Continuous process for ethanol production by bacterial fermentation

A continuous process for the production of ethanol by fermentation with strains of Zymomonas is provided. Metabolic processes are limited by the nutrients nitrogen, potassium and phosphorus. When growth is limited by one of these nutrients, the biomass expresses its maximum value for both q.sub.s and q.sub.p at any given value of D and S.sub.r. The process is conducted at a lower biomass concentration and a higher specific rate of ethanol formation than a similar process conducted with a nutrient medium that is not limited in nitrogen, potassium or phosphorus. A method of improving performance of Zymomonas in continuous ethanol fermentation at increased temperatures is also provided.

BACKGROUND OF THE INVENTION 
This invention relates to the bioconversion of a substrate by bacterial 
fermentation, and more particularly, to a continuous process for the 
production of ethanol by fermentation using strains of Zymomonas bacteria. 
Bacterial ethanol fermentation has been known in the art for many years, 
and in recent years fermentation using strains of Zymomonas mobilis has 
received increasing attention. The Z. mobilis strains convert a suitable 
substrate, such as glucose or another sugar, to ethanol. Significantly 
higher specific rates of sugar uptake and ethanol production and improved 
yield compared to traditional yeast fermentation have been reported for 
these Zymomonas strains. 
Fermentation by Z. mobilis has been carried out in batch and continuous 
culture. The fermentation product (ethanol) is dissolved in the liquid 
medium in the fermenter. The liquid medium is separated from solids 
(chiefly biomass) before the ethanol is recovered. Separation of these two 
phases early in the product-recovery process train is required. Before 
such a fermentation achieves commercial acceptance, however, productivity 
must be improved. The reported efforts to date have focused on developing 
more productive bacterial strains and modifying the configuration of the 
fermenter used in the fermentation. For example, improvement in ethanol 
productivity using a continuous culture with a cell recycle system has 
been reported with Z. mobilis strains. 
Recovery of the fermentation product can be a complex and multifaceted 
task. A significant proportion of the overall cost in a fermentation plant 
often must be spent for ethanol recovery. A recently reported technique 
involves the use of a flocculent strain of Z. mobilis that settles in the 
fermenter allowing the supernatant containing the ethanol to be withdrawn 
while leaving a majority of the cells in the fermenter. This method is 
based on the well-known gravity sedimentation principle for separating 
liquids and solids. The more conventional approaches for separating 
fermentation broth from biomass involve the withdrawal of a portion of the 
culture medium from the fermenter and separation of the two phases by 
centrifugation or filtration techniques. Regardless of the technique 
employed, for a particular ethanol recovery process, it is desirable to 
reduce the quantity of biomass in order to reduce the load of solids on 
the sedimentation, centrifugation or filtration apparatus. 
At the same time, however, the yield of ethanol from the fermentation must 
be maximized. Since product formation cannot occur in the absence of 
biomass, ethanol formation is dependent on cell mass concentration. In 
fact, the rate of ethanol production in the fermenter is directly 
proportional to the quantity of biomass in the fermenter under steady 
state conditions. Thus, within the limits of the metabolic regulatory 
controls of the microorganism and process dynamics, increasing the biomass 
in the fermenter while maintaining other conditions constant will shorten 
the time required to produce a given amount of ethanol. However, this 
seemingly simple approach for optimizing process performance will have an 
adverse effect on the ethanol recovery process because the load of solids 
on the separating equipment will be correspondingly increased. 
It is well known that the substrate, such as glucose, is the largest item 
of raw material cost in the fermentation. Therefore, the presence of 
substrate in the effluent from the fermenter is a continuous fermentation 
is to be avoided. The continuous fermentation should be conducted at 
optimum process product yield, which occurs when the substrate is 
completely converted to ethanol and when the substrate is minimally 
diverted from product (ethanol) formation to cell mass synthesis (i.e. 
when the growth yield with respect to carbon substrate is minimized). 
Thus, there exists a need in the art for a continuous process for the 
production of ethanol using strains of Zymomonas in which the substrate 
fed to the fermenter is converted to ethanol in as short a time as 
possible. The process should permit a reduction in the quantity of biomass 
in the fermenter in order to obtain a corresponding reduction in the load 
of solids on the ethanol recovery apparatus. In addition, the quantity of 
substrate in the effluent from the fermenter should be minimized. 
SUMMARY OF THE INVENTION 
This invention aids in fulfilling these needs in the art by providing a 
continuous process for the production of ethanol. The present invention 
relates to an improvement in the fermentation performance of strains of 
Zymomonas in continuous fermentations wherein metabolic processes are 
limited by various nutrients, these nutrients being nitrogen, potassium or 
phosphorus. When growth is limited by one of these nutrients, the biomass 
expresses its maximum value for both specific rate of substrate uptake 
(q.sub.s) and specific rate of product formation (q.sub.p) at any given 
value of dilution rate (D) and substrate concentration (S.sub.r). One of 
the criteria for assessing the improvement in performance of Zymomonas 
according to this invention is the increase in the specific rate of 
ethanol (product) formation (q.sub.p). The response of Zymomonas with 
respect to nutrient limitation by nitrogen, potassium or phosphorus could 
not be predicted from prior teachings. That Zymomonas expresses a maximal 
value fo q.sub.p (and q.sub.s) under conditions of nutrient limitation was 
not a predictable phenomenon. The process of this invention is conducted 
at a lower biomass concentration and a higher specific rate of ethanol 
formation than a similar process conducted with a nutrient medium that 
contains the nutrient in excess. 
In one embodiment of this invention, a method of improving continuous 
ethanol production by bacterial fermentation with strains of Zymomonas is 
provided without changing the fermentation temperature. These results are 
achieved by carrying out the fermentation under nutrient-limiting 
conditions. The limiting nutrient is either nitogen, potassium or 
phosphorus. The imposition of nutrient limitation makes it possible to 
conduct the fermentation at a lower biomass concentration at a given 
substrate concentration in the feed stream to the fermenter or at a given 
dilution rate than under conditions of nutrient-excess. Consequently, for 
a given substrate concentration in the feed stream to the fermenter or a 
given dilution rate, the fermenter can be operated at a higher specific 
rate of product formation under the nutrient-limiting conditions than 
under the nutrient-excess conditions. Moreover, as the concentrations of 
substrate in the feed stream is increased or as the dilution rate is 
increased, the biomass in the fermenter also increases, but the biomass 
level is less under nutrient-limiting conditions than under conditions of 
nutrient-excess. Furthermore, nutrient-limitation does not appreciably 
affect product yield when compared with a similar fermentation carried out 
under nutrient-excess; the yield at a given substrate concentration in the 
feed stream or a given dilution rate is substantially the same under 
nutrient-limiting conditions as under nutrient-excess conditions. In 
addition, product yield is substantially unaffected under 
nutrient-limiting conditions when either the concentration of substrate in 
the feed stream or the dilution rate is increased. At the same time, 
substantially all the substrate is converted to ethanol; unconverted 
substrate in the effluent can be avoided. 
The amount of the limiting nutrient in the culture medium required to 
achieve nutrient-limited fermentation according to this invention is 
proportional to the concentration of substrate in the feed stream to the 
fermenter and to the dilution rate. In order to maintain a specific rate 
of product formation, the amount of the limiting nutrient in the 
fermentation medium must be increased as the concentration of substrate in 
the feed stream is increased. Similarly, when the dilution rate is 
increased, the amount of limiting nutrient in the fermentation medium must 
be increased to maintain the specific rate of product formation. 
In a situation where high cell density is artificially maintained, such as 
in a single-stage recycle system, a higher overall productivity could be 
maintained with the nutrient-limited fermentation of this invention. In 
such a system, the upper limit to the productivity of the fermenter is 
often determined by the capacity of the recycle device to handle a maximum 
biomass load. Nutrient limitation results in a reduction in the biomass 
level without significantly altering the capacity of the fermenter to 
handle the same substrate load. 
In another embodiment of this invention, a method of improving performance 
of Zymomonas in continuous ethanol fermentation at increased temperatures 
has now been discovered. It has surprisingly been found that the specific 
rate of substrate uptake can be maintained, and even increased, at 
fermentation temperatures of about 33.degree. to about 37.degree. C. even 
though there is a lower biomass concentration in the fermenter. These 
results can be achieved without substantial amounts of substrate in the 
effluent from the fermenter. These advantages have a positive impact on 
product recovery and process economics. These results are achieved with 
this invention by carrying out continuous ethanol fermentation with 
Zymomonas strains under nutrient-limited conditions, where the limiting 
nutrient is nitrogen, potassium or phosphorus. The concentration of the 
limiting nutrient in the fermentation medium is increased with increasing 
temperature and decreased with decreasing temperature.

