WO2007032265A1

WO2007032265A1 - Continuous culture apparatus for alcohol producing bacterium and method of culturing the bacterium

Info

Publication number: WO2007032265A1
Application number: PCT/JP2006/317831
Authority: WO
Inventors: Keishi Shimazaki; Akihiko Kouno; Ayaaki Ishizaki
Original assignee: New Century Fermentation Research Ltd.
Priority date: 2005-09-15
Filing date: 2006-09-08
Publication date: 2007-03-22
Also published as: JPWO2007032265A1; CN101268179A

Abstract

A continuous culture apparatus wherein in an ethanol continuous fermentation using an anaerobic bacterium, even with a substrate containing impurities in high proportion, the substrate concentration of fermented liquid is held constant at a low level, ensuring reduced substrate loss. There is provided a continuous culture apparatus comprising fermenter (1) for culturing of an alcohol producing bacterium; supply pump (12) for feeding a substrate liquid to the fermenter; flow meter (4) for detecting of the value of flow rate of carbon dioxide gas emitted from the fermenter; and control unit (5) capable of controlling the supply pump on the basis of output of the flow meter, wherein when the value of flow rate of carbon dioxide gas is below a given range, the control unit causes the supply means to supply the substrate liquid to the fermenter with the employment of the flow rate value as a trigger. The rate per time of supply of the substrate liquid to the fermenter and time of supply of the substrate liquid by the supply means are constant.

Description

Specification

Apparatus and method for continuous culture of alcohol-producing bacteria

Technical field

[0001] The present invention relates to a continuous culture apparatus for alcohol-producing bacteria (including anaerobic bacteria such as Zymomonas mobillis and Escherichia coli imparted with alcohol fermentation ability, excluding yeast) and a method for the same. More specifically, the present invention predicts the consumption of the substrate in the fermentation using the change in the flow rate of carbon dioxide gas as exhaust as an index when the substrate supply solution is supplied to the fermentor continuously or intermittently, and the fermenter The present invention relates to a technology for controlling the supply of the substrate solution and the bowing of the fermentation solution so that the substrate concentration becomes a constant value at a low concentration.

Background art

[0002] In the fermentation industry, for the fermentation of various amino acids, organic acids, ethanol, acetone'butanol, nucleic acid-related substances, etc. by microorganisms and the production of microbial cells themselves (for example, production of yeast cells), Fermentation by microorganisms is used. In these industrial productions, almost all of them are carried out by batch fermentation using a substrate such as sugar as the main raw material. Therefore, preparations other than fermentation itself, preparation of equipment, seed fermentation, and cleanup after the fermentation is completed • The washing must be repeated and the net operating time of the fermenter is very short and the productivity is low .

[0003] In order to solve this problem, a cascade method in which several fermenters are arranged in series to perform fermentation production is used for ethanol fermentation using yeast, but this is not a radical solution. Batch fermentation also requires frequent manual sugar concentration analysis to monitor the progress of fermentation, and securing human resources for this is a burden for factory operation management.

[0004] By the way, in all fermentation methods, it is necessary to prevent inhibition by high-concentration substrates, effectively use the substrate, keep the residual sugar concentration of the fermentation broth as low as possible, and reduce substrate loss. is required. In addition, in order to facilitate the separation of hydrolyzed products that have been fermented and minimize the cost burden of the waste liquid treatment process, the outflow of uneaten substrate of bacteria into the product separation process is minimized. There is a need to.

[0005] The residue concentration is controlled to be low and constant, and the substrate is supplied and the fermentation broth is extracted. If it is possible to perform a continuous fermentation, the productivity of the fermentation process will be dramatically improved. In addition, such a process can eliminate almost no analysis work for sugar concentration management, thereby eliminating unnecessary labor, greatly reducing labor costs, and eliminating night work.

[0006] In the patent document 1 (Japanese Patent Laid-Open No. 2003-274934), the present inventors proposed a method for continuous ethanol fermentation using anaerobic bacteria such as Zymomonas mobillis. This method makes it possible to perform continuous fermentation using the principle that the amount of change in pH of the fermentation broth associated with bacterial metabolism corresponds to the amount of substrate consumed and the amount of ethanol produced.

[0007] That is, in order to correct changes in the pH of the fermentation broth, a certain amount of substrate corresponding to the integrated value of the added amount of alkali (ammonia, caustic soda, etc.) is supplied, and the corresponding fermentation broth is withdrawn. Continuous fermentation is performed while keeping the amount of fermentation liquid in the tank constant. However, if this method is applied to inexpensive industrial raw materials containing many unknown impurities such as molasses, the amount of change in pH does not necessarily correspond to substrate consumption, making continuous fermentation difficult by this method. There was a case that it might become.

Patent Document 1: Japanese Patent Laid-Open No. 2003-274934

Disclosure of the invention

Problems to be solved by the invention

[0008] Therefore, the present invention provides accurate sugar consumption and residual amount even in low-cost but high-impurity substances such as molasses food waste and food factory effluent in continuous ethanol fermentation using anaerobic bacteria such as Zymomonas mobillis. An object of the present invention is to provide a continuous culture apparatus for alcohol-producing bacteria and related technology, which can predict the sugar concentration, keep the substrate concentration of the fermentation broth constant at a low level, and reduce the substrate loss.

