Method of continuously producing ethanol from sugar-containing substrates

In a method of continuously producing ethanol from sugar-containing substrates by fermentation of sugars by means of a flocculating strain of Zymomonas mobilis under anaerobic conditions and at a pH of from 4.5 to 7 a substrate is led commonly with Zymomonas mobilis cells through at least three fermentation stages without preceding sterilization, a concentration of at least 4% by volume of ethanol is maintained in each fermentation stage, a residence time of the fermentation medium in the entire system of maximally 31/3 h corresponding to a dilution rate of the fermentation medium in the entire system of at least 0.3 h.sup.-1 is adjusted, the Zymomonas mobilis cells are separated by sedimentation after the final fermentation stage, the Zymomonas mobilis cells are recycled into the first fermentation stage, and the ethanol-containing substrate separated from the Zymomonas mobilis cells is drawn off.

The invention relates to a method of continuously producing ethanol from 
sugar-containing substrates by fermentation of the sugars by means of a 
fluctuating strain of Zymomonas mobilis under anaerobic conditions and at 
a pH of from 4.5 to 7. 
A method of this kind is known from U.S. Pat. No. 4,413,058. Therein, an 
slantedly positioned tube reactor is used, at whose lower end the 
sugar-containing substrate is fed and in whose upper end region the 
fermented ethanol-containing substrate is discharged. The flow rate is 
adjusted such that the conversion of sugar to ethanol is ensured, yet that 
the used culture of a fluctuating strain of Zymomonas mobilis, i.e. 
Zymomonas mobilis "f" NRRLB 12526 is by no means discharged from the 
reactor. Almost over its entire length, the tube reactor is provided with 
a series of CO.sub.2 discharge openings, conduits each leading away 
therefrom. The free ends of these conduits open into the surrounding air 
approximately at the height of the upper end of the tube reactor or 
sightly thereabove. The mouths of the conduits are closed, e.g., by cotton 
wads. 
Since the microorganism culture must not be discharged, with the known 
method the flow rate of the substrate must be very closely monitored. 
The construction of the tube reactor used is so complex that its 
application on a technical scale is not possible at all or with very great 
difficulties only. Furthermore, plants already existing and having 
conventional fermentors would have to be completely replaced, resulting in 
especially high investment costs. 
Also according to German Offenlegungsschrift No. 1 48 329, a flocculating 
strain, i.e. Zymomonas mobilis ATCC 31822, obtained by selection from 
strain ATCC 31821, is used for producing ethanol from a carbohydrate 
substrate. The method disclosed is, however, only semi-continuous. At 
first, an inocculated fermentation medium is shaken in a fermentor. When 
the carbon dioxide development has stopped, the bacteria cell colonies are 
allowed to settle, the ethanol-containing supernatant is drawn off and 
replaced by fresh fermentation medium. 
A two-step fermentation process for producing ethanol by using Zymomonas 
mobilis strains, i.e. ATCC 29191 and ATCC 10988, is furthermore disclosed 
in European patent specification No. 0 047 641, wherein a bacteria cell 
suspension is produced in the first step, and ethanol is produced in the 
second step by adding fermentable sugar to that suspension. In the second 
step, only a slight bacteria cell reproduction is to occur. Flocculating 
Zymomonas mobilis strains are not considered, and thus a separation of the 
bacteria cultures used, from the fermentation medium by sedimentation is 
not possible on an industrial scale, but energy and time consuming 
centrifugation or filtering methods must be utilized for this purpose. 
A serious disadvantage common to all the known methods listed is that it is 
absolutely necessary to use sterilized substrates. The expenditure 
connected with a fermentation procedure under sterile conditions very much 
reduces the economy of such a method, a fact that is also clearly 
expressed in the article 37 Ethanol production by Zymomonas and 
Saccharomyces, Advantages and Disadvantages" in Eur. Jour. of Applied 
Microbiology and Biotechnology, 18, 1983, pp. 387-391. 
If one were to work in accordance with the known methods without 
sterilization, there would be the great risk of an infection, particularly 
by lactic bacteria or by yeasts, which - similar to Zymomonas mobilis - 
are ethanoltolerant even up to concentrations of approximately 15 percent 
by volume. 
Zymomonas mobilis has a specific ethanol production rate that is two to 
three times that of yeast, with simultaneously higher yields, and 
furthermore it does not require oxygen for its growth, whereas yeast must 
be at least slightly aerated for a sufficient production of biomass. 
For enabling a utilization of these attractive advantages of Zymomonas 
mobilis also for the production of ethanol on an industrial scale, the 
invention has as its object to overcome the above-mentioned disadvantages 
and difficulties of the known methods and to provide an operationally safe 
method which does without a sterilization of the substrates and for whose 
execution also existing fermentation plants are excellently suited after 
only slight adaptations thereof. 
