Continuous fermentation process for the production of metabolities using a moving filter

A metabolite, e.g., ethanol, is continuously produced from low cost carbohydrate substrates by a process which comprises pulverizing the carbohydrate substrate; liquefying and saccharifying the pulverized substrate; continuously fermenting the lique-saccharified substrate in a fermentor equipped with a moving filter, in the presence of flocculent biological cells maintained at a concentration ranging from 90 to 160 g/l by using the moving filter and a culture medium to produce a fermentation product mixture; and recovering the desired metabolite from the fermentation product mixture.

FIELD OF THE INVENTION 
The present invention relates to a process for producing metabolites from 
low cost substrates; and, more particularly, to a highly efficient and 
economical continuous fermentation process for producing metabolites by 
employing flocculent biological cells and a fermentor equipped with a 
moving filter. 
BACKGROUND OF THE INVENTION 
A wide range of metabolites including alcohol have been used for various 
purposes such as bioenergy, e.g., vehicular fuel; pharmaceuticals, e.g., 
antibiotics, anticancer drugs, proteins and hormones; food, e.g., amino 
acids, liquor, nucleotides and related products, lactic acid, citric acid 
and organic acids; industrial chemicals; and environment-related products. 
The commercial production of metabolites, e.g., ethanol, has been carried 
out by conventional batch fermentation processes which generally suffer 
from low productivity. Therefore, there have been keen interests in 
developing an efficient continuous fermentation process capable of 
substantially lowering the production cost, and particularly using a 
low-cost raw material to make the price of the final product competitive. 
Hitherto, there have been reported several types of continuous fermentation 
processes, including a process for producing ethanol in a tower-type 
fermentor packed with flocculated yeast cells. In theses processes, 
however, high-cost raw materials such as purified molasses and glucose are 
used as the carbohydrate substrate, while the reported reactor 
performances are generally unsatisfactory due to various deficiencies such 
as low productivity, low cell density, instability of the packed bed 
caused by hydrodynamic factors, and the tendency of cell aggregates to 
break up under a poor nutrient condition. 
The performance of a continuous metabolite production reactor may be 
evaluated based on three criteria: productivity, product stream quality 
and long-term stability. 
The productivity, expressed by grams of the metabolite produced per hour 
per liter of working reactor volume, may be calculated by multiplying the 
metabolite product concentration in the product stream (g/l) by the 
dilution rate which is defined as the volume of substrate introduced to 
the reactor per hour per working volume of the reactor. The key for 
increasing the productivity lies in increasing the cell density, i.e., the 
concentration of the active cell aggregates used for carrying out the 
fermentation. 
The product stream quality is determined by the metabolite product 
concentration as well as the concentration of unreacted substrate in the 
effluent. As the cost for separating the metabolite product from the 
product stream increases with a decrease in the product concentration, it 
is desirable to raise the metabolite concentration in the product stream 
as high as possible. On the other hand, the presence of unreacted 
substrate not only reduces the overall process efficiency but also induces 
the problem of cell bleeding which is caused by CO.sub.2 generated by 
fermentation of the residual substrate. 
The long-term stability, which is perhaps the most important criterion in 
deciding the viability of a particular reactor system, may be achievable 
only when a high cell density can be sustained over a sufficient period of 
time under a set operational condition of the reactor. If the cell density 
decreases or fluctuates with time for any reason, commercial 
implementation of the system may become difficult. 
The prior art processes using the tower-type continuous reactor have failed 
to meet one or all of the above criteria, rendering them unsuitable for 
commercial applications. For instance, Netto et al. attempted continuous 
ethanol production from corn hydrolysate using a tower fermentor packed 
with flocculated yeast for a period of 300 hours. However, they could not 
raise the dilution rate beyond 0.3 hr.sup.-1 because of a rapid fall of 
the cell density with the lapse of time from the initial level of 80-90 
g/l. The maximum productivity achieved was shown to be only 18.4 g of 
ethanol per hour per liter of reactor volume while the residual glucose 
concentration amounted to 2.3 g/l. Even under this poor performance 
condition, they still experienced the problem of an operational 
instability due to the deflocculation of cells (see Netto et al., "Ethanol 
Fermentation by Flocculating Yeast: Performance and Stability Dependence 
on a Critical Fermentation Rate", Biotechnology Letters, 7, 1985, pp 
355-360). 