Means for solving the problem

[0009] As a result of diligent research aimed at solving the above problems, the present inventors have determined the amount of carbon dioxide flowing out of the fermenter as exhaust in continuous ethanol fermentation by anaerobic bacteria using industrial raw materials such as molasses. We focused on the fact that the substrate consumption in the fermenter can be accurately grasped if the changes are accurately captured. Furthermore, the decrease in carbon dioxide gas generation that occurs when the substrate in the fermentation broth is detected as a decrease in the flow rate of carbon dioxide gas. Thus, we have completed a continuous culture method that enables stable continuous fermentation over a long period of time while maintaining the substrate concentration in the fermenter at a low and constant level by controlling the substrate supply and withdrawal of the fermentation broth.

[0010] In the continuous culture method for alcohol-producing bacteria according to the first invention, a substrate solution is supplied to a fermenter for culturing alcohol-producing bacteria, and an amount of the fermentation solution is fermented according to the amount of substrate solution supplied. In this method, the substrate liquid is drawn out from the fermenter when the flow rate of carbon dioxide discharged from the fermenter falls below a predetermined range.

[0011] According to this configuration, since the substrate solution is supplied and the fermentation solution is extracted while the substrate in the fermenter is almost exhausted, substrate loss due to uneaten bacteria hardly occurs. In addition, as will be apparent from the examples described later, even when inexpensive industrial raw materials containing many unknown impurities such as molasses are used as substrates, the substrate concentration in the fermenter is kept at a low level. It can be continuously fermented stably for a long time.

[0012] Furthermore, it is desirable that the time for supplying the substrate liquid to the fermenter and the rate per time for supplying the substrate liquid to the fermenter are constant.

[0013] That is, the supply amount of the substrate solution at one time is calculated based on the substrate consumption rate several hours before the start of supply, and the added substrate is consumed by the microorganisms based on the calculated substrate consumption rate. It is desirable to give the supply rate and supply time to do the rest.

[0014] The predetermined range is preferably determined by a threshold value of the carbon dioxide flow rate value.

The invention's effect

[0015] According to the present invention, the substrate loss due to leftovers of bacteria is almost eliminated, and the substrate concentration in the fermenter can be reduced even when inexpensive industrial raw materials containing many unknown impurities such as molasses are used as the substrate. While maintaining a low and constant level, continuous fermentation can be performed stably for a long time. Brief Description of Drawings

FIG. 1 is a block diagram of a continuous culture apparatus according to an embodiment of the present invention.

FIG. 2 is a flowchart showing supply control processing in an embodiment of the present invention.

FIG. 3 is a graph showing changes in bacterial concentration, residual sugar, carbon dioxide flow rate, and substrate supply state in one embodiment of the present invention.

FIG. 4 is a graph showing changes in turbidity, ethanol accumulation, carbon dioxide flow rate, and residual sugar in an embodiment of the present invention. FIG. 5 is a graph showing the culture results of Comparative Example 1

FIG. 6 is a graph showing the culture results according to Example 5 of the present invention.

FIG. 7 is a graph showing the culture results according to Example 6 of the present invention.

Explanation of symbols

[0017] 1 Fermenter

2 Stirrer

3 Bubble trap

4 Flow meter

5 Control unit

6 Interface

7 CPU

8 ROM

9 Turbidimeter

10 pH meter

11 Substrate liquid tank

12 Substrate liquid supply pump

13 Neutralizer tank

14 Neutralizer supply pump

15 Circulation pump

16 Cross flow filter

17 Switching valve

18 Drawing pump

19 Drawer tank

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described with reference to the drawings. First, prior to the description of the specific configuration, the main points of this embodiment are summarized as follows.

[0019] This embodiment relates to a continuous ethanol fermentation method for anaerobic bacteria such as Zymomo nas mobillis that can use any assimilated fermentation resource as a substrate solution. The start of continuous fermentation is By adding the substrate solution for the first time, and adding the substrate solution for a certain period of time at the feed rate determined in the early stage of fermentation. The feed rate obtained in advance is determined from the relationship between the strain, medium composition, and fermentation conditions, and by measuring the bacterial concentration and sugar consumption rate through a fermentation test using batch fermentation. Subsequent substrate liquids are supplied by detecting the decrease in carbon dioxide generation that occurs when the substrate in the fermenter is depleted with a mass flow sensor, etc., so that the bacteria do not enter the state of starvation. The substrate concentration is always kept within a certain range of low levels.

Based on the above, the continuous culture apparatus in this embodiment will be described below with reference to FIG. Fermenter 1 cultivates anaerobic bacteria such as Zymomonas mobillis inside. The fermenter 1 of this embodiment is a small tank with a volume of 3 liters and is not a pressure vessel, so it is difficult to control the internal pressure. However, it is also possible to use a fermenter that is a pressure vessel, and in that case, it is also preferable to control the internal pressure. The temperature in the fermenter 1 is controlled to be a predetermined value, and the contents in the fermenter 1 are slowly stirred at a constant speed by the stirrer 2 provided in the fermenter 1.