With the method of the initially defined kind, according to the invention 
this object is achieved by a combination of the following measures: 
that the substrate, without a preceding sterilization, is led commonly with 
Zymomonas mobilis cells through a plurality of fermentation stages, i.e. 
at least three, 
that in each fermentation stage a concentration of at least 4 % by volume 
of ethanol is maintained, 
that the residence time of the fermentation medium consisting of a 
substrate and Zymomonas mobilis cells in the entire system is adjusted to 
a maximum of 3 1/3 h, preferably to from 0.8 to 2.5 h, and that the 
dilution rate of the fermentation medium in the overall system is adjusted 
to a minimum of 0.3 h.sup.-1, preferably to from 0.4 to 1.25 h.sup.-1, 
that the Zymomonas mobilis cells are separated by sedimentation after the 
last fermentation stage and are recycled to the first fermentation stage, 
and 
that the ethanol-containing substrate separated from the Zymomonas mobilis 
cells is drawn off. 
For the individual fermentation stages, fermentors connected in series, 
preferably three to six fermentors, are provided. In this manner it is 
possible to individually adjust the alcohol and substrate concentrations 
for the individual stages. Also the temperature is separately controllable 
in each stage. The sugar-containing substrate continuously flows through 
all fermentation stages, the sugars being gradually fermented. Additional 
substrate and an agent for controlling the pH-value, e.g. lye, may be 
added in doses to each fermentor or each fermentation stage, if necessary, 
so that a maximum productivity is achieved by accurately adapting the 
ratio of microorganism population to the alcohol and sugar concentrations 
in each stage. For this purpose, a certain excess of substrate may be 
maintained in all the fermentors. That is just what is not possible in a 
one-stage fermentation procedure, since in that case the sugars contained 
in the substrate must be fermented as far as possible so as to avoid 
losses. 
Suitably, the substrates are supplied having high sugar contents, which has 
the advantage that the former are storable over longer periods of time 
without having to be afraid of a microbial infection. Aqueous dilution 
medium is added in the first fermentation stage only. A resulting sugar 
concentration of the substrate of approximately 15 % has proved to be 
particularly favorable. 
Zymomonas mobilis is able to ferment sugars such as glucose, fructose and 
sucrose that are, e.g., contained in molasses, starch and cellulose 
hydrolysates. 
The substrate furthermore contains nutritive salts, such as ammonium 
sulfate, as well as vitamins in a known manner and amount. As a further 
positive side effect of the method of the invention it has shown that one 
can do almost completely without the usual addition of very expensive 
yeast extract and can use corn steep liquor in its place. 
At a pH of between 4.5 and 7, Zymomonas mobilis has the best growth 
conditions (cf. "Ethanol production by Zymomonas mobilis" in Advances in 
Biochemical Engineering, Vol. 23, p. 37). 
Below a pH of 4, a pronounced growth inhibition already occurs, and at a pH 
of approximately 3, the bacterium completely stops reproducing. The most 
favorable pH range for a fermentation with Zymomonas mobilis thus lies 
between 4.5 add 6.0; a pH of approximately 5.0 is optimal. In such a 
slightly acidic medium practically only lactic bacteria and yeasts have 
favorable living conditions. An infection by other microorganisms is very 
little likely if pH values of 7 or slightly therebelow are avoided. Lactic 
bacteria, however, are already clearly damaged from an ethanol 
concentration of approximately 4 % by volume onwards, although 
heterofermentative types themselves excrete ethanol as metabolic product. 
Yeasts grow the best at pH values of from 4 to 6, and their ethanol 
tolerance and temperature sensitivity are almost the same as those of 
Zymomonas mobilis. 
However, Zymomonas is strictly anaerobic and is damaged by oxygen. Although 
yeast can also grow and ferment completely without oxygen, it tolerates 
oxygen on the other hand and is stimulated to an increased cell formation 
thereby. This can be seen quite clearly from the specific growth rates of 
aerobically and anaerobically grown yeasts. While a yeast of the type 
Saccharomyces grown without oxygen has a specific growth rate of 
approximately 0.15 h.sup.-1, the same yeast has a growth of 0.25 h.sup.-1 
under aeration, thus growing almost twice as fast. Anaerobically, 
Zymomonas mobilis grows approximately as fast as aerated yeast; with a 
longer access of oxygen, the bacterium even dies. 
For taking advantage of the reduced growth rate of yeasts relative to 
Zymomonas mobilis under anaerobic conditions for preventing infections, a 
further combination characteristic according to the invention is the 
adjusting of a short residence time or a high dilution rate of the 
fermentation medium in the overall system of the fermentation stages or 
fermentors. Due to the high flow rates, the occurrence of yeasts is 
prevented by practically washing out the infecting cells. For instance, at 
a residence time of 2 h in the entire system the flow rate is more than 
three times too high for the occurrence of yeast. Furthermore, by this 
procedure an especially high production of ethanol is obtained. 