DETAILED DESCRIPTION 
The accompanying FIGURE will provide a background for the following 
discussion in which the method and apparatus for carrying out this 
invention are described in detail. Referring to the FIGURE, nutrient 
medium 1 in reservoir 2 is fed by a pump 3 to a fermenter 4 containing a 
fermentation medium 5. The medium is maintained at a constant volume in 
the fermenter by means of an overflow weir 6 that empties into a container 
7. Carbon dioxide formed during the fermentation is vented at 8. 
The fermenter 4 is provided with an agitator 9 for mixing the fermenter 
contents. A pH probe 10 is immersed in the fermentation medium 5 and is 
connected to a pH controller 11 for regulating the amount of pH regulating 
agent 12 in reservoir 13 added by a pump 14 to the fermentation medium 5. 
The temperature of the fermentation medium is monitored by temperature 
probe 15. The fermentation medium is heated or cooled as required through 
a coil 16 and regulated by the temperature controller 17. 
The fermentation medium is formulated so that all but a single essential 
nutrient are available in excess of the amount required to synthesize a 
desired cell concentration. The single growth-limiting nutrient controls 
the size of the steady-state cell population. 
Conventional expressions are used throughout this description when the 
kinetics of the fermentation process are discussed. The abbreviations 
identified in the following list have been used in order to facilitte the 
discussion. Units of measure have been included where appropriate. 
S.sub.r =Substrate concentration in the feed stream to the fermenter; 
g/liter, i.e. g/L. 
S.sub.o =Substrate concentration in the effluent from the fermenter; g/L. 
V=Volume of fermentation medium in the fermenter; L. 
X=Concentration of biomass in the fermentation medium (dry basis); g/L. 
[P]=Concentration of ethanol in the fermentation medium; g/L. 
u=Specific growth rate (mass); hr.sup.-1. 
D=Dilution rate; hr.sup.-1. 
N*=Amount of a given nutrient; g. 
q.sub.s =Specific rate of substrate uptake; g substrate/g 
biomass-hr.sup.-1. 
q.sub.s.sup.max =Maximum observed specific rate of substrate uptake; g 
substrate/g cell-hr.sup.-1. 
q.sub.p =Specific rate of ethanol formation; g ethanol/g biomass-hr.sup.-1. 
q.sub.p.sup.max =Maximum observed specific rate of ethanol formation; g 
ethanol/g cell-hr.sup.-1. 
VP=Volumetric Productivity; g ethanol/L-hr.sup.-1. 
Y.sub.n =Growth yield coefficient for a specified limiting nutrient, n; g 
dry biomass/g-atom nutrient. 
n=Limiting nutrient, i.e., nitrogen, potassium or phosphorus. 
Y.sub.p/s =Product Yield Coefficient=q.sub.p /q.sub.s ; g ethanol 
produced/g substrate consumed. 
Y.sub.x/s =Growth yield coefficient; g biomass/g-atom of substrate 
consumed. 
YE=yeast extract (Difco) in aqueous medium; g/L. 
AC=High grade anhydrous NH.sub.4 Cl in aqueous solution; g/L. 
AS=High grade anhydrous (NH.sub.4).sub.2 SO.sub.4 in aqueous solution; g/L. 
Eth=ethanol 
Glu=glucose 
Nutrient-Limited Fermentation 
As used herein, the expression "nutrient-limited fermentation" and similar 
expressions mean a fermentation of an organic substrate by a Zymomonas 
strain, where the fermentation is carried out in a continuous process 
under steady-state conditions in a medium in which one or more nutrients 
are present in an amount such that the rate of growth is limited by the 
availability of one or more of the essential nutrients. 
As used herein the expression "limiting nutrient" means nitrogen, potassium 
or phosphorus. 
The process of this invention is carried out as a continuous fermentation. 
The term "continuous" is used in its conventional sense and means that 
nutrients are fed to a fermenter substantially continuously and that an 
output, or effluent, stream is substantially constantly withdrawn from the 
fermenter. The nutrient stream usually comprises an aqueous organic 
substrate solution. The effluent stream comprises biomass and the liquid 
phase from the fermentation medium. 
Fermentation can be carried out in a bioreactor, such as a chemostat, tower 
fermenter or immobilized-cell bioreactor. Fermentation is preferably 
carried out in a continuous-flow stirred tank reactor. Mixing can be 
supplied by an impeller, agitator or other suitable means and should be 
sufficiently vigorous that the vessel contents are of substantially 
uniform composition, but not so vigorous that the microorganism is 
disrupted or metabolism inhibited. 
Fermentation is carried out with a submerged culture and under 
substantially anaerobic conditions. While the invention is described in 
the Examples hereinafter with freely mobile cells, it will be understood 
that immobilized cells can also be employed. The fermenter is preferably 
enclosed and vented to allow the escape of carbon dioxide evolved during 
the fermentation. Oxygen at the surface of the fermentation medium is to 
be avoided. This may inherently occur as the heavier carbon dioxide 
evolved during the fermentation displaces the oxygen in the gas phase 
above the medium. If necessary, the gas phase above the medium can be 
purged with an inert gas to remove oxygen and maintain substantially 
anaerobic conditions. 
The fermenter can be operated with or without cell recycle. Cell recycle 
makes it possible to increase the productivity of the system by operating 
at a higher steady-state cell concentration compared to a similar system 
without cell recycle. When cell recycle is employed, a portion of the 
fermenter contents is withdrawn from the fermenter, the ethanol-containing 
phase is separated from the effluent, and the resulting concentrated cells 
are returned to the fermenter. The separation is typically carried out by 
microfiltration or centrifugation. Since the process of this invention is 
carried out at reduced biomass concentration in the fermenter, the load of 
solids on the cell recycle apparatus is reduced and ease of ethanol 
recovery is increased. 
The composition of the effluent stream can vary and will usually be the 
same as the composition of the fermentation medium. When a flocculent 
strain of Zymomonas is employed, however, or if partial separation of 
biomas from the liquid phase otherwise occurs in the fermenter, the 
effluent can contain a larger proportion of biomass or liquid phase 
dependding upon the location where the efflent is withdrawn from the 
fermenter. 
The microorganism employed in the process of this invention is a 
gram-negative, faculative anaerobic bacterium of the genus Zymomonas 
capable of fermenting an organic substrate to ethanol under substanially 
anaerobic continuous culture conditions. Typical strains are Zymomonas 
mobilis and Zymomonas anaerobia. Suitable strains of Zymomonas are 
available from microorganism depositories and culture collections. 
Examples of suitable Z. mobilis strains are those identified as ATCC 
10988, ATCC 29191, ATCC 31821 and ATCC 31823 [ex ATCC 31821]. Examples of 
other strains of Z. mobilis are those identified as NRRL B-14023 [CP 4] 
and NRRL B-14022 [CP 3]. Flocculent strains can also be employed. These 
strains include ATCC 35001 [ex ATCC 29191], ATCC 35000 [x NRRL B-14023], 
ATCC 31822 [x ATCC 31821], and NRRL B-12526 [x ATCC 10988]. Z. mobilis 
strains are occasionally referred to in the literature by the following 
alternate designations: 
TABLE 1 
______________________________________ 
Culture Collection 
Accession No. Literature Designation 
______________________________________ 
ATCC 10988 Strain ZM 1 
ATCC 29191 Strain ZM 6 (or Z6) 
ATCC 31821 Strain ZM 4 
ATCC 31822 Strain ZM 401 
ATCC 31823 Strain ZM 481 
NRRL B-14022 Strain CP3 
NRRL B-14023 Strain CP4 
______________________________________ 
It will be understood that other Zymomonas strains can be obtained by 
selective cultivation or mutation as well as by genetic engineering 
techniques to provide microorganisms with desired metabolic properties. 
The substrate employed in the process of this invention is an organic, 
fermentable sustrate for the Zymomonas strain. As the carbon source for 
both the growth and fermentation stages of the process, various 
carbohydrates cand be employed. Examples of suitable carbohydrates are 
sugars, such as glucose, fructose and sucrose; molasses; starch 
hydrolysates; and cellulose hydrolysates. Other suitable substrates will 
be apparent to those skilled in the art. The organic substrate can be 
employed either singly or in admixture with other organic substrates. 
The substrate is fed to the fermenter in aqueous solution. The 
concentration of organic substrate in the fermentation medium will depend 
upon the culture conditions. The substrate is employed in an amount 
sufficient for cell growth and product formation. Typically, the 
concentration of fermentable substrate in the feed stream to the fermenter 
will be about 100 to about 180 g/L. 
The flow rate of the substrate solution to the fermenter will depend upon 
the size and configuration of the fermenter, the amount of biomass in the 
fermenter and the rate at which substrate is consumed, and can be 
determined with a minimum of experimentation. The flow rate should be 
below the rate at which a substantial amount of substrate appears in the 
effluent from the fermenter. Preferably, the flow rate of the substrate 
solution to the fermenter should be such that the effluent substrate 
concentration is less than about5% S.sub.r, and should be such that the 
effluent is substantially free of substrate under optimum operating 
conditions. 
