolicus (ATCC 31936) strain JW200Fe(3) of the microorganism
Thermoanaerobacter ethanolicus (ATCC 31937), and JW200L-Fe(7) of the
microorganism Thermoanaerobacter ethanolicus (ATCC 31938), produce ethanol
at levels in the fermentation medium greater than 1.5% (w/v).
The following are microorganisms, culture methods, fermentations,
strain maintenance procedures, morphology and taxonomy of the derivatives,
isolation of the parent strain, strain JW200 of the microorganism
Thermoanaerobacter ethanolicus.
(a) Organisms. The organisms are cultures of the parent strain,
strain JW200 of the microorganism Thermoanaerobacter thanolicus
(ATCC 31550), the parent strain, and its high ethanol producing derivatives~
specifically, strain JW200L-Large of the microorganism Thermoanaerobacter
ethanolicus (ATCC 31936), strain of JW200Fe(3) of the microorganism
Thermoanaerobacter ethanolicus (ATCC 31937), and strain JW200L-Fe(7) of
the microorganism Thermoanaerobacter ethanolicus (ATTC 31938).
(b) Culture Methods. The foregoing strains are routinely grown or
cultivated under anaerobic and thermophilic conditions, specifically 68DC
and pH between 5.7 to 8.6 in anaerobic tubes (Bellco Glass Co., Vineland,
N.J., no. 2046 - 18142) in the aqueous nutrient medium described in U.S.
patent No. 4,292,407 referred to above and Arch. Microbiol. 128, 343 - 348,
1981 and which is known in the art. Extruded cracked corn was used at
concentrations of 20% (w/v) as a rapid indicator of hydrolysis of starch
by the derivative strains. When concentrations of substrates exceeded 5%
(w/v) yeast extract in the aqueous nutrient medium was increased to .6%
(w/v). All of the aforementioned derivative strains produce mainly ethanol
and only small amounts of lactate and acetate as fermentation products.
Additionally, all the aforementioned derivative strains produce ethanol
at a substrate concentration in the fermentation medium greater than 1%
(w/v). Routine growth includes but is not limited to the following
substrates: soluble and insoluble starch, pectin, disaccharides sucll as
cellobiose, sucrose, lactose, and maltose, monosaccharides sucil as glucose,
fructose, mannose, galactose, xylose, and ribose, and glycerol and pyruvate
wherein the substrates are fermented to cthanol in the aqueous nutrient
~;
~L
37
--6--
medium.
(c) Fermentations. Fermentations of the aforementioned substrates
using the derivative strains continuously produce recoverable amounts of
ethanol. More specifically, substrate can be added to a fermentation using
the derivative strains, and ethanol can be removed therefrom during the
same continuous fermentation. Fermentations are conducted under anaerobic
and thermophilic conditions or under anaerobic and extreme thermophilic
conditions. Extreme thermophilic conditions are understood to mean that
fermentations are at 70C or higher. Fermentations are conducted at a pH
range of about 5.7 to 8.6 and at a temperature range between about 40C
and 78C. Thus a process for continuously producing recoverable amounts
of ethanol such that a substrate can be added to a fermentation and the
ethanol can be removed therefrom during a fermentation is provided. This
process comprises subjecting the aqueous nutrient medium containing the
substrate at a substrate concentration in the fermentation medium, greater
than 1% (w/v), wherein the substrate includes but is not limited to the
following substrates: soluble and insoluble starch, pectin, disaccharides
such as cellobiose, sucrose, lactose, and maltose, monosaccharides such
as glucose, fructose, mannose, galactose, xylose, and ribose, and glycerol
and pyruvate, to the fermentation action of a derivative of strain JW200
of the microorganism Thermoanaerobacter ethanolicus (~TTC 31550) wherein
the derivative is derived through both induced and spontaneous mutagenesis.
The process includes recovering ethanol from the fermentation medium.