A part of the Zymomonas mobilis floc remains in the individual fermentation 
stages, even if the entire system has reached the state of equilibrium 
with a constant continuous flow. A smaller portion of the floc that 
depends on the residence time observed is delivered from the stages and 
moved to the respective next stage. After the final fermentation stage, 
the floc carried on by the substrate is separated, the separation from the 
substrate being effected in the simplest way, by sedimentation. With this 
separation made feasible by the use of flocculent Zymomonas mobilis 
strains, a large amount of undesired microorganisms possibly contained in 
the fermentation medium is removed from the system with the supernatant 
ethanol-containing substrate, whereas with a separation by centrifugation 
or filtration, all the foreign organism would be recycled together with 
Zymomonas mobilis. 
According to a preferred embodiment of the method of the invention, at 
least a part of the CO.sub.2 formed in the individual fermentation stages 
is circulated in each stage. 
Therein, the CO.sub.2 is withdrawn from the gas space of the fermentors and 
preferably is re-fed into the fermentors finely distributed from below, in 
which fermentors the gas flows through the fermentation medium and there 
causes a uniform distribution of the Zymomonas mobilis floc. Furthermore, 
any possible deposits in the bottom region of the fermentors are prevented 
in this manner. 
For ensuring as uniform a distribution of the floc in the entire 
fermentation space as possible, is has also proved favorable to draw off 
the fermentation medium from the bottom of each of the individual 
fermentors and to feed it to the top of the next fermentor. 
A further preferred embodiment of the method of the invention resides in 
that at least a part of the distillery slops remaining after recovery of 
the ethanol from the drawn-off ethanol-containing substrate is recycled 
into the first fermentation stage. 
The ethanol recovery is effected, e.g., by rectification of the drawn-off 
fermented substrate whose ethanol content in most cases amounts to 
approximately 9 to 10 % by volume. The distillery slops remaining, which, 
due to the heating occurring during the rectification, are nearly sterile, 
still contains nutrient residues that can be used by the microorganisms if 
the distillery slops are recycled. Furthermore, in this manner the fresh 
water demand is lowered. The recycling is, however, limited in that the 
content of the substrate of unusable substances carried along in the 
distillery slops must not rise too high.

The method according to the invention will now be explained in more detail 
by way of the following examples and the drawing. 
Example 1: 
The fermentation process was carried out in three stages in a plant 
schematically illustrated by the drawing. 
Three closed, substantially cylindrical fermentors connected in series are 
denoted by 1a, 1b and 1c in the drawing. Slightly above the bottom of the 
fermentors, a gas distributing means, e.g. a glass frit 2a, 2b and 2c, is 
each installed. Upon the third fermentor 1c, a sedimentation vessel 3 
follows, which has the same construction as the fermentors, yet which has 
a conically downwardly tapering bottom part. 
A concentrated sugar-containing substrate can be fed to the fermentors 1a, 
1b and 1c via a duct 4, the branch ducts being each provided with closing 
means 5a, 5b and 5c. Furthermore, a lye duct 6 is provided, from which 
also branch ducts lead to the fermentors 1a, 1b and 1c. 
In each of these branch ducts, there is a feed control 7a and 7b and 7c. As 
the feed controls, e.g. solenoid valves are suited, which may be 
controlled by pH-measuring probes (not illustrated) in the fermentors. The 
first one of the fermentors connected in series, 1a, in addition contains 
a feed 8 for fresh water or recycled distillery slops. From the gas space 
of each of the fermentors 1a, 1b and 1c, a gas duct leads to the gas 
distribution means 2a, 2b and 2c provided in the bottom part of the 
respective fermentor, via a compressor 9a, 9b and 9c. 
For discharging excessive gas amounts, a gas discharge charge duct also 
provided with closing means 10a, 10b, 10c and 10d is installed in the top 
part of each fermentor 1a through 1c and the deposit container 3. The 
biomass collecting in the conically designed bottom part of the deposit 
container 3 is recycled to the top part of the first fermentor 1a by means 
of a pump 11 via the thick matter duct 12. The fermentation medium is each 
conducted away from the bottom part of the fermentors 1a and 1b and fed 
through ducts 13 and 13' to the top part of the next fermentor 1b and 1c, 
c, respectivley, and from the bottom part of the fermentor 1c to the top 
part of the sedimentation vessel 3, via duct 13". The ethanol-containing 
substrate is drawn off from the sedimentation vessel 3 via the overflow 
duct 4. For thermosetting, all the fermentors and the sedimentation vessel 
may be provided with a double shell not illustrated, through which a heat 
transfer medium flows. 