Limtong et al. also employed flocculating yeast to produce ethanol 
continuously from glucose at a relatively high cell density of 70-90 g/l. 
However, they had to lower the glucose substrate concentration in the feed 
in order to suppress the residual glucose concentration below 1 g/l, thus 
limiting the attainable ethanol concentration to below 6.6%. They also 
experienced a rapid drop in the cell concentration when the dilution rate 
was increased beyond 0.5 hr.sup.-1 (see Limtong et al., "Continuous 
Ethanol Production by a Concentrated Culture of Flocculating Yeast", J. 
Ferment. Technol., 62, 1984, pp 55-62). 
Further, Chen produced ethanol at a concentration of 7.7% and 3.1% from 
glucose and molasses, respectively, by using a tower-type fermentor. 
Although Chen achieved a relatively high cell concentration at a high 
dilution rate, e.g., 90-100 g/l at 0.7 hr.sup.-1, these were attained only 
in short-term studies and the issue of the long-time stability was not 
addressed. Moreover, the residual glucose concentration in the case of 
using molasses was very high, i.e., 62 g/l (see C. S. Chen, "Ethanol 
Fermentation Using Self-aggregating Yeast", Proceedings of YABEC '95, 
1995, pp 24-38). 
Thus, despite the use of high-cost raw materials such as purified molasses 
and glucose, the prior studies have not succeeded in developing an 
efficient continuous process for producing ethanol because of the 
occurrence of the variety of problems described above. 
In order to make the process more economical, therefore, there has 
continued to exist a need to develop a commercially viable continuous 
fermentation process which is capable of utilizing low-cost raw materials, 
e.g., untreated molasses, starch-containing materials such as tapioca, 
sweet potato and grains, and cellulose- and xylose-containing materials 
such as wood, corn and other plant stovers. When such material is used as 
the feed substrate, however, each of the problems mentioned above is 
expected to become even further aggravated, and, moreover, there may 
emerge additional problems caused by suspended solid particles remaining 
in the lique-saccharified substrate. Complete removal of such suspended 
particles is not achievable by a conventional centrifugation or filtration 
method, and a substrate solution containing such solid particles, when fed 
to a tower-type continuous reactor, would cause such problems as increased 
culture medium viscosity due to the buildup of solid particles, increased 
mass transfer resistance, channeling, deflocculation of cell aggregates, 
low cell density by cell bleeding and lowering of the decanter efficiency. 
SUMMARY OF THE INVENTION 
Accordingly, it is an object of the present invention to provide a process 
which is substantially free of the problems mentioned above and capable of 
continuously producing a metabolite from low-cost substrates, in a high 
productivity and economic fashion, by way of employing flocculent 
biological cells and a fermentor equipped with a moving filter. 
In accordance with one aspect of the present invention, there is provided a 
continuous process for producing a metabolite from a carbohydrate 
substrate which comprises: (A) pulverizing the carbohydrate substrate; (B) 
liquefying and saccharifying the pulverized substrate; (C) fermenting the 
lique-saccharified substrate in a fermentor equipped with a moving filter, 
in the presence of flocculent biological cells maintained at a 
concentration ranging from 90 to 160 g/l by using the moving filter and a 
culture medium to produce a fermentation product mixture; and (D) 
recovering the metabolite from the fermentation product mixture.

DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the present invention, there is provided an economical 
process for continuously producing a metabolite by fermenting a low cost 
carbohydrate substrate in a continuously stirred tank reactor equipped 
with a moving filter wherein a high concentration of flocculent biological 
cells is maintained by using the moving filter. 