Distillation means may be used to recover ethanol from tile fermentation
medium under anaerobic and thermophilic conditions, or an inert carrier
gas such as nitrogen bubbled through the fermentation vessel may carry the
ethanol from the fermentation medium into a condensation unit.
(d) Strain ~laintenance Procedure. Stock cultures of all of the
aforementioned strains were maintained Eor 1 to 2 months at room
tempcrature, about 25C, after growth at 68C in the aqueous nutriellt mediurn
supplemented with 20% (w/v) cracked corn or by adding an equal amoullt of
sterile anaerobic glycerol to a growing culture and storing the mixture
at 20C for at least 18 months.
--7--
(e) Morphology and Taxonomy of the Derivatives. All of the derivative
strains exhi~it the morphologic and taxonomic characteristics of the parent
strain, strain JW200 of the microorganism Thermoanaerobacter ethanolicus
(ATCC 31550) described in U.S. patent No. 4,292,407 referred to above.
(f) Isolation of the Parent Strain, Strain JW200 of the microorganism
Thermoanaerobacter ethanolicus (ATCC 31550). The parent strain, strain
JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550) can
be isolated by the methods described in U.S. patent No. 4,292,407 referred
to above and Arch. Microbiol. 1 , 343 - 348, 1981, and which is known in
the art. Samples of the parent strain, strain JW200 of the miCroOrganiSm
Thermoanaerobacter ethanolicus (ATCC 31550) can also be obtained from the
American Type Culture Collection (ATCC) Rockville, 12301 Parklawn Drive,
Maryland, 20852, U.S.A.
Example I
Ultraviolet Light Mutagenesis
Strain JW200 of the microorganism Thermoanaerobacter ethanolicus (ATCC
31550), the parent strain, was grown in the aqueous nutrient medium with
1% (wtv) soluble corn starch to early log phase, then transferred to a 3
ml quartz cuvette pregassed with argon and rinsed with reducing solution.
The cells were held under a bacteriostatic UV light source at 10 cm for
times up to 3 minutes. Samples were withdrawn every 30 seconds and diluted
and rolled out (a type of plating technique for anaerobic microorganisms
known in the art) in 2% (w/v) agar containing 2% (w/v) soluble corn starch.
Individual colonies were analyzed randomly since most produced alcohol
Strain JW200L of the microorganism Thermoanaerobacter ethanolicus was
derived from a UV treatment of 60 seconds. It produced in excess of 80
mM ethanol at 2% (w/v) starch. This strain was further selected for
spontaneous mutations by iron deprivation and low growth on pyruvate to
give strain JW200L-Fe(7) of the microorganism Thermoanaerbacter ethanolicus
(ATCC 31938). Strain JW200L-Large of the microorganism Thermoanaerobacter
ethanolicus (ATCC 31936) was derived from strain JW200L of the microorganism
Thermoanaerobacter _thanolicus and further selected for good growth on
pyruvate.
~ AJ3~7
Example II
Iron Deprivation
Ferredoxins, which are iron containing proteins, have been
reported to be involved in ethanol production in some thermophilic
anaerobic bacteria; therefore, 0.1 ml of a culture of strain JW200
of the microorganism Thermoanaerobacter ethanolicus (ATCC 31550)
was transferred to 1 ml of the aqueous nutrient medium supplemented
with 1% (w/v) starch and containing no iron. The culture was
incubated at 60C for 30 minutes. At that time 250 ug/ml FezS34
was added and incubation was continued for three days. Growth
after 3 days was low. These cells were diluted 1/100 into the
aqueous nutrient containing 2% (w/v) starch grown at ~8C and
assayed for ethanol after 24 hours. Cells from the culture strain
JW200Fe of the microorganism Thermoanaerobacter ethanolicus,
derived as described above, from 2CG (w/v) starch produced 80 mM
ethanol and 4 mM acetate over 24 hours.