[0021] The discharge pipe from the fermenter 1 is connected to a flow meter 4 via a foam trap 3 that removes foam. The flow meter 4 measures the flow rate value of the carbon dioxide gas discharged from the fermenter 1, and is composed of, for example, a mass port sensor, an exhaust gas flow meter, a pressure gauge, and the like. The flow rate value of carbon dioxide gas measured by the flow meter 4 is output to the interface 6 of the control unit 5. Of course, other physical quantities that uniquely correspond to the flow rate value of carbon dioxide gas may be measured.

[0022] The control unit 5 controls the entire continuous culture apparatus shown in FIG. The interface 6 of the control unit 5 includes a turbidimeter 9 (preferably a laser turbidimeter) that measures the turbidity inside the fermenter 1 in addition to the flow rate of carbon dioxide flow rate of 4 flowmeters. The turbidity value and the pH value from pH meter 10 that measures the pH value inside fermenter 1 are input. The CPU 7 is the core of the control unit 5 and executes the control program stored in the ROM 8. This control program includes a supply control program according to the flow chart of FIG. In addition, the CPU 7 outputs control signals to the substrate solution supply pump 12, neutralizing agent supply pump 14, circulation pump 15, switching valve 17, and drawing pump 18 via the interface 6 as appropriate, thereby controlling the entire continuous culture apparatus. To do.

[0023] The substrate solution tank 11 stores a substrate solution as shown in Examples described later. Substrate solution supply pump 1 When a substrate supply signal is input to the interface 2 from the interface 6, the substrate solution is supplied from the substrate solution tank 11 into the fermenter 1 at a predetermined rate for a predetermined time. As a result, the amount of residual sugar in fermenter 1 increases.

[0024] The neutralizer tank 13 stores a neutralizer such as ammonia, for example. When fermentation proceeds in fermenter 1, the pH in fermenter 1 decreases as bacteria grow. In this embodiment, the CPU 7 outputs a neutralizing agent supply signal to the neutralizing agent supply pump 14 so that the pH value is always a constant value of about 4.5 to 6 (depending on conditions), and fermenter 1 Control the pH value inside.

[0025] The fermenter 1 is connected with a circulation pump 15 that draws out the fermented liquid in the fermenter 1 and pumps it to the crossflow filter 16 in accordance with a circulation instruction signal from the interface 6. The cross flow filter 16 returns a part of the extracted fermentation broth into the fermenter 1 and connects the filtrate to one input port of the switching valve 17. The fermented liquid in the fermenter 1 communicates with the other input port of the switching valve 17.

The switching valve 17 connects one of the other Z input ports to the output port in accordance with a switching signal from the interface 6. The extraction pump 18 uses the bow I extraction instruction signal from the interface 6 !, but pumps out the filtrate Z fermentation broth from the output port of the switching valve 17 to the extraction liquid tank 19. As a result, the fermented liquid containing ethanol is taken out into the drawing liquid tank 19. Instead of the cross flow filter 16, a centrifuge may be used.

Next, each process of substrate liquid supply control by the control unit 5 will be described with reference to FIG. In this process, when the flow rate of carbon dioxide gas discharged from the fermenter 1 falls below a predetermined range, the control unit 5 outputs a substrate supply signal to the substrate liquid supply pump 12 (supply means) as a trigger. It is important to supply the substrate solution to the fermenter 1.

[0028] The circulation pump 15, the cross flow filter 16, the switching valve 17 and the extraction pump 18 correspond to extraction means for extracting the fermentation liquid from the fermentation tank 1, and the control unit 5 includes the substrate liquid supply pump 12 This drawing means is controlled so that this drawing means draws out from the fermenter 1 an amount of fermentation liquid that matches the supply amount of the substrate liquid supplied to the fermenter 1. The CPU 7 refers to the turbidity value from the turbidimeter 9 and pulls out the filtrate from the cross flow filter 16 if the fermenter concentration in the fermenter 1 is low (the switching valve 17 is in the state shown in FIG. ), If fermenter concentration is high, from fermenter 1 (switch The valve 17 is in a state opposite to that in Fig. 1) The fermentation broth is drawn out, and the concentration of the fermentation broth in the fermenter 1 is kept constant.

[0029] A description will be given with reference to FIGS. First, as shown in Step 1 of FIG. 2, the inoculum and substrate solution are adjusted to the fermenter 1, and in Step 2, the waiting time TO, the carbon dioxide flow rate value threshold Th, the supply time T1, and the supply Rate R1 is set. These values are set in advance according to the conditions, and are corrected as necessary. Fermentation starts at time t = tO in FIG.

[0030] The feed rate R1 is set to a value given by the relational expression between the bacterial cell concentration and the sugar consumption rate obtained in advance so that the substrate concentration of the fermentation broth can be maintained at a low level immediately before the withering, Store it in ROM8.

[0031] At the beginning of fermentation, the concentration of bacteria is low in the fermenter 1 (Fig. 3 (a)), and the substrate is abundant (Fig. 3 (b)). After that, as a characteristic of alcohol-producing bacteria, growth stops even if the substrate remains. Therefore, in order to increase the bacterial concentration!], The culture solution is circulated, the filtrate is drawn out, concentrated culture is performed while supplementing the corresponding medium, and the bacterial concentration is increased. When the bacterial concentration reaches the set value, turbidity control is started and continuous fermentation is started.

