Sugars are converted to a mixture of solvents including butanol by a fermentation process employing a coculture of microorganisms of the Clostridium genus, one of said microorganisms favoring the production of butyric acid and the other of which converts the butyric acid so produced to butanol. The use of a coculture substantially increases the yield of butanol over that obtained using a culture employing only one microorganism

Bergstrom, S. L.

Foutch, G. L.

English

NASA Technical Reports Server

United States Patent 
Bergstrom et al. 
1111 Patent Number: 4,539,293 
[45] Date of Patent: Sep. 3, 1985 
[54] PRODUCTION OF BUTANOL BY 
FERMENTATION IN THE PRESENCE OF 
COCULTURES OF CLOSTRIDIUM 
[75] Inventors: Sheryl L. Bergstrom, Duarte, Calif.; 
Gary L. Foutch, Stillwater, Okla. 
[73] Assignee: The United states of America as 
represented by the Administrator of 
the National Aeronautics and Space 
Administration, Washington, D.C. 
[21] Appl. No.: 493,179 
[22] Filed: May 10, 1983 
[51] Int. Cl.3 ......................... C12P 7/16; C12R 1/145 
[52] US.  C1. ..................................... 435/160; 435/842 
[58] Field of Search ................... 435/41, 42, 150, 155, 
435/160, 161, 162, 813, 820, 842 
~561 References Cited 
U.S. PATENT DOCUMENTS 
4,400,470 8/1983 Zeikus et al. ....................... 435/162 
4,424,275 1/1984 Levy ................................... 435/160 
Primary Examiner-Sidney Marantz 
Assistant Examiner-C. Lee Foulke 
Attorney, Agent, or Firm-Paul F. McCaul; John R. 
Manning; Thomas H. Jones 
[571 ABSTRACT 
Sugars are converted to a mixture of solvents including 
butanol by a fermentation process employing a cocul- 
ture of microorganisms of the Clostridium genus, one of 
said microorganisms favoring the production of butyric 
acid and the other of which converts the butyric acid so 
produced to butanol. The use of a coculture substan- 
tially increases the yield of butanol over that obtained 
using a culture employing only one microorganism. 
4 Claims, No Drawings 
https://ntrs.nasa.gov/search.jsp?R=19850026914 2020-03-22T20:23:13+00:00Z
4,539,293 
1 
PRODUCTION OF BUTANOL BY 
FERMENTATION IN THE PRESENCE OF 
COCULTURES OF CLOSTRIDIUM 
BACKGROUND OF T H E  INVENTION 
Origin of the Invention 
The invention described herein was made in the per- 
formance of work under a NASA Contract and is sub- 
ject to the provisions of Section 305 of the National 
Aeronautics and Space Act of 1958, Public Law 85-568 
(72 S T A T  435; 43 USC 2457). 
Field of the Invention 
This invention relates to the production of butanol by 
fermentation of sugars in the presence of two different 
microorganisms of the Clostridium genus. 
Background Discussion 
Butanol may be produced by the fermentation of 
carbohydrates which break down into sugars, for exam- 
ple, sugars containing five and six carbon atoms such as 
glucose. This process was developed by Dr. Charles 
Weizmann during World War I and was the principal 
source of butanol until this chemical was produced 
from low-priced hydrocarbon feedstocks. T o  a large 
extent this process has been replaced, but it is still used 
on a commercial scale in the Union of South Africa. 
Nevertheless, with the increasing cost of hydrocarbon 
feedstocks plus the possible additional uses of butanol 30 
in, for example, oil field tertiary recovery processes, the 
demand for butanol is increasing and the Weizmann 
Process now has potential commercial application in 
the United States. 
a suitable feedstock such as molasses sugars in the pres- 
ence of Clostridium microorganisms which convert the 
sugar to a solvent mixture of butanol, acetone and etha- 
nol. Typically, from 29 to 33 weight percent of the 
fermentable carbohydrates are converted to these sol- 40 
vents. In the solvent mixture the usual ratio of butanol 
to  acetone to ethanol is approximately 6:3:1. The pro- 
cess is a batch process as opposed to a continuous pro- 
cess, and, when fermentation is completed, the solvent 
SUMMARY OF THE INVENTION 
We have discovered that the yield of butanol from 