The production of ethanol by strain J~200 of the microorgan-
ism Thermoanaerobacter ethanolicus (ATCC 31550), by the strain
.
JW200L (described in Example I) oF the microorganism Thermoanae-
robacter ethanolicus and by strain JW200Fe of the microorganism
Thermoanaerobacter ethanolicus with increasing starch concentra-
tions is given in Table I below.
9 1~ 3~
TABLE 1
.
Ethanol production from starch at increasing concentrations by two
derivatives of strain JW200 of the microorganism Thermoanaerobac-
ter ethanolicus (ATCC 31550) and the parent strain, strain JW200
of the microorganism Thermoanaerobacter ethanolicus (~TCC 31550).
Strains of Thermoanaerobacter ethanolicus
. .
(ATCC 31550)
JW200 JW200L JW200Fe
10STARCH g/l ETHANOL mM ETHANOL mM ETHANOL mM
58 59 59
82 98 82
210 90
8 320 110
100 5 242 - 245
140 11 142 360
Example III
Pyruvate Selection
Strain JW200L (derived as described in Example I) of the
microorganism Thermoanaerobacter ethanolicus, strain JW200Fe
(derived as described in Example II) of the nicroorganism Thermo-
anaerobacter ethanolicus, and strain JW200L-Fe (derived by treat-
ment described in Example I followed by treatment described in
Example II) of the microorganism Thermoanaerobacter ethanolicus,
were grown in the aqueous nutrient medium with 2% (w/v) solid agar
supplemented with 1% (w/v) pyruvate and both 0.05% (w/v) glucose
and xylose. Large and small colonies of strain JW200L of the
microorganism Thermoanaerobacter ethanolicus, of strain JW200Fe of
the microorganism Thermoanaerobacter ethanolicus, and of strain
JW200L-Fe of the microorganism Thermoanaerobacter ethanolicus were
selected. Each colony was grown in the aqueous nutrient medium
supplemented with both 1% (w/v) cellobiose and 1% (w/v) xylose.
--10--
The best strains were then transferred to the aqueous nutrient medium with
20~ (w/v) cracked corn to test for the ability to grow at high substrate
concentrations. The best cultures were transferred to the aclueous nutrient
medium with increasing concentrations of soluble corn starch. The ethanol
production was measured after four days and is shown in Table II below for
representative strains (derivative strains). Samples of these derivative
stralns, specifically strain JW200L-Large of the microorganism
Thermoanaerobacte ethanolicus (ATCC 31936), strain JW200Fe(3) of the
microorganism Thermoanaerobacter ethanolicus (ATCC 31937) and strain JW200L-
Fe(7) of the microorganism Thermoanaerobacter ethanolicus (ATCC 31938),
can be derived as described in Example I, II and III above or samples can
be obtained from the American Type Culture Collection (ATCC) Rockville,
12301 Parklawn Drive, Maryland, 20852, U.S.A.
Table II
E~hanol production from starch for derivatives of strain JW200 of themicroorganism Thermoanaerobacter ethanolicus (ATCC 31550).
Derivative Strains of Thermoanaerobacter ethanolicus
(ATCC 31937)(ATCC 31936)(ATCC 31938)
JW200Fe(3) JW200L-LargeJW200L-Fe(7)
STARCH g_ ETHANOL mM ETHANOL mM ETHANOL mM
31 336 l75 315
62 470 246 195
200 416 210 150
25 242 272 205 180
-
~ L~L8 ~3~3~
Strain JW200L-Large of the microorganism Thermoanaerobacter
ethanolicus tATCC 31q36), strain JW200Fe(3) of the microorganism
Thermoanaerobacter ethanolicus (ATCC 31937), and strain JW200L-Fe-
(7) of the microorganism Thermoanaerobacter ethanolicus (ATCC-
31938) have been maintained without reversion for a period of over
six months.