(i) Pulverization and liquefaction-saccharification 
The carbohydrate substrate may be preferably pulverized to a substrate 
powder. The carbohydrate substrates which may be used in the present 
invention include molasses, industry-grade glucose, starch-containing 
materials such as tapioca, potato, sweet potato and grains, and cellulose- 
and xylose-containing materials such as wood, corn stovers, cane stovers 
and other agricultural wastes. It is preferred to pulverize the 
carbohydrate substrate until the particle size thereof lies within the 
range of 10 to 1,000 .mu.m using a conventional equipment, e.g., ball 
mill. 
The powdered carbohydrate substrate may be then liquefied and saccharified 
by using a conventional method known to ordinary persons skilled in the 
art. For example, the liquefaction-saccharification may be carried out 
with water and enzymes, which are capable of hydrolysing the substrate to 
glucose, at an elevated temperature, e.g., 50.degree. to 95.degree. C., 
for several hours. Representatives of such useful enzyme include amylase, 
glucoamylase, cellulase, cellobiase and a mixture thereof. The 
lique-saccharified substrate may have a glucose concentration ranging from 
140 to 300 g/l and a solid particle concentration of less than 60% (v/v), 
wherein the suspended solid particles may have a particle size 
distribution ranging from 1 to 1,000 .mu.m, more preferably 1 to 100 
.mu.m. 
Subsequently, the lique-saccharified substrate may be directly introduced 
into a fermentor, or if necessary, may be subjected to a liquid/solid 
separation procedure to remove relatively large solid particles present 
therein before being introduced to the fermentor. This solid particles 
separation may be carried out by employing a simple device such as a 
decanter, low speed centrifuge or membrane. The feed to the fermentor 
preferably has a suspended solid content of less than 20% (v/v). 
(ii) Fermentation 
In accordance with the present invention, the fermentation process is 
carried out in a fermentor equipped with a moving filter in the presence 
of flocculent biological cells and a culture medium. The flocculent cells 
which may be used in the present invention include those having the 
flocculating property, which may be procaryotic or eucaryotic and be found 
in nature or produced by a gene manipulation technique. Suitable cells may 
be selected depending on the desired metabolic product; and 
representatives of such cells include Streptomyces species, Aspergillus 
species, yeast and the like. In the inventive process, the concentration 
of the cells in the fermentor during the continuous fermentation procedure 
is maintained at a range of 90 to 160 g/l. 
The moving filter in the fermentor may be of a screen or a membrane with an 
opening size ranging from 10 to 4,000 mesh, preferably 50 to 2,000 mesh, 
and may be placed in a proper position inside the fermentor. The filter in 
accordance with the present invention makes it possible to maintain the 
cell concentration at a high level by way of blocking the passing of the 
flocculent cells while allowing the suspended solid particles to pass 
freely therethrough. 
The moving filter may be installed, if desired, outside the fermentor, 
e.g., in a sedimentor suitably connected to the fermentor. The filter may 
be shaped in a variety of form such as cylinder, basket, plate or disk. 
Multiple filters may also be installed in an arrangement suitable for a 
particular need. 
The moving filter may be equipped with an agitator e.g., a screw, ribbon or 
intermig type, to generate a various mode of liquid motions including 
rotation, reciprocation, vibration, swirling, gyration, and a combination 
thereof. Further, the filter may be optionally equipped with backflushing 
accessories to deal with a process upset that might cause a clogging 
problem. The cells and solid particles that may deposit on the surface of 
the filter may be removed by a conventional method, i.e., application of 
vibration, ultrasonic waves, gas jets and the like. 
The fermentation process may be carried out at a condition suitably chosen 
depending on the desired metabolite product, types of biological cells, 
culture medium and the like, as is well known to the ordinaly person 
skilled in the art. 
The culture medium may be employed to adjust the growth, the flocculating 
property and the aggregate size of the cells. The medium may include a 
nitrogen containing compound and, if required, additional compounds in 
trace amounts depending on the glucose concentration of the 
lique-saccharified substrate. Illustratives of the nitrogen containing 
compounds include ammonium phosphate mono- or dihydride, ammonium sulfate, 
ammonium nitrate, ammonium acetate, urea, yeast extract, peptone or a 
mixture thereof, and representatives of the additional compounds include 
sodium or potassium dihydrogen phosphate, disodium or dipotassium hydrogen 
phosphate, sodium chloride, calcium chloride, ferrous sulfate, magnesium 
sulfate or a mixture thereof. 