the Weizmann Process may be substantially increased, 
for example, by as much as 20% or even more, by using 50 
a coculture of at least two different Clostridium species. 
One of these species should favor the production of 
butyric acid and the other species should favor the 
conversion of this butyric acid to butanol. The two 
different species could be used concurrently in the same 55 
reaction vessel or in a multiple stage system wherein (1) 
the species which favors the production of butyric acid 
is in a first stage and acts on the feedstock to convert the 
fermentable carbohydrates to butyric acid and (2) in a 
second stage products from the first stage are fed into a 60 
second reaction vessel wherein the second species acts 
on the butyric acid to  convert it to  butanol. At  equilib- 
rium in the single stage system, the number ratio of the 
butyric acid forming species to butanol conversion spe- 
cies should range between about 1:3 and about 3:l. 
Although several species may be employed, we have 
found that Clostridium pasteurianum is particularly 
suited to  form the butyric acid. Also, but to a lesser 
5 
15 
20 
25 
Briefly, the Weizmann Process comprises fermenting 35 
mixture is separated from the spent materials. 45 
65 
2 
degree, the species Clostridium butyricum is also useful 
in forming the butyric acid. Two different species of the 
second microorganism which converts the butyric acid 
to butanol have been employed. The most preferred is 
Clostridium butylicum. In the preferred mode of practic- 
ing this invention, a coculture of the C. pasteurianum 
and C. butylicum species is employed in a single stage 
reactor to convert the feedstock to butanol, acetone and 
ethanol. Clostridium acetobutylicum may also be used in 
place of, or concurrently with, the C. butylicum species. 
In accordance with the most preferred way of prac- 
ticing the invention, a coculture of the C. pasteurianum 
and C. butylicum species are mixed wit11 glucose as the 
feedstock in the presence of a suitable nutrient and al- 
lowed to ferment until gas production terminates. Pref- 
erably, the mixture is buffered so that the pH of the 
reaction mixture remains in the range of from about 4.5 
to about 5.2. The most preferred temperature for incu- 
bation of the reaction mixture is about 37" C. This tem- 
perature permits the microorganisms to be most active. 
The amount of butanol produced by the process of 
this invention was compared with the amount of buta- 
nol produced by conventional processes. Results indi- 
cated that the process of this invention yielded an in- 
crease of at least 2070, which is very significant and 
represents a substantial economic benefit. For  example, 
the solvent mixture of butanol, acetone, and ethanol 
usually has, at a maximum, 60 weight percent butanol. 
With the process of this invention, the percentage of 
butanol is typically 70 weight percent or more in the 
solvent mixture. 
EXAMPLES 
The following presents experimental results using the 
cocultures of this invention to produce butanol by the 
Weizmann Process. The results of these experiments are 
set forth in Tables 1 and 2. Both tables present test data 
based on conducting the Weizmann Process using 
cocultures and single microorganism systems. Table 1 
presents data in which the reaction mixture was buff- 
ered with calcium carbonate. Table 2 presents data in 
which the reaction mixture was not buffered. Buffering 
is highly desirable in order to maintain the pH of the 
reaction mixture in the desired range. If the pH of the 
reaction mixture is outside of this range, the yield of 
butanol is substantially reduced. 
The fermentation process was conducted using con- 
ventional procedures, with the exception that cocul- 
tures were employed in accordance with the principle 
of this invention. The cocultures were grown and main- 
tained on Cooked Meat Media provided by the 
Baltimore Biological Laboratories and identified as No. 
11 128. This media was maintained in Brewer jars under 
anaerobic conditions. After an initial growth of approxi- 