The process of this invention can be carried out over a moderate range of 
temperatures. The effects of temperature changes on fermenter performance 
are discussed below, but generally speaking, the process of this invention 
is carried out at a temperature of about 27.degree. C. to about 37.degree. 
C., preferably about 30.degree. C. 
Zymomonas fermentations have been reported at pH values ranging from about 
4 to about 8 in the culture medium. The process of this invention can be 
carried out over a moderate range of pH values in the culture medium, but 
rapid metabolism of the organic substrate with high product yield occurs 
over a narrower range. The process of this invention is preferably carried 
out at a pH of about 4.5 to about 6.5. At pH values above about 6.5, 
product yield decreases and the formation of undesirable products 
increases. The particularly preferred pH is about 5.5, which was the pH 
used in all of the fermentations described herein. 
The pH in the culture medium often falls and rises during the fermentation. 
To restrict pH changes during fermentation, the medium can be buffered. In 
addition, the pH of the medium can be intermittently or continuously 
monitored and acidic or basic substances added to adjust pH during the 
course of fermentation. A buffering agent or a pH regulating agent that is 
non-toxic and substantially non-inhibitory to the microorganism can be 
employed for this purpose. The pH regulating agent is typically a 
hydroxide or an organic or inorganic acid. Examples of suitable pH 
regulating agents are potassium hydroxide, sodium hydroxide and 
hydrochloric acid. 
The process of this invention is carried out under sufficiently sterile 
conditions to ensure cell viability and metabolism. This requires careful 
selection of the microorganism, sterilization of the apparatus for the 
fermentation and of the liquid and gaseous streams fed to the fermenter. 
Liquid streams can be sterilized by several means, including radiation, 
filtration and heating. Small amounts of liquids containing sensitive 
vitamins and other complex molecules can be sterilized by passage through 
microporous membranes. Heat-treatment processes are preferred for 
sterilizing the substrate feed stream and can be carried out by heating 
the stream in a batch or continuous flow vessel. The temperature must be 
high enough to kill essentially all organisms in the total holding time. 
Water utilized in the preparation of the substrate solution and in the 
preparation of the fermentation broth in the fermenter can be sterilizer 
in a similar manner or by other conventional techniques. 
After the fermenter has been inoculated with the Zymomonas microorganism, 
the quantity of biomass is multiplied. The growing culture is allowed to 
complete the lag phase and substantially the entire exponential phase of 
growth before flow to the fermenter is initiated. The fermentation is 
allowed to proceed under substantially steady state conditions with the 
continuous introduction of fresh substrate and the continuous withdrawal 
of product from the fermenter. While product formation is not solely 
associated with growth, it will be understood that a portion of the 
substrate fed to the fermenter goes into cell maintenance. Thus, in the 
case of direct conversion of glucose to ethanol 
1 mole glucose.fwdarw.2 moles ethanol +2 moles CO.sub.2. The maximum 
conversion is 2 mole ethanol per mole glucose or 0.51 g ethanol/g glucose, 
but theoretical yield cannot be achieved in practice since some of the 
substrate goes into cell mass. The process of this invention is carried 
out at a yield of at least about 80%, preferably at least about 94%, of 
theoretical yield. In this context, complete fermentation means that 
greater than 95% of the sugar substrate has been converted to ethanol 
product. 
Viable cell concentration in the fermenter will depend upon several 
factors, such as dilution rate, substrate concentration, maximum growth 
rate and growth yield coefficient. The fermenter can be operated over a 
range of biomass concentrations and the optimum concentration can be 
determined without undue experimentation. The practical range of values 
will generally depend upon process economics. For example, a continuous 
chemostat culture without cell recycle at maximum substrate concentration 
in the feed stream can typically be operated at a maximum biomass 
concentration (DWB) of about 3.5 g/L. A practical range of biomass 
concentrations is about 0.8 to about 3.2 g/L. 
The concentration of ethanol in the fermentation medium should be maximized 
in order to reduce the cost of product recovery. The process of this 
invention is carried out at ethanol concentrations up to about 85 g/L, 
preferably about 28 g/L to about 70 g/L, especially about 50 g/L to about 
60 g/L, in the fermentation medium. 
Z. mobilis is sensitive to ethanol concentration, and at concentrations in 
excess of about 50 g/L (5% w/v), at T.ltoreq.33.degree. C. cell growth and 
metabolism are retarded. This can be caused by a high concentration of 
substrate in the feed steam to the fermenter. Thus, as the value for 
S.sub.r is increased, the maximum dilution rate for substantially complete 
conversion of substrate to ethanol should be decreased. The process of 
this invention is carried out at a dilution rate of about 0.05 hr.sup.-1 
to about 0.35 hr.sup.-1, preferably about 0.1 hr.sup.-1 to about 0.2 
hr.sup.-1. 
These features of this invention will be more fully understood from the 
following discussion. The effects of the limiting nutrient on fermener 
performance at various concentrations of substrate in the feed stream and 
different dilution rates are summarized below. The results obtained in a 
series of experiments with varying operating conditions are reported in 
the following Tables. 
Nitrogen-Limited Fermentation 
Table 2 shows the effect of increasing the concentration of glucose in the 
feed stream on the performance of a continuous fermentation of Z. mobilis 
strain ATCC 29191 in a chemostat at a constant dilution rate of 0.15 
hr.sup.-1 under either nitrogen-limiting conditions or conditions of 
nitrogen-excess. The amount of assimilable nitrogen was varied by changing 
the amount of either yeast extract (YE), ammonium chloride (AC) or 
ammonium sulphate (AS). The amount of the nitrogen-limiting additive was 
the minimal amount required to achieve maximal rate of sugar utilization 
and ethanol production by the Z. mobilis strain under the fermentaion 
conditions. 
TABLE 2 
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Amount of Assimilable Nitrogen as either Yeast Extract 
(Difco), Ammonium Chloride or Ammonium Sulphate Required 
to Achieve Maximal Rate of Sugar Utilization and Ethanol 
Production by Z. mobilis ATCC 29191 in Continuous Culture 
at Fixed Dilution Rate (0.15 hr.sup.-1) as a Function of 
Feed Sugar Concentration (S.sub.r) 
Excess Nitrogen Nitrogen Limitation 
S.sub.r 
[P] X X YE AC AS 
g/L g/L g/L q.sub.s 
q.sub.p 
g/L q.sub.s 
q.sub.p 
g/L g/L g/L 
______________________________________ 
20 9.6 0.58 5.2 2.5 0.36 8.3 3.9 0.8 0.19 0.23 
60 28 1.73 5.2 2.5 1.1 8.3 3.9 2.4 0.59 0.72 
110 52 3.17 5.2 2.5 2.0 8.3 3.9 4.4 1.10 1.32 
______________________________________ 
Units: 
q.sub.s = g glu/g cellhr.sup.l ; 
q.sub.p = g eth/g cellhr.sup.-1. 
The data in Table 2 show that a fermenter can be operated according to this 
invention at a lower biomass concentration under nitrogen-excess at a 
given glucose concentration. For example, with a glucose concentration 
(S.sub.r) of 20 g/L in the feed stream, the biomass concentration (X) in 
the fermenter was only 0.36 g/L under nitrogen-limiting conditions, 
whereas the biomass concentration was 0.58 g/L under conditions of 
nitrogen-excess. A similar comparison can be made for the other glucose 
concentrations shown in Table 2. 
The data in Table 2 also show that the fermenter can be operated at a 
higher specific rate of product formation (q.sub.p) under 
nitrogen-limiting conditions than under conditions of nitrogen-excess at a 
given glucose concentration. The observed specific rate of product 
formation of 3.9 under nitrogen-limiting conditions was approximately 1.56 
times greater than observed q.sub.p under conditions of nitrogen-excess. 
In addition, the data in Table 2 demonstrate that as the concentration of 
glucose in the feed stream (S.sub.r) is increased, biomass concentration 
(X) also increases under both nitrogen-limiting and nitrogen-excess 
conditions, but the biomass level is less under nitrogen-limiting 
conditions than under conditions of nitrogen-excessfor a given S.sub.r. 
For near complete conversion of glucose to ethanol, the amount of limiting 
nitrogen was increased as the glucose concentration was increased. For 
example, in order to achieve complete fermentation of added substrate, the 
quantity of yeast extract in the nutrient medium was increased from 0.8 
g/L to 4.4 g/L when the glucose concentration in the feed stream was 
increased from 20 g/L to 110 g/L. The culture expressed maximal values for 
q.sub.p and q.sub.s under these conditions. 