(g) Aqueous Nutrient Medium. The aqueous nutrient medium
referred to in (b) Culture Methods, above, used for isolation,
growth or cultivation, and to maintain the isolated parent and
derivative strains of Thermoanaerobacter ethanolicus has the
following preferred composition: KH2P04, 1.5 9/l; Na2HP04 12H20,
4.2 9/1; NH4Cl, 0.5 9/1; ~IgC12, 0.18 gil; yeast extract (Difco),
2.0 9/1; glucose, 8.9 g/l; and Wolfe's mineral solution, 5 ml.
The medium is prepared under anaerobic conditions and must be
stored under an atmosphere of an inert gas, such as nitrogen or
argon. The pH of the medium is in the range of about 6.~ and 7.8,
preferably 7.3, and is adjusted as required with a sterile, anae-
robic NaOH or HCl solution.
(h) Thermoanaerobacter ethanolicus Morphology and Toxonomy.
The following more particularly describes (e) ~lorphology and Tox-
onomy, abcve:
(i) Morphology: Cells grown at 60 C. show tumbling
motility. Older cells, or grown in liquid or at higher tempera-
tures, such as 75 C., show less motility although they are flag-
ellated. The flagellation is of the retarded peritrichous type,
with between 1 and 10 flagella that are up to 80 ~m long. Young
cell rods during logarithmic growth often show pointed ends. These
rods are 4 to 8 ~m long and 0.6 to 0.9 ~m thick. Cell rods of the
late logarithmic growth phase can grow up to 200 ~m long, which
then may divide into chains of bacteria during the beginning of
the stationary growth phase;
(ii) Spores: Spores are not formed;
(iii) Other Characteristics: The strains are strictly
anaerobic, gram-variable, catalase-negative, pyruvate is metabo-
-12- ~L~L~ o~ ~
lized via pyruva-te-ferredoxin reductase system. Two anaerobic
ferredoxins and a rubredoxin are present; and
(iv) Taxonomy: The strains have some characteristics
similar to Clostridiuln thermohydrosulFllricum, the only other known
extreme thermophilic, anaerobic, glycolytic bacteria. However,
the new strains do not form spores, thus excluding them from the
genus Clostridium. Other properties described in this application
appear to exclude the new strains from previously identified gen-
era that are described in the 8th edition of Bergey's t~anual ofDeterminative Bacteriology (Williams and Wilkins Comp. Baltimore)
1974.
Therefore, the new strains represent a new genus and new
species that has been named Thermoanaerobacter ethanolicus, wherein
ATCC 31550 is representative of these strains.
(i~ Isolation of Thermoanaerobacter ethanolicus. The spe-
cific method of isolation, (f) Isolation of the Parent Strain,
Strain JW200 of the microorganism Thermoanaerobacter ethanolicus
(ATCC 31550), referred to above, is more particularly described as
follows: samples from the hot sprinss of Yellowstone National
Park were collected under sterile anaerobic conditions; one g of
samples were used to incubate 100 ml of the nutrient medium
described hereinabovei the samples were incubated at 74 C.; after
3 days dilution rolls were made using anaerobic tubes in accord-
ance with the Hungate technique as modified by Bryant and Robinson;after 2 days incubation at 74 C., agar shakes were made from the
tubes of the highest dilution which exhibited growth; the nutrient
medium, supplemented with 2% agar was used for the agar cultures;
the agar shakes were rolled out and incubated at 60 C. in a 30
slanted position; and final strain cultures were obtained by
selecting single colonies and repeating the agar shake procedure
several times.
3~
-13-
The foregoing examples illustrate specific embodiments within
the scope of this invention and are not to be construed as limi-ting
said scope. While the invention has been described herein with
regard to certain specific embodiments, it is not so limited. It
is to be understood that variations and modifications thereof may
be made by those skilled in the art without departing from the
scope of the invention.
Industrial Applicability
This invention is useful in fermenting starch~ cellobiose,
glucose, xylose, and other sugars to ethanol.