mately 48 hours, the cultures were transferred to 30 
milliliter serum bottles containing Thioglycollate Me- 
dium without added dextrose. Two and one-half weight 
percent glucose was added to the Thyioglycollate 
media without dextrose. Also, a chip of calcium carbon- 
ate was added for the purpose of preventing a decrease 
in pH. The cultures were incubated at 37" C., with the 
sample being ventilated daily to  prevent buildup of gas. 
Correct conditions were maintained so that the micro- 
organisms grew under anaerobic conditions. The fol- 
lowing lists the microorganisms tested, which were 
supplied by the Northern Regional Research Center of 
the Department of Agriculture in Peoria, Illinois. 
3 
4,539,293 
4 
TABLE 2-continued 
MICROORGANISM TESTED 
Strain No. 
5 
1. Clostridium pasteurianum NRRL B598 
2. Clostridium butyricum NRRL B1092 
3. Clostridium butylicum NRRL B592 and 
NRRL B593 
4. Clostridium acetoburylicum NRRL B527 10 
Gas chromatography analytical procedures were 
employed to determine the quantities of ethanol, ace- 
tone, acetic acid, butyric acid, and butanol produced. A ,5 
Hewlett Packard Model 5880A was used, employing 
Porapak QS as the absorbent medium in a 5-footx 4- 
inch diameter stainless steel column. The total sugars 
were analyzed using dinitrosalicylate reagent supplied 
by Kodak Chemical Company. The following Tables 1 
and 2 present the test data obtained. 
20 
TABLE 1 
25 (With Buffer) 
Weight percent 
ETH- ACE- ACETIC BUTA- BUTYRIC 
STRAIN ANOL TONE ACID NOL ACID 
NRRL B593 0.01 0.26 neg. 0.45 0.24 
NRRL B598 neg.' neg. 0.23 neg. 0.63 30 
NRRL B593 0.03 0.32 neg. 0.54 0.17 
and B598 
NRRL B592 0.05 0.16 0.28 0.46 0.13 
NRRL B598 neg. neg. 0.23 neg. 0.63 
NRRL B592 0.06 0.20 0.35 0.70 0.16 35 
and B598 
'neg - none detected 
TABLE 2 40 
(Without Buffer) 
ETH- ACE- ACETIC BUTA.. BUTYRIC 
STRAIN ANOL TONE ACID NOL ACID 
NRRL 8593 0.03 0.09 0.2.5 0.40 0.20 45 
NRRL 8598 neg.' neg. 0.12 neg. 0.55 
NRRL B593 0.02 0.1 I 0.24 0.47 0.13 
(Without Buffer) 
ETH- ACE- ACETIC BUTA- BUTYRIC 
STRAIN ANOL TONE ACID NOL ACID 
and B598 
'neg: none detected 
The test results, particularly Column 5 ,  substantiates 
that the amount of butanol produced is substantially 
increased by employing a coculture is accordance with 
the teachings of this invention. By the proper selection 
of cocultures, the yield of butanol can be substantially 
increased over what is conventionally obtained. 
The above description presents the best mode con- 
templated of carrying out the present invention. This 
invention is, however, susceptible to modifications from 
the embodiments discussed in the above examples. Con- 
sequently, it is not the intention to limit this invention to 
the particular examples disclosed. On the contrary, the 
intention is to cover all modifications falling within the 
spirit and scope of the invention as expressed in the 
appended claims. 
What is claimed is: 
1. A process for the production of butanol from sugar 
comprising the steps of: 
mixing sugar with a coculture of a species of Clostrid- 
ium pasteurianum and a species of Clostridium bu- 
tylicum: 
fermenting the sugar with the coculture to produce 
an organic solvent mixture containing butanol in 
which the amount of butanol is increased by at least 
20 percent as compared to a fermentation process 
using Clostridium butylicum, and 
recovering the butanol. 
2. The process of claim 1 wherein a solvent mixture 
of butanol, acetone and ethanol is produced by the 
coculture cultivation and said solvent mixture contains 
at least approximately 70 weight percent butanol. 
3. The process of claim 1 wherein the pH of the 
coculture is maintained in the range of from about 4.5 to 
about 5.2. 
4. The process of claim 1 wherein the process is con- 
ducted in a single stage system in which, at equilibrium, 
the weight ratio of Clostridium pasteurianum to Clostrid- 
ium butylicum ranges between about 1:3 and about 3:l. * * * * *  
50 
55 
60 
65 


Production of butanol by fermentation in the presence of cocultures of clostridium

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