The data in Table 2 also show that nitrogen-limitation in the fermentation 
medium does not appreciably affect product yield when compared with a 
similar fermentation carried out under conditions of nitrogen-excess. The 
product yield was about 92% of the theoretical maximum yield in all cases. 
[[e.g., [28/(60.times.0.51)].times.100=92%]]. These results were entirely 
unexpected. 
Another series of fermentations similar to those summarized in Table 2 was 
carried out, except that the concentration of glucose in the feed stream 
(S.sub.r) to the fermenter was maintained at 110 g/L while the dilution 
rate (D) was varied. Table 3 shows the amount of assimilable nitrogen as 
either yeast extract (YE), ammonium chloride (AC) or ammonium sulphate 
(AS) required to achieve maximal rate of sugar utilization and ethanol 
production under nitrogen-limiting conditions and conditions of 
nitrogen-excess. 
TABLE 3 
______________________________________ 
Amount of Assimilable Nitrogen as either Yeast Extract (Difco) 
Ammonium Chloride or Ammonium Sulphate Required to 
Achieve Maximal Rate of Sugar Utilization and Ethanol Pro- 
duction by ATCC 29191 in Continuous Culture at Fixed Feed 
Glucose Z. mobilis Concentration (S.sub.r = 110 g/L) as a Function 
of the Dilution Rate (D) 
Excess 
Nitrogen Nitrogen Limitation 
D S.sub.r 
X X YE AC ASS.sub.o 
hr.sup.-1 
g/L g/L q.sub.s 
q.sub.p 
g/L q.sub.s 
q.sub.p 
g/L g/L g/L g/L 
______________________________________ 
0.1 110 2.53 4.4 2.1 1.33 8.3 3.9 3.0 0.71 0.88 5 
0.15 110 3.17 5.2 2.5 2.00 8.3 3.9 4.4 1.07 1.32 5 
0.2 110 3.63 6.1 2.9 2.65 8.3 3.9 5.9 1.42 1.74 5 
0.2 110 2.00 8.3 1.07 30 
______________________________________ 
Units: 
q.sub.s = g glu/g cellhr.sup.31 1 ; 
q.sub.p = g eth/g cellhr.sup.-1. 
The data in Table 3 demonstrate that a fermenter can be operated according 
to this invention at a lower biomass concentration under nitrogen limiting 
conditions than under conditions of nitrogen-excess at a given dilution 
rate. The fermenter can be operated at a higher specific rate of product 
formation under nitrogen-limiting conditions than under conditions of 
nitrogen-excess at a given dilution rate. As the dilution rate is 
increased, biomass concentration in the fermenter also increases with 
increasing amounts of nitrogen, but the biomass level is less under 
nitrogen-limitation than under nitrogen-excess. 
The amount of assimilable nitrogen required for nitrogen-limited 
fermentation is proportional to the dilution rate and must be increased as 
the dilution rate is increased to achieve complete substrate conversionto 
ethanol. The significance of this feature of the invention can be more 
fully appreciated by comparing the data in Table 3. When the fermentation 
was carried out under nitrogen-limiting conditions and the dilution rate 
was doubled, say from 0.1 hr.sup.-1 to 0.2 hr.sup.-1, the amount of 
ammonium chloride (AC), or its equivalent, had to be about doubled, e.g., 
from 0.71 to 1.42, in order to ensure substantially complete fermentation 
of the glucose substrate. By comparison, when the amount of assimilable 
nitrogen as ammonium chloride was maintained at 1.0 g/L while the dilution 
rate was increased 0.15 hr.sup.-1 to 0.2 hr.sup.-1, the biomass 
concentration in the fermenter remained constant at 2.00 g/L, but the 
fermentation was incomplete as evidenced by the appearance of unfermented 
glucose in the fermenter effluent. The glucose concentration in the 
effluent (S.sub.o) increased from a level of less than 0.5% (w/v) to a 
level of about 3.0%(w/v) when the concentration of assimilable nitrogen 
was not adequately controlled. 
Nitrogen limitation according to this invention does not appreciably affect 
the efficiency of conversion of substrate to ethanol, expressed as the 
amount of ethanol produced per amount of substrate utilized, rather than 
the amount of ethanol produced per the amount of substrate added to the 
fermenter. In the fermentations reported in Table 3 that were carried out 
according to this invention, the observed ethanol concentration was about 
52 g/L. This corresponded to a product yield of about 93% of the 
theoretical maximum yield. 
Under conditions of excess-nitrogen, the specific rate of glucose uptake 
and the specific rate of ethanol production increases with increasing 
dilution rates. In the case of nutrient-limited fermentation, the biomass 
expresses maximal rate of sugar uptake and maximal rate of ethanol 
production. The rates are higher in all cases with nutrient-limitation 
than with nutrient-excess. The data in Table 3 show that a more pronounced 
effect on the specific rate of glucose uptake and the specific rate of 
product formation can be obtained at lower dilution rates by carrying out 
the fermentation under nitrogen-limiting conditions according to this 
invention. 
Data demonstrating that there is typically a maximum imposed on both the 
concentration of glucose in the feed stream (S.sub.r) to the fermenter and 
the dilution rate (D) when complete conversion (95% or more) of sugar to 
ethanol is desired at relatively high ethanol concentrations can be found 
in Table 4. 
TABLE 4 
______________________________________ 
Amount of Assimilable Nitrogen as Either Yeast Extract (Difco) 
Ammonium Chloride or Ammonium Sulphate Require to Achieve 
Maximal Rate of Sugar Utilization and Ethanol Production 
by Z. mobilis ATCC 29191 at High Product Concentration 
D = 0.08 hr.sup.-1 
Excess Nitrogen Nitrogen Limitation 
S.sub.r 
Eth X X YE AC AS 
g/L g/L g/L q.sub.s 
q.sub.p 
g/L q.sub.s 
q.sub.p 
g/L g/L g/L 
______________________________________ 
150 67 2.16 5.1 2.4 1.37 8.3 3.9 3.1 0.73 0.90 
______________________________________ 
Units: 
q.sub.s = g glu/g cellhr.sup.-l ; 
q.sub.p = g product/g cellhr.sup.-1. 
When the concentration of glucose in the feed stream was elevated to 150 
g/L, the maximum dilution rate that could be maintained for complete 
fermentation was 0.08 hr.sup.-1. The ethanol concentration in the 
fermentation medium was 67 g/L for this glucose level. Table 4 compares 
the results for nitrogen-limited fermentation according to this invention 
with fermentation carried out under conditions of nitrogen-excess. The 
data show that even with a high concentration of sugar in the feed stream 
and a high ethanol concentration in the fermentation medium, this 
invention makes it possible to operate at a reduced biomass level, an 
increased rate of substrate uptake and an increased rate of product 
formation. 
Potassium-Limited Fermentation 
This invention can also be carried out by potassium-limited fermentation of 
Zymomonas in continuous culture in a manner analogous to the 
nitrogen-limited fermentation previously described and with comparable 
results. The range of tolerable amounts of potassium in the nutrient 
medium is rather narrow. For this reason, and for the additional reason 
that the amount of the limiting nutrient must be known with substantial 
precision, potassium-limited fermentation is conducted in a defined salts 
medium. An example of a suitable medium is described hereinafter with 
reference to Table 7. 
The amount of potassium, as potassium chloride, required to achieve maximum 
rate of sugar utilization and ethanol production by Z. mobils strain ATCC 
29191 in continuous culture in a chemostat at fixed dilution rate of 0.15 
hr.sup.-1 was determined as a function of feed sugar concentration. The 
results are reported in Table 5. 
TABLE 5 
______________________________________ 
Amount of Potassium (as Potassium Chloride) Required to 
Achieve Maximal Rate of Sugar Utilization and Ethanol Pro- 
duction by Z. mobilis ATCC 29191 in Continuous Culture at Fixed 
Dilution Rate (0.15 hr.sup.-1) as a Function of Feed Sugar 
Concentration D = 0.15 hr.sup.-1 
Excess Potassium 
Potassium Limitation 
S.sub.r 
Eth X X KCl 
g/L g/L g/L q.sub.s 
q.sub.p 
g/L q.sub.s 
q.sub.p 
g/L 
______________________________________ 
20 9.6 0.58 5.2 2.5 0.40 7.5 3.6 0.024 
60 28 1.73 5.2 2.5 1.2 7.5 3.6 0.071 
110 52 3.17 5.2 2.5 2.2 7.5 3.6 0.129 
______________________________________ 
Units: 
q.sub.s = g glu/g cellhr.sup.-1 ; 
q.sub.p = g eth/g cellhr.sup.-1. 