[0032] As the growth of bacteria increases, the concentration of bacteria increases and the amount of residual sugar in fermenter 1 decreases. As the fermentation progresses, the flow rate of carbon dioxide increases (Fig. 3 (c)). At this time, CPU7 checks whether the waiting time TO has elapsed (step 3) and does not supply the substrate until it has elapsed.

[0033] As shown in FIG. 3 (c), after the initial substrate solution addition at time tO, after the waiting time TO has elapsed, the CPU 7 determines in step 4 that the flow rate value of the carbon dioxide gas has fallen below the threshold value Th. Check for it. For example, at time t = tO + TO, the flow rate value of the carbon dioxide gas exceeds the threshold value Th. At this time, the CPU 7 confirms that the processing should not be terminated in step 8. Return to step 4 and perform the above check again.

[0034] At time t = tl, when the substrate in the fermenter 1 is depleted, the amount of carbon dioxide generated suddenly decreases and falls below the threshold Th. With this as a trigger, the process moves to step 5 for step 4 force. That is, the CPU 7 outputs a substrate supply signal to the substrate solution supply pump 12 via the interface 6, and the substrate solution supply pump 12 sets the substrate solution from the substrate solution tank 11 to the supply rate R1. Then, supply to fermenter 1 starts (step 5), and this state continues for a predetermined time T1 (step 6). As a result, as shown in FIG. 3 (b), the amount of residual sugar in the fermenter 1 that has been almost zero turns to increased calories. In other words, if the substrate supply is resumed at time t = tl, the operation can be continued at a residue concentration close to the limit substrate concentration and low.

[0035] Now, as is well known, the relational expression between carbon dioxide generation amount and fermentation in ethanol fermentation using glucose as a substrate is given by the following theoretical formula.

[0036] C H O → 2C H OH + 2CO (1)

6 12 6 2 5 2

That is, when 2 moles of ethanol is produced per 1 mole of glucose, 2 moles of carbon dioxide gas is generated. Therefore, if the sugar is depleted and ethanol production is stopped, carbon dioxide gas is not generated. Therefore, by using the decrease in carbon dioxide emission as an index, it is possible to detect substrate depletion with high reliability.

[0037] In the fermentation system of this continuous fermentation, the concentration of bacteria is controlled by the turbidimeter 9 at a constant level. Actually, the substrate consumption becomes faster or slower than the predicted value input to the control unit. Sometimes it is preferable to finely adjust the timing of supply.

[0038] Then, when the predetermined time T1 elapses for the time tl force, the CPU 7 outputs a signal to stop the substrate supply to the substrate solution supply pump 12 (step 7), and the substrate solution supply pump 12 supplies the substrate. To stop. Therefore, as shown in FIG. 3 (b), the amount of residual sugar starts to decrease again, and at time t2, the carbon dioxide flow rate again falls below the threshold value Th.

[0039] Thereafter, similar processing is repeated at times t2, t3, t4 '(steps 3 to 8).

[0040] Substantially, in the second and subsequent times (after time tl), the flow meter 4 detects a sudden decrease in carbon dioxide generation that occurs when the substrate in the fermenter 1 depletes, It starts when Th is reached.

[0041] In this way, it is possible to continuously add a substrate solution without depleting a substrate such as sugar while sequentially checking the carbon dioxide gas generated by microorganisms, thereby easily increasing the substrate concentration in the fermenter. It becomes possible to keep it at a low level of 5gZ or less. Even if it is difficult to continuously ferment by relying on the amount of change in pH based on inexpensive industrial raw materials that contain a lot of unknown impurities such as molasses, this point is appropriate, so it is wider than the conventional technology. Fermentation can be carried out at a low cost. Example

[0042] Hereinafter, the present invention will be described in more detail with reference to Examples. Of course, the present invention is not limited by the following examples.

[0043] (Example 1)

The inoculum was prepared by using YM broth (Difco Laboratories, Detroit) as a medium, adjusting to the specified concentration, dispensing 10 milliliters into a test tube, and heat-sterilizing at 115 ° C for 10 minutes.

. This was inoculated with Zymomonas mobillis NRRL B-14023, and used after standing still at 30 ° C for 18 hours.

[0044] The main fermentation used was Brazilian molasses diluted 5.3 times with tap water and autoclaved at 120 ° C for 10 minutes. Place 2 liters of this medium in a small (3 liter) fermenter that has been sterilized in advance, add 100 milliliters of the seed fermentation solution prepared as described above, and start fermentation at a temperature of 30 ° C and stirring at lOOrpm. did. Since the pH of the fermentation broth decreased with the growth of the bacteria, pH was kept at 5.5 with 1N ammonia water while monitoring the pH online with a pH meter.