Although the inventive fermentation may be carried out in a continuous 
mode, a fill-draw type sequencing batch reactor(SBR) operation may also be 
used during the start-up period, or when it is necessary to rapidly raise 
the cell density and/or to adjust the cell floc size during the steady 
state operation. To accelerate the fermentation rate, air may be 
introduced to the fermentor by a suitable means, e.g., an air sparger, 
which may be installed in the fermentor. 
The SBR mode of operation may be conducted as follows. The feed solution 
and the nutrients are introduced into a space external to the filter and 
the fermentation product is taken out from the inside of the moving 
filter. The fermentor is first inoculated with suitable biological cells 
at a concentration ranging from 3 to 4 g/l. The cell culture may be 
performed in a batch mode followed by 3 to 5 cycles of fill-draw SBR 
operations, thereby increasing the cell concentration to a desired level, 
e.g., 15 to 20 g/l. At this point, the cells flocculate to form cell 
flocs, which continue to grow. Recycling of the cells from inside to 
outside of the moving filter may be conducted during the SBR operation. 
When the cell concentration reaches the desired level, the operation is 
switched to a continuous mode and the dilution rate of the substrate is 
increased gradually until the cell concentration reaches above 100 g/l. 
Finally, a steady-state continuous fermentation operation is carried out 
at a dilution rate of the substrate ranging from 0.5 to 2 hr.sup.-1, while 
maintaining the cell concentration at a range of about 90 to 160 g/l. 
In the fermentation process, multiple fermentors may be employed in order 
to further increase the productivity of the desired metabolite product. 
(iii) Purification 
The fermentation product mixture is then subjected to a series of 
separation processes to recover the desired metabolite product. For 
example, the mixture may be first introduced into one or more sedimentors 
wherein bled cells are allowed to settle. Such cells may be removed or 
recycled to the fermentor, and the supernatant which may contain solid 
materials is drawn out for a conventional separation process, e.g., 
distillation, adsorption or the like. 
Preferred Embodiment of the Invention 
In accordance with the preferred embodiment of the present invention, 
ethanol may be continuously produced from a low cost carbohydrate 
substrate in a high productivity. 
Specifically, the carbohydrate substrate is pulverized, and then 
lique-saccharified. The lique-saccharified substrate preferably has a 
glucose concentration ranging from 14 to 30% and solid particles content 
of less than 60% (v/v), and, more preferably, less than 20% (v/v), wherein 
the size of solid particles ranges from 10 to 1,000 .mu.m. 
The lique-saccharified substrate is then fermented in a fermentor equipped 
with a moving filter in the presence of flocculent biological cells 
maintained at a concentration ranging from 90 to 160 g/l by using the 
moving filter and a culture medium to produce a fermentation product 
mixture. 
In the ethanol fermentation process, as the biological cells, a yeast, 
preferably Saccharomyces cerevisiae, Saccharomyces diastaticus and 
Saccharomyces uvarum, may be used, while Saccharomyces uvarum is most 
preferred. 
The ethanol fermentation may be carried out at a temperature ranging from 
4.degree. to 40.degree. C., more preferably from 15.degree. to 35.degree. 
C., and at a pH ranging from 3.5 to 9, more preferably from 4 to 8, while 
the filter may be rotated at a rate less than 1,000 rpm, more preferably 
less than 500 rpm. The components of the culture medium may be employed in 
their respective amount ranging from 0 to 40 g/l. 
In the case of ethanol production in accordance with the inventive 
fermentation process, the ethanol concentration reaches a level of 7 to 
10% by a single stage fermentation, which may be increased further beyond 
10% by a suitable combination of a multiple number of fermentors. 
Subsequently, ethanol is easily recovered from the supernatant of the 
fermentation product mixture by a suitable separation method, e.g., a 
distillation or adsorption process. 