As shown in Table 5 and as in the nitrogen-limited fermentation, 
potassium-limited fermentation can be carried out at lower biomass 
concentration than under conditions of potassium-excess at a given glucose 
concentration. Also, the fermenter can be operated at a higher q.sub.p 
under potassium-limitation than under potassium-excess at a given glucose 
concentration. As glucose in the feed is increased, biomass in the 
fermenter also increases, but the biomass level is less under 
potassium-limitation than under potassium-excess. As with nitrogen-limited 
fermentation, the amount of potassium required for potassium-limited 
fermentation is proportional to the concentration of glucose in the feed 
stream; the amount of potassium as KCl must be increased as glucose 
concentration is increased to maintain q.sub.p. Once again, the nutrient 
limitation does not appreciably affect product yield when compared with a 
similar fermentation carried out under nutrient-excess. 
When this invention is carried out with potassium-limited fermentation and 
when it is necessary to control the pH of the fermentation medium, a pH 
regulating agent or buffering agent other than potassium hydroxide or 
other potassium-containing compound should be employed, otherwise it is 
practically impossible to control the amount of potassium in the 
fermentation medium. The use of sodium hydroxide as the pH regulating 
agent in a potassium-limited fermentation has been found to be 
advantageous. Sodium apparently acts as a potassium antagonist, and the 
resulting elevated level of sodium after the addition of sodium hydroxide 
to the fermenter potentiates the effect of potassium-limitation on the 
specific activity of the biomass. A buffering agent, such as NaH.sub.2 
PO.sub.4, can also be employed. 
Phosphorus-Limited Fermentation 
This invention can be carried out by phosphorus-limited fermentation of 
Zymomonas in continuous culture in a manner analogous to the 
nitrogen-limited fermentation previously described and with comparable 
results. As with potassium-limited fermentation, the range of tolerable 
amounts of phosphorus in the nutrient medium is rather narrow. For this 
reason, and for the additional reason that the amount of the limiting 
element must be known with substantial precision, phosphorus-limited 
fermentation is conducted in a defined salts medium. Once again, an 
example of a suitable medium is described hereinafter with reference to 
Table 7. 
The amount of assimilable phosphorus as potassium dihydrogen phosphate 
required to achieve maximal rate of sugar utilization and ethanol 
production by Zymomonas mobilis strain ATCC 29191 in continuous culture in 
a chemostat at fixed dilution rate of 0.15 hr.sup.-1 was determined as a 
function of feed sugar concentration. The amount of potassium dihydrogen 
phosphate employed and the results obtained are reported in Table 6. 
TABLE 6 
______________________________________ 
Amount of Assimilable Phosphorus, as KH.sub.2 PO.sub.4, Required to 
Achieve Maximal Rate of Sugar Utilization and Ethanol 
Production by Z. mobilis ATCC 29191 in Continuous Culture 
at Fixed Dilution Rate (0.15 hr.sup.-1) as a Function of Feed 
Sugar Concentration 
D = 0.15 hr.sup.-1 
Excess Phosphate 
Phosphate Limitation 
S.sub.r 
Eth X X KH.sub.2 PO.sub.4 
g/L g/L g/L q.sub.s 
q.sub.p 
g/L q.sub.s 
q.sub.p 
g/L 
______________________________________ 
20 9.6 0.58 5.2 2.5 0.42 7.2 3.4 0.04 
60 28 1.73 5.2 2.5 1.25 7.2 3.4 0.13 
110 52 3.17 5.2 2.5 2.29 7.2 3.4 0.23 
______________________________________ 
Units: 
q.sub.s = g glu/g cellhr.sup.-1 ; 
q.sub.p = g eth/g cellhr.sup.-1. 
The data in Table 6 show that the results obtained with phosphorus-limited 
fermentation are analogous to the results obtained with nitrogen-limited 
and potassium-limited fermentations. Phosphorus-limited fermentation can 
be carried out at lower biomass concentration, higher specific rate of 
glucose uptake, higher specific rate of product formation and comparable 
product yield as compared to fermentation carried out under conditions of 
phosphorus-excess. The data show that the amount of phosphorus required 
for phosphorous-limited fermentation is proportional to the concentration 
of glucose in the feed stream. 
When this invention is carried out with phosphorus-limited fermentation and 
when it is necessary to control the pH of the fermentation medium, a pH 
regulating agent or buffering agent other than a phosphorus-containing 
compound should be employed. The additional source of phosphorus from a 
phorphorus-containing pH regulating agent may prevent the degree of 
control of nutrient-limitation required by the invention. For 
phosphorus-limited fermentation KH.sub.2 PO.sub.4 can be employed as a 
buffer and NaOH titrant diluted to 0.5N in order to avoid large changes in 
pH during automatic titration. 
Formulating the Nutrient Medium 
The identity of the chemical constituents in the nutrient medium and the 
amount of each constituent should be sufficient to meet the elemental 
requirements for cell mass and ethanol production and should supply 
appropriate energy for synthesis and maintenance. The nutrient medium 
should contain carbon, nitrogen, potassium, phosphorus, sulfur, magnesium, 
calcium and iron in required amounts. The chemical constituents should 
also meet specific nutrient requirements including vitamins and trace 
minerals. 
As the assimilable source of nitrogen, various kinds of inorganic or 
organic salts or compounds can be included in the nutrient medium. For 
example, ammonium salts, such as ammonium chloride or ammonium sulfate, or 
natural substances containing nitrogen, such as yeast extract, peptone, 
casein hydrolysate or corn steep liquor, or amino acids, such as glutamic 
acid, can be employed. These substances can be employed either singly or 
in combination of two or more. 
Examples of inorganic compounds that can be included in the culture medium 
are magnesium sulfate, potassium monohydrogen phosphate, potassium 
dihydrogen phosphate, sodium chloride, magnesium sulfate, calcium 
chloride, iron chloride, magnesium chloride, zinc sulfate, cobalt 
chloride, copper chloride, borates and molybdates. 
Organic compounds that may be desirable in the fermentation include, for 
example, vitamins, such as biotin, calcium pantothenate, and the like, or 
organic acids, such as citric acid, or amino acids, such as glutamic acid. 
It has been found, however, that biotin is not required in the growth 
medium. 
Fermentation aids that are non-toxic to the microorganism can be included 
in the nutrient medium and fermentation broth. For example, an 
anti-foaming agent in a minor amount has been found to be advantageous. 
Examples of nutrient media that have been found suitable for use in this 
invention are described in Table 7. 
TABLE 7 
______________________________________ 
Chemical Composition of Growth Media for 
Continuous Culture of Z. mobilis 
SEMI-SYNTHETIC DEFINED 
MEDIUM SALTS MEDIUM 
INGREDIENT (g/L) (g/L) 
______________________________________ 
D-Glucose 100 100 
(anhydrous) approx. 
Yeast Extract 
5 -- 
(Difco) 
NH.sub.4 Cl 2.4 2.4 
KH.sub.2 PO.sub.4 
3.48 3.48 
MgSO.sub.4.7H.sub.2 O 
1.0 1.0 
FeSO.sub.4.7H.sub.2 O 
0.01 0.01 
Citric Acid 0.21 0.21 
Vitamins 
Ca-pantothenate 
0.001 0.001 
Biotin 0.001 0.001 
Antifoam 
as required 
______________________________________ 
The semi-synthetic medium is suitable for use in the nitrogen-limited 
fermentation according to this invention. It will be understood that the 
composition of this medium will depend on the technical quality of the 
nitrogen source. For example, yeast extract from a batch from one 
commercial source may exhibit a different potency with respect to the 
content of assimilable nitrogen than a yeast extract from a different 
batch or from another commercial source. Also, the amount of the nitrogen 
source required in the medium will depend on the degree of hydration; 
anhydrous chemicals are preferred and were employed in the Examples and in 
the fermentations reported in the Tables. 
The defined salts medium is suitable for use in carrying out the 
potassium-limited and phosphorus-limited fermentations. Only inorganic 
sources of nitrogen, such as ammonium salts, are employed in defined salts 
media. In the experiments reported in the foregoing Tables and Examples, 
the defined salts medium was used in the potassium- and phosphorus-limited 
fermentations, with the following exceptions. 
Ordinarily, the phosphorus in the nutrient medium is supplied as a salt 
having an anion that is substantially non-toxic to the microorganism and 
that does not substantially inhibit normal metabolic processes. While a 
potassium salt, such as potassium dihydrogen phosphate, is typically 
employed for nitrogen-limited and phosphorus-limited fermentations, a 
sodium salt is preferably substituted for the potassium salt in 
potassium-limited fermentation. For example, sodium dihydrogen phosphate 
instead of potassium dihydrogen phosphate can be utilized. Since the 
amount of available potassium must be precisely known under 
potassium-limited conditions, this substitution makes it easier to control 
the relative proportions of nutrients. 