[0045] Since the growth of the bacteria reached its peak 15 hours after the start of the fermentation, the concentration of the bacteria was increased by circulating concentration of the fermentation broth. Circulate the fermentation broth through a cross-flow filter (Nippon Pole, 0.45m, 0.1 lm ² cartridge) installed outside the fermenter, extract the filtrate out of the fermenter, and use the same medium as at the start of fermentation. Replenishment to increase the bacterial concentration while maintaining a constant fermentor volume

[0046] In this way, the bacterial concentration reached about 4 gZ Ritter in about 24 hours from the start of fermentation. Thereafter, the bacterial concentration could be controlled to 4.0 ± 0.2 gZ litter using a laser turbidimeter (ASR Model LA-301 type) for a total time of 255 hours as shown in FIG. When the bacterial concentration became constant, the residual substrate concentration in the fermentation broth was measured with a glucose Z sucrose analyzer, and the total glucose Z sucrose was 10 g / liter. Was diluted 5.7 times and autoclaved at 120 ° C for 10 minutes, and continuously supplied for 4 hours at a supply rate of 950 gZh as a substrate solution.

[0047] When the supply was stopped after 4 hours of supply, the bacteria remained in the fermentation broth! The exhausted carbon dioxide gas suddenly declined about 15 minutes after the supply was stopped. When the amount of exhaust carbon dioxide reached the set value of 0.08 liter Zmin, it was determined that the substrate was depleted and the supply was resumed. Actually, the residual sugar concentration when the carbon dioxide generation reaches the set lower limit is almost OgZ liter according to the analyzer. This operation is performed automatically by the control program.

[0048] After that, supply for 4 hours at a supply rate of 1, OOOgZh, stop the temporary supply, confirm a sudden decrease in the exhaust carbon dioxide gas, and resume the supply when the exhaust carbon dioxide flow rate reaches the threshold. The program was activated and continuous operation was performed. The concentration of residual sugar in the fermentation broth during the supply can be estimated from the time until the sudden decrease in carbon dioxide generation occurs after the supply is stopped. If it is determined that the residual sugar concentration is high from this length of time, the feed rate is slightly reduced.

[0049] However, the set value of the substrate supply here may differ depending on the fermentation conditions such as the fermentation method and the type of substrate, the performance of the strain used, and the like. In this example, the addition time of the substrate solution was set to 4 hours each time. However, as long as the activity of the bacteria is not significantly changed as described above, it is not necessary to make the addition time constant. It is desirable to take longer gradually to reduce time loss.

[0050] In Fig. 4, the progress of the fermentation is shown in the state of turbidity control (upper), the generation of exhaust carbon dioxide by the mass flow sensor (lower), and the ethanol concentration in the fermenter and the residual sugar concentration in the fermenter are shown. Also shown. Stable continuous operation continued for 255 hours by the above-mentioned supply program. During this time, the concentration of residual sugar in the fermentation broth during the supply was maintained at about 10.0 ± 5 gZ litter as the total amount of glucose Z sucrose. The average ethanol concentration of the fermentation broth was about 68 gZ liters. In a continuous continuous operation for 230 hours, 220 liters of fermentation broth containing 68 gZ of ethanol was obtained.

[0051] (Example 2)

The fungus used for the seed (seed), the medium, the fermentation method, the fermentation conditions, and the apparatus used for the main fermentation are the same as in Example 1.

[0052] The main fermentation medium and substrate solution used were corn starch sugar broth added with CSL (corn steep liquor). [0053] After suspending cornstarch 200gZ litter in water and making a slurry, adjust ρΗ to 6.0 with stirring, and add Termamyl 120L (Novo) 0.5 litter Zg starch to 90 ° C on the water bath. , lh liquefaction treatment. After cooling, the pH was adjusted to 4.5, and Dextrozyme (Novo) 0.6 / z litter Zg starch was subjected to saccharification treatment at 60 ° C for 24 hours in a thermostatic bath. As a result, a sugar solution having a glucose concentration of 178 gZ Ritter was obtained.

[0054] This was diluted with water to a glucose concentration of 130 gZ litter, CSL (Seiei saccharification KK) 5 gZ litter from which solid content had been removed by centrifugation was added in advance, and after autoclaving after pH adjustment Was used as a medium / substrate solution.

[0055] The fermentation procedure and control program are exactly the same as in Example 1.

[0056] The supply rate is 950 gZh, the duration of one supply is the same 4 hours as in Example 1, and the threshold value of exhaust carbon dioxide is 0.08 liters / min as in Example 1.

[0057] Fermentation was stably continued for a long time, and a 180-liter fermented liquid containing 58 g of Z-liter ethanol was obtained by continuous operation for 200 hours.

[Example 3]

[0059] The main fermentation medium and the substrate solution were prepared by adding molasses to cassava starch sugar solution.

[0060] Cassava starch (Thailand) 200gZ Ritter suspended in water to make slurry, adjust ρΗ to 6.0 with stirring, Termamyl 120L (Novo) 0.5 Ritter Zg starch and water bath Perform 90 ° C, lh liquefaction treatment above. After cooling this, the pH was adjusted to 4.5, and Dextrozyme (Novo) 0.6 Litter Zg starch was added, and the mixture was treated with sugar at 60 ° C for 24 hours. As a result, a sugar solution having a glucose concentration of 175 gZ Ritter was obtained. This was diluted with water to a dulcose concentration of 130 g / liter, and used in Example 1. 5. 7-fold diluted solution was mixed 1: 1, and after autoclaving after pH adjustment. Medium · Used as a substrate solution.