The productivity of ethanol in accordance with the inventive fermentation 
process can reach the level of 70 g/hr/liter of the fermentor volume or 
more while retaining the yield close to the theoretical value, i.e., about 
94%, which is much higher than the productivity achievable by the prior 
art methods. The cell concentration in the fermentor can be maintained at 
a level as high as about 110 to 130 g/l; the continuous operation can be 
sustained for a long period time, e.g., 2 months or longer; and the 
process is substantially free from the risk of contamination by foreign 
microorganisms. 
Examples 
The following Examples are intended to illustrate the present invention 
more specifically, without limiting the scope of the invention. 
Reference Example 1: Dependency of the cell flocculation property on the 
type of nitrogen source 
A lique-saccharified substrate solution containing 150 g/l of glucose and 
1.5% (v/v) of solid particles having a size distribution ranging from 10 
to 1,000 pm was mixed with distilled water in a ratio of 1:3. 100 ml of 
the resultant solution was charged into a 300 ml flask and a nitrogen 
source to be tested was added thereto in an amount corresponding to a 
concentration of 3 g/l. Thereafter, to the flask were added 3 g/l of 
Saccharomyces uvarum (ATCC 28097) cell and 1 g/l of KH.sub.2 PO.sub.4. The 
cells were cultivated at 30.degree. C. on a shaking incubator at 200 rpm. 
After 14 hours, the flocculation activity of the cell was determined by 
measuring the light transmittance at 7 cm height of a test sedimentor 
using a cadmium sulfide (CdS) photoconductivity cell. As shown in FIG. 1, 
the best result was obtained with ammonium acetate, i.e., the 
sedimentation was completed within 15 seconds. 
Reference Example 2: Effect of pH on flocculation 
A lique-saccharified substrate solution containing 150 g/l of glucose and 
1.5% (v/v) of solid particles having a size distribution ranging from 10 
to 1,000 .mu.m was mixed with distilled water in a ratio of 1:3 at 
30.degree. C. 100 ml of the resultant solution was charged into a 300 ml 
flask, and added thereto were 3 g/l of ammonium sulfate as a nitrogen 
source, 3 g/l of Saccharomyces uvarum (ATCC 28097) cell, and 1 g/l of 
KH.sub.2 PO.sub.4. After 16 hours, pH of the solution was changed from 
2.36 to 4.49 by adding an aqueous KOH solution, and then the flocculation 
activity was observed using a cadmium sulfide(CdS) photoconductivity cell. 
The results illustrated in FIG. 2 show that the pH upshift greatly 
improves the flocculation activity of the cells. 
Reference Example 3: SBR (Sequencing Batch Reactor) operation 
A lique-saccharified substrate solution containing 150 g/l of glucose and 
1.5% (v/v) of solid particles was mixed with 12 g/l of ammonium acetate 
and 3 g/l of KH.sub.2 PO.sub.4. The resulting mixture was added to a 2 L 
fermentor (BioFlo Ilc, NBS, USA) whose basic features are schematically 
shown in FIG. 5. Batch and SBR operations were conducted at 30.degree. C., 
while rotating the agitator at 200 rpm, aerating at 0.5 vvm, and 
maintaining pH at 4.5. A pH upshift was observed upon the depletion of the 
glucose in the reaction mixture during the batch culture. Based on this 
characteristic pattern of pH change, the timing for the drawing-filling 
procedure for an SBR operation was determined. The results of carrying out 
four drawing-filling cycles are shown in FIG. 3, wherein the exchange 
volume was 1 l and the flocculent cell used was Saccharomyces uvarum (ATCC 
28097). 