The potassium in the nutrient medium for potassium-limited fermentation is 
supplied by a salt having an anion that is substantially non-toxic to the 
microorganism and that does not substantially inhibit normal metabolic 
processes. The source of potassium is preferably potassium chloride, 
although similar water-soluble, inorganic salts can be employed. 
The amount of limiting nutrient in the nutrient medium mainly depends on 
two factors: The concentration of substrate in the feed stream to the 
fermenter and the dilution rate. As the substrate concentration at a 
constant dilution rate is increased, the amount of limiting nutrient is 
increased. Similarly, at a constant substrate concentration, the amount of 
limiting nutrient is increased as the dilution rate increases. These 
relationships apply to nitrogen-limited, potassium-limited and 
phosphorus-limited fermentations, since the fermentations are analogous to 
each other. The concentrations of inorganic salts other than the N-, K- 
and P-containing salts are relatively invariant with the formulations 
shown in Table 7. 
The nutrient media in Table 7 can also be employed in a fermentation 
carried out under conditions of nutrient excess. For example, a 
nitrogen-excess medium based on yeast extract (Difco) and ammonium ion can 
contain about 5 to about 10 g/L of the yeast extract and about 15 to about 
34 mM, preferably about 30 mM, ammonium ion. Molar values are given 
because the weight depends on the particular ammonium salt chosen. For 
ammonium chloride the corresponding concentrations would be about 0.8 to 
about 2.4 g/L, preferably about 1.6 g/L. The nutrient medium used in the 
fermentations carried out under conditions of nitrogen excess and reported 
in Tables 2, 3 and 4 was the semisynthetic medium of Table 7 containing 5 
g/L yeast extract (Difco) and 1.6 g/L NH.sub.4 Cl. 
The amount of limiting nutrient, namely nitrogen, potassium or phosphorus, 
expressed as the concentration in the growth medium being fed to the 
fermenter, required to achieve a condition of growth limitation can be 
determined from Equations (1) and (2) and a knowledge of values for the 
growth yield with respect to the particular limiting nutrient and the 
maximum specific rate of substrate utilization (q.sub.s.sup.max). While 
these values may be strain specific, they can be experimentally determined 
and examples are given below. 
The equations used to calculate the amount of limiting nutrient required to 
achieve a condition of nutrient deficiency or limitation are: 
EQU N*=X*/Y.sub.n (1) 
where 
N*=the amount of source of nutrient, g; 
X*=the dry mass of cells (dry wt biomass), g; and 
Y.sub.n =growth yield coefficient for a specified limiting nutrient, n; g 
dry biomass/g-atom nutrient source (see Table 9). 
The value for X* in Equation (1) is determined by Equation (2): 
EQU X*=S.sub.r (D)/q.sub.s.sup.max (2) 
where 
S.sub.r =the concentration of substrate in the feed stream to the 
fermenter, g/L; 
D=the dilution rate, hr.sup.-1 ; and 
q.sub.s.sup.max =the maximum observed specific rate of substrate uptake for 
the strain of Zymomonas being used in the continuous fermentation, g glu/g 
biomass-hr.sup.-1 ; (see Table 10). 
Experimentally determined values of various growth yield coefficients 
(Y.sub.n) for Z. mobilis strain ATCC 29191 with respect to different sole 
sources of assimilable nitrogen are given in Table 8. 
TABLE 8 
______________________________________ 
Observed Values of Growth Yields 
(Z. mobilis ATCC 29191) 
Nitrogen Source Growth Yield Y.sub.n 
______________________________________ 
Yeast Extract (Difco) 
0.45 g dry biomass/g YE 
Ammonium Chloride 
1.87 g dry biomass/g NH.sub.4 Cl 
Ammonium Sulphate 
1.52 g dry biomass/g (NH.sub.4).sub.2 SO.sub.4 
______________________________________ 
Growth yield coefficients (Y.sub.n) with respect to nitrogen, phosphorus 
and potassium were calculated from steady state biomass concentrations in 
respectively limited chemostat cultures at a fixed dilution rate of 0.15 
hr.sup.-1, a constant temperature of 30.degree. C. and a pH of 5.5. In 
each case the entering glucose concentration was approximately 100 g/L. 
The results are summarized in Table 9. The value of growth yield with 
respect to potassium in influenced by the [Na.sup.+ ] such that Y.sub.K 
decreases with increasing concentration of Na.sup.+ in the culture 
medium. The titrant used to maintain pH was NaOH. Observed values for 
different growth yields for Z. mobilis are in good agreement with general 
values cited in the literature with respect to the elemental composition 
(% w/w) of bacteria. 
TABLE 9 
______________________________________ 
Calculated Values of Growth Yields 
(Z. mobilis ATCC 29191) 
Y.sub.n Composition 
Type of Growth Yield of Biomass 
Limiting Nutrient, n 
(g biomass/g atom) 
% w/w 
______________________________________ 
Nitrogen (N) 7.1 14 
Phosphorus (P.sub.i) 
44 2.3 
Potassium (K.sup.+) 
33 3.0 
______________________________________ 
The values given in Table 9 can be substituted in Equation (1). It will be 
understood that these values may vary with the fermentation system and 
operating techniques and should be confirmed by experimentation in the 
system under study or in question. 
It has been suggested in the literature that the values for q.sub.s.sup.max 
and q.sub.p.sup.max are stain specific traits in Z. mobilis. In any event, 
q.sub.s.sup.max and q.sub.p.sup.max for the strain of interest can best be 
determined by means of non-carbon limitation under steady-state conditions 
in continuous culture in a chemostat. The value for q.sub.s.sup.max may 
vary depending on the nature of the limiting nutrient and the particular 
strain of Z. mobilis chosen, but q.sub.s.sup.max is generally about 7 to 
about 10 g glucose/g cell-hr.sup.-1. Experimentally determined values of 
q.sub.s.sup.max for nutrient-limited fermentations by the strain ATCC 
29191 are given in Table 10. 
TABLE 10 
______________________________________ 
Observed Average Values of Maximum Specific Rate of Glucose 
Uptake for Nutrient-Limited Fermentation by Strain 
ATCC 29191 in Continuous Culture in a Chemostat 
Type of 
Limiting 
Chemical Identity 
q.sub.s.sup.max 
Nutrient, 
of Limiting (g glucose/ 
Y.sub.X /n 
n Nutrient biomass-hr.sup.-1) 
(g glu/g of n) 
______________________________________ 
Nitrogen 
NH.sub.4 Cl 8.3 1.87 
(NH.sub.4).sub.2 SO.sub.4 
8.3 1.52 
Yeast Extract (Difco) 
8.3 0.45 
Potassium 
KCl 7.5 17 
Phosphate 
KH.sub.2 PO.sub.4 
7.2 10 
______________________________________ 
The values in Table 10 can be substituted in Equation (2), once again 
subject to confirmation in the system under study. 
The observed q.sub.s.sup.max for potassium-limited Z. mobilis strain ATCC 
29191 has been found to be 7.5 g glucose/g biomass-hr.sup.-1, and the 
value for the growth yield (Y.sub.K) with respect to KCl has been found to 
be 17 g biomass/g KCl. The observed value for Y.sub.K is 33 g biomass/g 
K.sup.+, which is equivalent to saying that the biomass is 3% w/w 
potassium. Equations (1) and (2) can also be used to predict the amount of 
potassium (e.g. as KCl) required to achieve a condition of potassium 
limitation in continuous culture at various values for the concentration 
of glucose in the feed stream (S.sub.r) and dilution rate (D). 
The observed average value for q.sub.s.sup.max for a phosphorus-limited 
culture of Z. mobilis is 7.2 g glu/g biomass-hr.sup.-1 and the growth 
yield with respect to phosphorus (as potassium dihydrogen phosphate) is 10 
g biomass/g KH.sub.2 PO.sub.4. The observed Y.sub.p is 44 g biomass/g P, 
which is equivalent to biomass being 2.3% w/w with respect to its 
phosphorus content. These values can be substituted appropriately into 
Equations (1) and (2) in order to predict the amount of phosphorus 
required to achieve a condition of phosphorus-limitation at various values 
of S.sub.r and dilution rate. 
The values in Tables 8 and 9 were determined at various values for S.sub.r 
and D. As mentioned previously, because Z. mobilis is sensitive to ethanol 
at concentrations in excess of about 5% (w/v), there is an upper limit to 
the practical value of S.sub.r, namely about 150 g fermentable sugar/L. 
The following Examples illustrate working embodiments of this invention. 