[0061] The fermentation procedure and control program are exactly the same as in Example 1.

[0062] The supply rate is 1,000 g / h, the duration of one supply is the same as in Example 1, 4 hours, and the exhaust carbon dioxide The threshold value of the sample is 0.08 litter Zmin as in the first embodiment.

[0063] Fermentation was continued stably for a long time, and 175 L of fermentation broth containing 58 g of Z L of ethanol was obtained by continuous operation for 200 hours.

[0064] (Example 4)

[0065] The main fermentation medium and the substrate liquid are model garbage saccharified liquefied liquids that are assumed to be fermented from raw garbage.

[0066] Rice (white rice) 250g, cabbage 59g, boiled carrot 50g, banana (edible part) 50g, sashimi 50g, squeezed into a juicer mixer and crushed juice into a total amount of 1 liter Dilute with water. While stirring, the pH was adjusted to 4.5 mm, and Dextrozyme (Novo) 0.6 liter Zg starch was subjected to sugar treatment at 60 ° C. for 24 hours in a thermostatic bath. A sugar solution having a glucose concentration of 85 g / liter after sugar was obtained. This was autoclaved as it was and used as a medium'substrate solution.

[0067] The fermentation procedure and control program are exactly the same as in Example 1.

[0068] The supply rate is 1,200 gZh, the duration of one supply is 4 hours, the same as in Example 1, and the exhaust carbon dioxide threshold is 0.08 litter Zmin, the same as in Example 1.

[0069] Fermentation was continued stably for a long time, and 150 litter of fermentation broth containing 41 gZ litter of ethanol was obtained by continuous operation for 150h.

[0070] According to the above embodiment, even when an inexpensive industrial raw material containing a large amount of unknown impurities such as molasses is used as a substrate, the substrate concentration in the fermenter is kept at a low and constant level, and it is stably continuously for a long time. It is clear that fermentation is possible, and the present invention has a remarkable IJ point compared to the prior art.

[0071] Hereinafter, Comparative Example 1 and Examples 5 and 6 relating to β-ratata antibiotics will be described. Items common to Examples 5 and 6 are as follows.

[0072] In ethanol production by continuous ethanol fermentation using Zymomonas mobilis, the medium initially charged in the fermenter is sterilized by heating and steaming and then cooled to obtain the initial medium. In addition, penicillin G potassium dissolved in sterilized cold water is added to the initial medium in a final concentration of 5 — Add to 20 IUZ milliliters, add seeds and start fermentation.

[0073] As in Example 1, the bacterial concentration is increased by batch culture, and the bacterial concentration is performed to increase the cell concentration to the target value. When the cell concentration reaches the target, dissolve penicillin G potassium in the same manner as the initial medium, and continuously add the substrate solution added to a final concentration of 5-20 IUZ milliliters.

[0074] Also, for cross flow filter 16 used for concentration of bacteria, penicillin G potassium was added to a final concentration of 5-20 IUZ milliliter and incubated at 30 ° C for 2 hours. Cross flow filter 16 The contamination bacteria remaining in the membrane and piping details are killed.

[0075] These points are the differences between Examples 1 to 4 and Examples 5 and 6. As described below in more detail than in Examples 5 and 6, these treatments allow ethanol continuous fermentation by Zymomonas mobilis to undergo complete contamination and growth of contaminant bacteria over a long period of 350 hours. Without this, ethanol could always be produced by fermentation at the maximum speed. Of course, this method is effective not only in Zymomonas mo bilis but also in fermentation using genetically modified E. coli to which alcoholic fermentation ability is imparted.

[0076] Here, the same effect can be obtained by using ampicillin, which is not limited to penicillin, and all β-ratata antibiotics effective against Durham positive bacteria.

[0077] Next, prior to Examples 5 and 6, Comparative Example 1 will be described.

[0078] (Comparative Example 1)

The inoculum was prepared using ΥΜ broth (Difco Laboratories, Detroit) as the medium, adjusted to the specified concentration, dispensed 10 milliliters into a test tube, and heat-sterilized at 115 ° C for 10 minutes. This was inoculated with Zymomonas mobilis NRRL B-14023, and used after static fermentation at 30 ° C for 18 hours.

[0079] Main fermentation consists of crystalline glucose 140gZ Litter, yeast extract 5gZ Litter, taste liquid (soy flake acid hydrolyzate) 0.5vol% medium adjusted to pH 5.5, 120 ° C, autoclaved for 20 minutes. The 2 liters of this medium was placed in a pre-sterilized 3 liter single fermentor, and 100 milliliters of the seed fermentation solution prepared as described above was added, and fermentation was started at a temperature of 30 ° C. and stirring at lOOrpm. Fermentation solution pH decreases with bacterial growth Therefore, while monitoring the pH with a pH meter 10, the pH was maintained at 5.5 with IN ammonia water.

FIG. 5 is a graph showing the fermentation process in Comparative Example 1. After seeding, bacterial growth is started by batch culture, and when the cell concentration exceeds the lgZ litter, bacterial concentration by the crossflow filter is started, and the substrate is added with the substrate solution and the sterilization solution is cultured. The cell concentration was increased by extracting from the system.