The fermentors and the sedimentation vessel of the plant used had an inner 
diameter of approximately 12 cm and a height of approximately 55 cm; their 
filling volume amounted to 5 1 each. As the gas distribution means, a 
glass frit was installed slightly above the bottoms of the fermentors, and 
all the fermentors as well as the deposit container were provided with 
double shells. 
At the start of the fermentation process, a nutrient and vitamin containing 
substrate having 15 % of sugar was provided in all the fermentors and in 
the sedimentation vessel, glucose, inverted sucrose or a starch 
hydrolysate having been used equally successfully for preparing the 
substrate. The air contained in the containers was removed by flushing 
with CO.sub.2 or nitrogen, and the entire system was inocculated with 
approximately 70 g of Zymomonas mobilis floc. This bacterial culture had 
been grown in a prefermentation, because according to experience it takes 
a few days until the floc is actually formed after an inocculation of the 
substrate with a flocculating strain of Zymomonas mobilis. 
After the inocculation, the system was left without supply of substrate 
until an intensive gas development started and the ethanol concentration 
in each fermentor had reached approximately 4% by volume. The CO.sub.2 
formed in the fermentation stages was circulated in each stage or in each 
fermentor via the compressors 9a, 9b and 9c. 
Thereupon, the supply of concentrated substrate and dilution medium was 
started. The initial settings were 150 ml of substrate with 60 % of 
sugar/h and 750 ml of fresh water/h in the first fermentor 1a, as well as 
100 ml of substrate with 60 % of sugar/h in the second fermentor 1b, so 
that as a result a sugar concentration of 15 % was adjusted at a total 
passage of 1 1/h. Although the substrate had initially been adjusted to a 
pH of 5.0, lye had to be added continuously for maintaining that pH, 
because an acidification of the fermentation medium occurred as a 
consequence of the uptake of NH.sub.4 + from the ammonium sulfate 
contained in the substrate by the organisms. 
By an intensive recycling of the bacterial fLoc collecting in the bottom 
part of the sedimentation vessel 3, the biomass concentration was 
continuously increased so as to finally be at a balance of between 20 and 
25 g of dry substance/1. Due to the high specific productivity of 
Zymomonas mobilis it was thereupon possible to increase the total supply 
of substrate from 1 1/h to 6 1/h, without detecting unfermented sugar in 
the fermentation medium after the third fermentor 1c. In the fermented 
substrate from the overflow duct 14, 9.0 to 9.2 % by volume of ethanol 
were measured corresponding o a yield of from 93 to 95 % of theory. 
Thus, with an addition of substrate of 6 1/h and an effective fermentation 
volume of 15 1 (3.times.5 1; residence time in the entire system thus 2.5 
h) there results a volumetric productivity of approximately 36 1 of 
ethanol/m.sup.3. h. 
Even higher productivities are attainable with the plant disclosed. If, 
however, industrial scales are considered, the removal of the fermentation 
heat from the correspondingly larger containers at an even higher flow 
rate already constitutes a problem that is difficult to solve. 
In the pilot plant disclosed, the fermentation was continuously carried out 
for three weeks. No infection problems occurred, although neither the 
substrate concentrate nor the dilution liquid had been sterilized. 
COMATIVE EXAMPLE 1: 
As disclosed in the preceding Example, the system was run up and then, at a 
total supply of substrate of 6 1/h, the pH control was switched off. 
Because of the abovementioned acidification due to an NH.sub.4 + uptake of 
Zymomonas mobilis, the pH of the fermentation medium gradually sank and 
finally reached a value of 2.8. As a first reaction of the system, 
unfermented sugar occurred in the overflow 14, so that the flow rate had 
to be reduced. After approximately 30 h, the first yeast cells could be 
detected in the microscope, which cells reproduced continuously and 
finally could also be found in the bacterial floc. After the flow rate had 
to be reduced to only 0.6 1/h, the fermentation was stopped. 
COMATIVE EXAMPLE 2: 
As described above, the system was run up to an optimal output, and 
subsequently air was blown in via the frits 2a, 2b and 2c instead of the 
CO.sub.2 formed. As the first effect, it was found that the ethanol yield 
sank to clearly below 90 %, probably due to an increased formation of 
byproducts, such as acetic acid or acetic aldehyde. Subsequently, also the 
flow rate had to be reduced, which again had the consequence that - also 
aided by the supply of oxygen - yeast cells occurred in the fermentation 
medium. 
Contrary to the Comparative Example 1, the optimal operating condition of 
the system could be restored again by stopping the supply of air, and 
supplying CO.sub.2 again. This was possible because, contrary to the 
course of the method according to Comparative Example 1, the bacterium 
Zymomonas mobilis was not irreversibly damaged, but only subjected to less 
favorable conditions. After the supply of air had been stopped, at first 
the yield improved again, the flow rate could be increased, and after 
approximately 40 h all the yeast had been flushed out of the system again.