EXAMPLE 1 
Continuous ethanol fermentations were performed in apparatus similar to 
that described in the Figure. Bench-top chemostats (Model C30, New 
Brunswick Scientific Co. N.J.) were used in which the content working 
volume (V) of 350 ml was established and maintained by means of an 
attached overflow tube. The culture was agitated by means of a pair of 
turbine six blade impellers operating at 200 RPM. The temperature was 
controlled at 30.degree. C. and the pH was monitored using a combination 
Ingold electrode coupled to a Model pH-40 (New Brunswick Scientific) pH 
analyzer. The addition of titrant (KOH) was controlled automatically by 
the pH controller and maintained at 5.5. The vessel was not sparged with 
gas of any kind except during start-up when oxygen-free N.sub.2 was used 
at a rate of approximately 0.5 v/v/m. 
The chemical composition of the semi-synthetic culture medium is described 
in Table 7. The concentration of glucose were 20, 60 or 110 g/L. Yeast 
extract obtained from Difco was the sole source of assimilable nitrogen 
added to the culture medium (i.e., ammonium chloride was not added). In 
order to achieve a condition of nitrogen-excess growth, 5 g/L yeast 
extract were added to the basal salts medium (at all concentrations of 
glucose). For nitrogen-limitation the amount of yeast extract added to the 
salts medium depended on the amount of glucose in the medium such that for 
media containing 20, 60 and 110 g glucose/L, yeast extract in amounts of 
0.8, 2.4 and 4.4 g, respectively, were added per liter (L). 
Polypropylene glycol 2025 was added to the medium as an antifoaming agent 
at a concentration of 0.1 ml/L. Media were prepared and autoclaved in 13 L 
pyrex carbuoys. Sterile culture medium was fed to the fermenter at a 
constant rate (F) by means of a peristaltic pump such that the dilution 
rate (calculated as F/V) was 0.15/hr.sup.-1. The fermenter was inoculated 
(15% v/v) with Z. mobilis ATCC 29191, which had been grown overnight in 
medium of similar composition in a non-agitated flask incubated at 
30.degree. C. Flow to the fermenter was not commenced until the culture 
was in late-exponential phase of growth. Growth and biomass concentration 
were determined as dry weight of culture collected on preweighed 
microporous filters (Millipore Corp., 0.45 .mu.m pore size). Sampling the 
biomass and weighing the dry cells has been found to be much more accurate 
and reliable than turbidity measurement or measurements made by indirect 
methods. Steady-state was presumed to have occurred after a minimum of 4 
culture turnovers, a turnover being equivalent in time to the reciprocal 
of the dilution rate. Glucose was determined using a YSI Glucose Analyzer 
(Model 27, Yellow Springs Instrument Co., Ohio). Ethanol was measured by 
HPLC (HPX-87H Aminex, 300.times.7.8 mm column, from Bio-Rad, Burlington, 
Ont. Can.). The culture was routinely examined for contamination both by 
microscopic assessment and by plating on selective diagnostic agar media. 
The specific rate of glucose uptake, q.sub.s, (g glucose consumed/g 
biomass-hr.sup.-1), was calculated as follows: 
##EQU1## 
where S.sub.r and S.sub.o represent the concentration of fermentable sugar 
in the feed reservoir and fermenter effluent, respectively; 
D=the dilution rate (hr.sup.-1); and 
X=the dry weight culture biomass concentration (g/L). 
Similarly, the specific rate of ethanol formation, q.sub.p, (g ethanol/g 
biomass-hr.sup.-1) was calculated as follows: 
##EQU2## 
where [P] represents the steady state ethanol concentration. 
The results of this experiment are summarized in Table 2. 
EXAMPLE 2 
The same procedure was followed as in EXAMPLE 1 except the sole source of 
nitrogen was ammonium chloride (no yeast extract was added to the medium 
and as such it is referred to as a defined salts medium). Table 2 shows 
the amount of ammonium chloride (NH.sub.4 Cl) used at different values for 
S.sub.r with respect to glucose, these being 0.19, 0.59 and 1.10 g 
NH.sub.4 Cl/L for S.sub.r glucose values of 20, 60 and 110 g/L, 
respectively. 
EXAMPLE 3 
The same procedure was followed as in EXAMPLE 1 except that the sole source 
of assimilable nitrogen was ammonium sulphate (AS). The amounts added and 
the results obtained are shown in Table 2. 
EXAMPLE 4 
Experiments were performed with Z. mobilis strain ATCC 31821. The results 
were substantially the same as those observed with strain ATCC 29191. 
EXAMPLE 5 
Experiments were performed with Z. mobilis strain ATCC 10988. The results 
were substantially the same as those observed with strain ATCC 29191. 
EXAMPLE 6 
Experiments were performed to show the amount of assimilable nitrogen 
required to achieve nitrogen-limitation as a function of dilution rate. 
The results are summarized in Table 3. S.sub.r was constant at 110 g/L and 
the dilution rate was set at 0.1, 0.15 and 2.0 hr.sup.-1. The sole sources 
of nitrogen were yeast extract (Difco), ammonium chloride and ammonium 
sulphate. Although the results shown in Table 3 were obtained with strain 
ATCC 29191, substantially similar results were observed with both ATCC 
31821 and ATCC 10988. 
EXAMPLE 7 
Table 4 summarizes the results of an experiment with ATCC 29191 to 
illustrate the effect of end-product (ethanol) inhibition on the general 
formula for predicting fermentation performance under conditions of 
nitrogen excess and nitrogen limitation. Even when the continuous 
fermenter was operated near its upper limit with respect to ethanol 
concentration, the specific activities (q.sub.s and q.sub.p) of the 
culture were improved by imposing the condition of nitrogen limitation. 
EXAMPLE 8 
Experiments were performed to show the amount of potassium required to 
achieve potassium-limitation as a function of glucose concentration at a 
constant dilution rate for ATCC 29191. D was constant at 0.15 hr.sup.-1, 
the pH was controlled with NaOH at 5.5 and S.sub.r was set at 20, 60 and 
110 g/L. The sole source of potassium was KCl. The concentration of KCl 
and results obtained are summarized in Table 5. 
EXAMPLE 9 
Experiments were performed to show the amount of assimilable phosphorus, as 
KH.sub.2 PO.sub.4, required at various sugar concentrations to achieve 
maximal rate of sugar utilization and ethanol production by Z. mobilis 
strain ATCC 29191, in continuous culture in a chemostat at a fixed 
dilution rate of 0.15 hr.sup.-1. A pH of 5.5 was maintained with KOH. The 
concentrations of KH.sub.2 PO.sub.4 and the results obtained are shown in 
Table 6. 
Effect of Temperature on Nutrient-Limited Fermentation 
Microbial growth and product formation are the result of a complex series 
of biochemical reactions that are temperature dependent. Zymomonas strains 
have a broad range of temperatures within which metabolic processes will 
occur and an optimum temperature range within which the rate of product 
formation is maximized. As the temperature is increased toward the optimum 
temperature, the rate of product formation increases. Above the optimum 
temperature, the rate of product formation rapidly declines, due in part 
to an increasing cell death rate and reduced cell growth rate. Lower 
biomass level translates to incomplete fermentation in a continuous system 
unless the dilution rate is adjusted appropriately downwardly. This is 
because, at relatively high substrate concentrations in the fermenter 
feed, the specific rate of substrate uptake will increase with increasing 
temperature, but the reduced biomass decreases the capacity of the 
fermenter to process the same substrate load. Heretofore, the optimum 
temperature was not exceeded when the object was to obtain maximum 
conversion of substrate to product in the least possible time. 
The effect of temperature on the maximum specific growth rate (u.sub.max) 
of Z. mobilis strain ATCC 29191 in batch culture has been reported in the 
literature and is shown in Table 11. The culture medium contained 2% (w/v) 
glucose. 
TABLE 11 
______________________________________ 
The Effect of Temperature on the Maximum Specific 
Growth Rate of Z. mobilis ATCC 29191 in 
Batch Culture (2% glu and pH 5.5) 
Temperature 
max 
(.degree.C.) 
(hr.sup.-1) 
______________________________________ 
30 0.27 
33 0.38 
36 0.26 
______________________________________ 
The data in Table 11 show that as the temperature in the culture medium was 
increased from 30.degree. C. to 33.degree. C., the maximum specific growth 
rate increased, but with a further increase in temperature from 33.degree. 
C. to 36.degree. C., the maximum specific growth rate declined. Thus, the 
optimum fermentation temperature for this system was about 33.degree. C. 
Cell growth was inhibited above this temperature. 
While increasing temperature affects cell growth as shown in Table 11, it 
has now been found that increasing temperature also affects substrate 
conversion and product formation. Table 12 shows the effect of increasing 
temperature on performance of a carbon-limited continuous fermentation by 
Z. mobilis strain ATCC 29191 in a chemostat at a constant dilution rate of 
0.19 hr.sup.-1. The substrate was fed to the chemostat as an aqueous 
solution containing 2% (w/v) glucose. 