[0081] Since the cell concentration reached the target value of about 5 gZ litter at about 40 hours from the start of the culture, continuous fermentation mode was entered using the system of FIG. As shown in Fig. 5, until 80 hours after the start of culture (40 hours after continuous fermentation), the bacterial concentration was maintained at approximately 6 gZ litter, and the ethanol concentration was gradually decreased but maintained above 60 gZ litter. Although the amount of carbon dioxide generated is also decreasing, it is about 250 milliliters Zmin (about 1 OmmolZmin), and the ethanol production rate is maintained at about 30 gZh.

[0082] However, after 40 hours from the start of continuous fermentation, the generation rate of carbon dioxide gas suddenly decreases, the ethanol concentration also decreases sharply, and the ammonia consumption rate rapidly increases. At 120 hours of culture, the fermentation was completely uncontrollable, so the culture was discontinued. This is probably because lactic acid bacteria were contaminated for 40 hours of culture (almost simultaneously with the start of continuous fermentation), which grew rapidly after 80 hours. Incidentally, when the culture solution after 100 hours of actual culture was cultured on an agar plate medium, a large number of colonies of lactic acid bacteria were confirmed.

[0083] (Example 5)

The inoculum was prepared by using YM broth (Difco Laboratories, Detroit) as a medium, adjusting to the specified concentration, dispensing 10 milliliters into a test tube, and heat-sterilizing at 115 ° C for 10 minutes. This was inoculated with Zymomonas mobilis NRRL B-14023 and fermented by standing at 30 ° C for 18 hours.

[0084] The main fermentation was crystal glucose 140gZ Litter, yeast extract 5gZ Litter, taste liquid (soy flake acid hydrolyzate) 0.5 vol% medium adjusted to pH 5.5 and 120 ° C Autoclaved for 20 minutes. Initial culture medium used for cultivation 'Substrate solution used for concentration of bacteria' Continuously fermented substrate solution was cooled to a final concentration of 5 IUZ A penicillin G-potassium product was used so as to be a riliter.

[0085] Place 2 liters of this medium into a 3 liter small fermentor that has been sterilized in advance, add 100 milliliters of the seed fermentation solution prepared as described above, and start fermentation at a temperature of 30 ° C and stirring at lOOrpm. did. Since the pH of the fermentation broth decreased with the growth of the bacteria, the pH was kept at 5.5 with 1N ammonia water while monitoring the pH with a pH meter 10.

[0086] The system is the same as that shown in Fig. 1 according to the first embodiment. As described in Comparative Example 1, since contamination was caused in the past culture, there is a possibility that contaminants remain inside the filter 16 or the like. For this reason, thoroughly remove the contaminating bacteria remaining before the culture. The filter cartridge used for the culture that has been contaminated at the end should be thoroughly disassembled and washed after the fermentation and immersed in an alkaline solution for several days. Sterilized. After that, an attempt was made to eradicate residual contaminants by washing with penicillin. That is, every corner of the system in FIG. 1 was washed with penicillin, and as described above, the filter cartridge washed with penicillin was attached to fermenter 1 and used for bacterial concentration and circulation.

FIG. 6 shows the culture results. After seeding, bacterial growth is started by batch culture, and when the cell concentration exceeds the lgZ litter, concentration of the cells with the crossflow filter 16 is started, and the substrate solution is added with penicillin G potassium at a final concentration of 5 IUZ milliliters. To add substrate. Further, the sterilization solution was extracted from the culture system, and the cell concentration was increased.

[0088] Since the cell concentration reached the target value of about 5 gZ litter at about 30 hours from the start of culture, the system of Fig. 1 was used to enter the continuous fermentation mode.

[0089] Substrate solution for continuous fermentation was prepared with triglyceride 140gZ Litter, yeast extract (YE) 5gZ Litter, taste solution 5milliliter Z Litter adjusted to PH5.5, cooled to 120 ° C for 20 minutes, cooled and then penicillin G Potassium was added to 5 IU / milliliter.

FIG. 6 shows the results of continuous fermentation according to Example 5. Over the long period of 150 hours, ethanol could be continuously produced with a healthy fermentation without being violated by contamination. During all continuous operations, the bacterial concentration is maintained at 5 ± 0.5 gZ liters, the amount of C02 generated is about 350 milliliters Zmin (about 15 mmolZmin), and the ethanol production rate is about 42 gZh, maintaining a high production rate. It was. The supply rate of the substrate solution and the extraction rate of the culture solution are stable as shown in Fig. 6. We were able to run continuously. The ethanol concentration of the culture was 65 ± 5 gZ liters (approximately 8.2%) over the entire period. In this way, the effects of washing the penicillin in the filter 16 and penicillin-added caroten to the medium are remarkable, and it has become possible to carry out continuous ethanol fermentation for a long time without being damaged by contamination.

[Example 6]

The bacteria, medium, fermentation method, and fermentation conditions used for the seeds are the same as in Example 5.

[0092] Initial medium 'Substrate solution when concentrating bacteria · Continuous fermentation substrate solution had the same composition and the same adjustment method.