Example 1: Liquefaction-saccharification 
Tapioca was ball milled to obtain a powder having 10 to 1,000 .mu.m 
particle size. In 300 L tank, 25 kg of the tapioca powder was mixed with 
100 kg of hot water, and the resulting mixture was transferred to a 200 L 
tank and liquefied at 90.degree. C. for 2 hours after adding 15 ml of 
.alpha.-amylase (Termamyl 120L, Novo) to the mixture. The saccharification 
step was then conducted at 60.degree. C. for 10 to 12 hours by adding 37.5 
ml of glucoamylase (SPEZYME GA 300N, Genencor), 37.5 ml of cellulase 
(Celluclast 1.5L, Novo) and 7.5 ml of cellobiase (NOVOZYME 188, Novo) to 
the liquefied tapioca solution. The solid particles in the 
lique-saccharified tapioca solution amounted to 45% (v/v) and had a 
particle size in the range of 10 to 600 .mu.m, as measured using a 
particle size analyzer (a product of Malvern Instruments, United Kingdom). 
The glucose concentration in the lique-saccharified solution was 150 g/l. 
The lique-saccharified solution was then centrifuged using a low-speed 
laundry type decanter and the solid particles remaining in the filtrate 
had an average particle size of 20 .mu.m as shown in FIG. 4. 
This filtrate having a solid particle content of 10% (v/v) and a glucose 
concentration of 150 g/l was employed as a feed solution to the subsequent 
fermentation process. 
Example 2: Fermentation 
Referring to FIG. 5, 500 ml of the feed solution obtained in Example 1 was 
introduced to a 3 L fermentor (10) (BioFlo Ilc, NBS, USA) equipped with a 
cylindric screen filter (20) having an opening size of 325 mesh which was 
allowed to move with an agitator (30). The working volume of the fermentor 
was 2 L. To the fermentor was added 4% (w/v) of urea as nitrogen source 
and 1% (w/v) of KH.sub.2 PO.sub.4. 
The mixture in the fermentor was then inoculated with 0.4 g of 
Saccharomyces uvarum (ATCC 28097) and cultured in a batch mode at 
30.degree. C. for 16 hours, followed by conducting 4 SBR cycles to 
increase the cell concentration to 18 g/l, when cell pellets started to 
form. During the SBR operation, recycling of the cells from inside to 
outside of the moving filter was conducted. Subsequently, the operation 
was switched to a continuous mode by continuously introducing the feed 
solution obtained in Example 1 to outside of the filter in the fermentor 
under a condition of 30.degree. C., agitation rate of 200 rpm, aeration 
rate of 0.5 vvm, and pH 4.5, and the dilution rate of the substrate was 
gradually increased, as shown in FIG. 6. During the course of this 
experiment, it was confirmed that there was no ethanol or glucose 
concentration gradient across the filter. 
The results of this continuous fermentation operation are shown in FIG. 6. 
Example 3: Long-term continuous operation at high dilution rates 
The procedure of Example 2 was repeated except that a 6:1 mixture of urea 
and ammonium acetate, instead of urea, was employed as the nitrogen source 
to conduct a long-term continuous operation at a dilution rate of 0.5 
hr.sup.-1. The results (FIG. 7) show that a steady cell density in the 
range of 120 to 140 g/l as well as a steady ethanol concentration in the 
range of 6.0 to 6.5% (w/v), could be maintained throughout the length of 
the operation, while the residual glucose concentration was practically 
nill. 
Example 4: Productivity 
In order to investigate the productivity at a high dilution rate of the 
inventive process, the procedure of Example 2 was repeated except that the 
dilution rate was varied from 0.5 to 1.2 hr.sup.-1, and the results in 
FIG. 8 show that the productivity increases with the dilution rate of the 
substrate to reach 70 g of ethanol/l.hr at a dilution rate of 1.18 
hr.sup.-1, while a steady cell concentration of 110 to 130 g/l was 
maintained throughout the experiment. 
The results of foregoing Examples 1 to 4 demonstrate that the inventive 
process has all the desirable features of high productivity, excellent 
fermentation product quality and satisfactory long-term stability, and 
that these results are clearly superior to those achievable by the prior 
art methods. 
While the invention has been described in connection with the above 
specific embodiments, it should be recognized that various modifications 
and changes may be made by those skilled in the art to the invention which 
also fall within the scope of the invention as defined by the appended 
claims.