TABLE 12 
______________________________________ 
Effect of Increasing Temperature on Performance of 
Carbon-limited Continuous Fermentation by Z. mobilis 
ATCC 29191 at Constant Dilution Rate (D = 0.19 hr.sup.-1 ; 
2% glu; pH 5.5) 
30.degree. C. 
33.degree. C. 
36.degree. C. 
______________________________________ 
S.sub.r (g/L) 
20.7 20.7 20.7 
S.sub.o (g/L) 
-- 0.1 0.5 
X (g/L) 0.65 0.55 0.45 
q.sub.s (g/g-hr.sup.-1) 
5.9 7.25 8.8 
q.sub.p (g/g-hr.sup.-1) 
2.80 3.41 4.14 
Y.sub.p/s (g/g) 
0.47 0.47 0.47 
______________________________________ 
In the continuous fermentations reported in Table 12, the glucose 
concentration in the feed to the fermenter was maintained constant at 20.7 
g/L. When the temperature in the fermentation medium was increased from 
30.degree. C. to 33.degree. C., the biomass concentration (X) in the 
fermenter decreased, but this decrease was compensated for by an increase 
in the specific rate of glucose uptake (q.sub.s). It will be observed, 
however, that glucose began to appear in the fermenter effluent (i.e. 
S.sub.o =0.1 g/L) at 33.degree. C. 
When the temperature in the fermentation medium was further increased from 
33.degree. C. to 36.degree. C., once again the biomass concentration in 
the fermenter declined, this time from 0.55 g/L to 0.45 g/L, but the 
decline was offset by a further increase in the specific rate of glucose 
uptake (q.sub.s). The conversion of glucose to ethanol was incomplete and 
the concentration of glucose in the effluent (S.sub.o) increased to 0.5 
g/L. While the yield coefficient (Y.sub.p/s) remained constant at 0.47 
g/g, the presence of unconverted glucose in the effluent was unacceptable. 
The effect of increasing temperature on the performance of K.sup.+ -limited 
continuous fermentation by Z. mobilis strain ATCC 29191 in a chemostat at 
a constant dilution rate of 0.15 hr.sup.-1 is shown in Table 13. Potassium 
was supplied as KCl at a concentration of 0.13 g/L. 
TABLE 13 
______________________________________ 
Effect of Increasing Temperature on Performance of 
K.sup.+ -limited Continuous Fermentation by Z. mobilis 
ATCC 29191 at Constant Dilution Rate (D = 0.15 hr.sup.-1) 
30.degree. C. 
35.degree. C. 
______________________________________ 
S.sub.r g/L 110 110 
[KCl] g/L 0.13 0.13 
X (g/L) 2.2 1.48 
q.sub.s (g/g-hr.sup.-1) 
7.5 8.9 
Y.sub.KCl (g/g) 17 11.5 
S.sub.o (g/L) -- 22 
______________________________________ 
The data in Table 13 show that increasing the temperature in the 
fermentation medium from 30.degree. C. to 35.degree. C. at a constant 
glucose concentration in the feed stream of 110 g/L resulted in a decrease 
in the biomass concentration and an increase in the specific rate of 
glucose uptake (q.sub.s). In this case, however, the yield coefficient 
(Y.sub.n where n=KCl) declined from 17 g/g KCl to 11.5 g/g KCl and the 
glucose concentration in the effluent increased from 0 g/L to 22 g/L. 
These changes in system performance would adversely affect process 
economics in a commercial operation. 
Table 14 shows the effect of increasing temperature on performance of a 
K.sup.+ -limited continuous fermentation by strain ATCC 29191 in a 
chemostat at varying dilution rates. The substrate was fed to the 
fermenter as an aqueous solution containing 2% (w/v) glucose and at a 
fixed flow rate. Potassium was supplied as KCl at a concentration of 0.1 
g/L. 
TABLE 14 
______________________________________ 
The Effect of Temperature on Kinetics of 
K.sup.+ -limited Z. mobilis Strain ATCC 29191 
q.sub.s 
Temp. S.sub.r S.sub.o D X (g/g- q.sub.p 
(.degree.C.) 
(g/L) (g/L) (hr.sup.-1) 
(g/L) hr.sup.-1) 
(g/g-hr.sup.-1) 
______________________________________ 
30.0 100 18.3 0.155 1.70 7.40 3.48 
32.8 100 10.6 1.157 1.68 8.20 3.85 
35.0 100 34.8 0.160 1.15 8.90 4.0 
______________________________________ 
Increasing the temperature by 2.degree.-3.degree. C. resulted in more 
conversion of glucose to ethanol, i.e. from approximately 82% to 90%, as 
judged by the decrease in effluent glucose (S.sub.o). However, further 
increase in temperature caused more glucose to appear in the effluent and 
the conversion fell to 65%. At 35.degree. C. the morphology of the culture 
changed dramatically becoming very filamentous. The result of operation at 
35.degree. C. was a reduced cell density, and even though the q.sub.p was 
higher, the reduced biomass could not handle the sugar load. 
The effect of increasing temperature on the performance of a K.sup.+ 
-limited continuous fermentation by Z. mobilis according to this invention 
is shown in Table 15. The fermentation was carried out by Z. mobilis 
strain ATCC 29191 in a chemostat at a constant dilution rate of 0.15 
hr.sup.-1. Potassium was supplied as KCl at concentrations indicated. 
TABLE 15 
______________________________________ 
Effect of Increasing Temperature on Performance of 
K.sup.+ -limited Continuous Fermentation by Z. mobilis 
Strain ATCC 29191 at Constant Dilution Rate (D = 0.15 hr.sup.-1) 
30.degree. C. 
35.degree. C. 
35.degree. C. 
______________________________________ 
S.sub.r g/L 
110 110 110 
[KCl] g/L 0.13 0.13 0.16 
X (g/L) 2.2 1.48 1.85 
q.sub.s (g/g-hr.sup.-1) 
7.5 8.9 8.9 
Y.sub.KCl (g/g) 
17 11.5 11.5 
S.sub.o (g/L) 
-- 22 -- 
______________________________________ 
The concentration of substrate to the fermenter was the same in all cases, 
i.e. 110 glucose/L. Comparing column 2 with column 3 in Table 15, when the 
fermentation temperature was increased from 30.degree. C. to 35.degree. 
C., the biomass concentration (X) decreased from 2.2 g/L to 1.48 g/L, 
while the specific rate of glucose uptake (q.sub.s) increased from 7.5 to 
8.9 g/g-hr.sup.-1. The amount of glucose in the effluent (S.sub.o) also 
increased from 0 to 22 g glucose/L, which was commercially unacceptable. 
Comparing column 2 with column 4 in Table 15, it is seen that by increasing 
the concentration of the limiting nutrient according to this invention, 
i.e. [KCl] in this case, when the temperature was increased from 
30.degree. C. to 35.degree. C., the biomass concentration (X) in the 
fermenter decreased from 2.2 g/L to 1.85 g/L while the specific rate of 
glucose uptake (q.sub.s) increased from 7.5 to 8.9 g/g-hr.sup.-1. However, 
there was substantially no glucose in the effluent from the fermenter when 
the concentration of the limiting nutrient was properly controlled, i.e. 
S.sub.o =O. 
These results demonstrate that this embodiment of the invention makes it 
possible to improve the performance of Zymomonas in continuous ethanol 
fermentation at increased temperatures. The specific rate of substrate 
uptake can be maintained, and even increased, at fermentation temperatures 
of about 33.degree. to about 37.degree. C. even though there is a lower 
biomass concentration in the fermenter. These results can be achieved 
without substantial amounts of substrate in the effluent from the 
fermenter. These results are made possible by carrying out continuous 
ethanol fermentation with Zymomonas strains under nutrient-limited 
conditions, where the limiting nutrient is nitrogen, potassium or 
phosphorus. The concentration of the limiting nutrient in the fermentation 
medium is increased with increasing temperature and decreased with 
decreasing temperature. 
In summary, the processes of this invention make it possible to carry out a 
continuous fermentation by Z. mobilis at reduced biomass concentration in 
the fermenter, increased specific rate of substrate uptake, increased 
specific rate of product formation and substantially without substrate in 
the effluent by regulating the amount of limiting nutrient. The processes 
of this invention take into account the unexpected decrease in growth 
yield with respect to the limiting nutrient and adjust the amount of the 
nutrient in the medium accordingly. The fermentation system operates 
efficiently at a high temperature, such as 35.degree. C., with respect to 
fermentation capacity, and at a higher specific rate of substrate uptake 
and a higher specific rate of product formation with a lower biomass 
concentration in the fermenter. As previously discussed, this can be of 
considerable advantage in designing and operating a continuous 
fermentation with cell recycle where the productivity is typically limited 
by the concentration of biomass that can be handled by the recycle device 
be it a filtration system, centrifuge or settler.