In other words, the sugar solution is obtained by diluting corn starch enzyme sugar liquor with water and adding it to a CSL (corn steep liquor) concentration of 5 milliliters / liter to a pH of 5.5, followed by a sugar concentration of 140 gZ And then autoclaved at 120 ° C for 20 minutes. After sterilization and cooling, when the medium was sufficiently cooled, penicillin G potassium was added to 5 IUZ milliliters and used for culture. The procedure for washing the penicillin in the cross-flow filter 16 and removing the contaminants is the same as in Example 5.

FIG. 7 shows the culture results according to Example 6. The system is the same as in Example 1. After seeding, bacterial growth is started by batch culture, and when the cell concentration exceeds the lgZ litter, concentration of the bacteria with a crossflow filter is started, and a substrate solution with penicillin G potassium added to a final concentration of 5 IUZ milliliter is supplied. Add substrate. In addition, the sterilization solution was extracted from the culture system to increase the cell concentration.

[0094] Since the cell concentration reached the target value of about 6 gZ litter at about 27 hours from the start of culture, the continuous fermentation mode was entered using the system of Fig. 1. The substrate solution for continuous fermentation is the above-mentioned corn starch enzyme sugar solution (sugar concentration 140gZ litter) — CSL (corn 'steep' liquor) 5mm liter Z liter is adjusted to PH5.5, then 120 ° C for 20 minutes auto Cooled after crepe sterilization and added with pericillin G potassium to 5 IU / milliliter.

[0095] As shown in Fig. 7, ethanol could be continuously produced with a healthy fermentation throughout the entire 350 hours without being violated by contamination. During all continuous operations, the bacterial concentration is maintained at 6 ± 0.5 gZ liters, the amount of carbon dioxide generated is 500 milliliters Zmin (about 21 mMolZmin), the ethanol production rate is about 58 gZh, and a high production rate is achieved over the entire incubation time. I was able to maintain it. The supply rate of the substrate solution and the extraction rate of the culture solution were stable as shown in the lower part of FIG. 7, and the continuous fermentation during the culture maintained a steady state and could be operated continuously for a long time. The ethanol concentration of the culture was 65 ± 5 gZ liters (approximately 8.2%) over the entire period. Thus, the effects of washing the penicillin in the filter 16 and penicillin-supplemented on the medium were remarkable, and continuous ethanol fermentation could be carried out for a long time without being damaged by contamination.

Claims

The scope of the claims

[1] a fermentor for culturing alcohol-producing bacteria;

Supply means for supplying a substrate solution to the fermenter;

A measuring device that detects a flow rate value of carbon dioxide discharged from the fermenter or a function value thereof, and a control unit that controls the supply unit based on an output of the measuring device, the control unit comprising the fermenter A continuous culture apparatus for alcohol-producing bacteria, characterized in that when the flow rate value of carbon dioxide discharged from the water falls below a predetermined range, the feeding means is used as a trigger to feed the substrate solution to the fermenter.

[2] The time according to claim 1, wherein the time for supplying the substrate liquid to the fermenter by the supplying means and the rate per time for supplying the substrate liquid to the fermentor by the supplying means are constant. Continuous culture equipment for alcohol-producing bacteria.

[3] The continuous culture apparatus for alcohol-producing bacteria according to claim 1, wherein the predetermined range is determined by a threshold value of a carbon dioxide flow rate value.

[4] The fermenter is further provided with a drawing means for drawing out the fermentation broth,

The control unit controls the extraction unit such that the extraction unit extracts an amount of the fermentation liquid in accordance with the supply amount of the substrate liquid supplied to the fermenter by the supply unit from the fermentation tank. 2. A continuous culture apparatus for alcohol-producing bacteria according to item 1.

[5] A continuous culture method for alcohol-producing bacteria, in which a substrate liquid is supplied to a fermentor for culturing alcohol-producing bacteria, and an amount of the fermentation liquid in accordance with the amount of the substrate liquid supplied is withdrawn from the fermenter.

When the flow rate of carbon dioxide discharged from the fermenter falls below a predetermined range, a substrate liquid is supplied to the fermenter using the flow rate as a trigger.

[6] The continuous culture of alcohol-producing bacteria according to claim 5, wherein the time for supplying the substrate liquid to the fermenter and the rate per time for supplying the substrate liquid to the fermenter are constant. Nourishing method.

7. The method for continuously culturing alcohol-producing bacteria according to claim 5, wherein the predetermined range is determined by a threshold value of a carbon dioxide flow rate value.

[8] The continuous culture method for alcohol-producing bacteria according to claim 5, further comprising the step of adding a β-ratata antibiotic.

[9] The method for continuously culturing alcohol-producing bacteria according to claim 8, wherein the β-ratata antibiotic is penicillin.

10. The method for continuously culturing alcohol-producing bacteria according to claim 8, wherein the β-ratata antibiotic is added when all or part of the continuous culture apparatus including the fermenter is washed.

11. The method for continuously culturing alcohol-producing bacteria according to claim 8, wherein the β-ratata antibiotic is added during operation of a continuous culture apparatus including the fermenter.