Patent Application
20200017886
This disclosure relates to a method of producing an alcohol by continuous fermentation, and a continuous fermentation apparatus to be used therefor.
Fermentation methods, which are substance production methods involving cultivation of microorganisms or cultured cells, can be largely classified as (1) a batch fermentation method and a fed-batch fermentation method, and (2) a continuous fermentation method.
The batch fermentation and the fed-batch fermentation method have the advantages of simple equipment, short time to complete cultivation, and less damage from contamination. However, since the concentration of products in the culture increases with time, the productivity and yield decrease due to the influence of osmotic pressure, product inhibition or the like. Because of this, it is difficult to stably keep the yield and productivity high for a long period of time.
On the other hand, the continuous fermentation method is performed by continuously culturing microorganisms or cultured cells in a fermenter while continuously supplying a culture medium to the fermenter. For example, a continuous culture method used for fermentation of L-glutamic acid or L-lysine has been disclosed (Toshihiko Hirao et al., Appl. Microbiol. Biotechnol., 32, 269-273).
Such a continuous fermentation method should theoretically enable a long-term efficient production of target substances. However, despite such an advantage, it is considered practically difficult to use conventional continuous fermentation methods to realize a long-term continuous operation on an industrial scale due to an increase in contamination risk in fermenters or the like (P. F. Strambury and A. Whitaker, “Principles of Fermentation Technology, From Laboratory to Industrial Scale,” Gakkai Publication Center, Sep. 1, 1998 (1st edition).
Furthermore, in continuous culture methods, a culture containing microorganisms for use in fermentation is removed from the fermenter while a fermentation raw material is continuously supplied to the fermenter, resulting in dilution of the microorganisms in the culture, which may limit the improvement in the production efficiency. Thus, maintaining the concentration of microorganisms in the culture at a high level in continuous fermentation methods has been an important issue in the continuous fermentation field.
To address the above problems, we proposed a continuous fermentation method including filtering a fermented liquid containing a microorganism through a separation membrane, recovering a product from the filtrate while retaining or refluxing the filtered microorganism in the culture to maintain the concentration of the microorganism in the fermenter at a high level, and a continuous fermentation apparatus for use in the method (WO 2007/097260).
There is a continuing need to provide a continuous fermentation apparatus wherein a microorganism is retained or refluxed in a fermenter by using a separation membrane and wherein a plurality of independent fermentation raw material supply systems are used in rotation to supply a fermentation raw material to a fermenter can effectively prevent contamination and efficiently produce an alcohol by continuous fermentation for a long period of time.
We thus provide:
(1) A method of producing an alcohol by continuous fermentation using a fermentation raw material,
A continuous fermentation apparatus characterized by retaining or refluxing a microorganism and the like in a fermenter while using a separation membrane allows for effective prevention of contamination, and efficient production of an alcohol by continuous fermentation for a long period of time.
A continuous fermentation apparatus wherein a microorganism and the like are retained or refluxed in a fermenter by a separation membrane can efficiently utilize the microorganism, and thus is expected to enable a longer-term continuous fermentation than conventional continuous fermentation apparatus. We, as shown in Examples 1 and 2 below, examined how long production of an alcohol by continuous fermentation can be continued using a continuous fermentation apparatus wherein a microorganism and the like were retained or refluxed in a fermenter by a separation membrane and wherein a raw material and the apparatus were not sterilized, and revealed that only after about 300 hours a decrease in the fermentation efficiency due to contamination was observed. Thus, it is also an important issues in a continuous fermentation apparatus wherein a microorganism and the like are retained or refluxed in a fermenter by a separation membrane, to prevent contamination and continue alcohol production by continuous fermentation for a long period of time.
Our method of producing an alcohol by continuous fermentation using a fermentation raw material comprises steps of:
In the method of producing an alcohol, the fermentation raw material is first provided.
Preferred examples of the fermentation raw material include common liquid media containing organic micronutrients such as carbon sources, nitrogen sources, and inorganic salts as appropriate. The carbon sources that can be used include sugars such as glucose, sucrose, fructose, galactose, and lactose; starch saccharified solutions containing the sugars, sweet potato molasses, sugar beet molasses, syrup (molasses), Hi-test molasses, and cane molasses; organic acids; and glycerins. The nitrogen sources that can be used include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, other supplementary added organic nitrogen sources, for example, oil cakes, soybean hydrolysates, casein digests, other amino acids, vitamins, corn steep liquors, yeast or yeast extracts, meat extracts, peptides such as peptone, various fermentation microorganisms and hydrolysates thereof. Inorganic salts such as phosphates, magnesium salts, calcium salts, iron salts, and manganese salts can be added as appropriate.
As the fermentation raw material, a non-sterilized non-sterile fermentation raw material is preferably used to not destroy nutrients in the fermentation raw material by heat. The term “sterilization” means a process of completely destroying or eliminating proliferative microorganisms. Specific examples of the process include a process of killing microorganisms by keeping a fermentation raw material under a high temperature condition of 100° C. or higher or under high-temperature and high-pressure conditions; a process of removing microorganisms by filtering a fermentation raw material; a process of making the proliferative ability of microorganisms lost through irradiation of a fermentation raw material with UV, gamma-ray or the like. As the non-sterile fermentation raw material, a fermentation raw material not treated with the sterilization as described above is suitably used. The non-sterile fermentation raw material may be in the state where proliferative microorganisms are not completely destroyed or eliminated. Such a non-sterile fermentation raw material is preferably a fermentation raw material containing sugar, more preferably a fermentation raw material containing syrup (molasses) as a main component. As used herein, “a fermentation raw material containing molasses as a main component” means that 50 percent by weight or more of substances contained in the fermentation raw material (except water) is molasses. Continuous fermentation can be advantageously performed using a non-sterile fermentation raw material for a long period of time from the viewpoint of industrial production efficiency. In particular, a fermentation raw material containing a high concentration of sugar including molasses can be preferably used for continuing a continuous fermentation for a long period of time because microorganisms hardly propagate in the fermentation raw material.
The method of producing an alcohol comprises culturing a microorganism in the fermentation raw material as described above in a fermenter to obtain a fermented liquid while supplying the fermentation raw material into the fermenter using a plurality of fermentation raw material supply systems; filtering the obtained fermented liquid through a separation membrane module to obtain an alcohol-containing filtered liquid and an unfiltered liquid; and retaining or refluxing the obtained unfiltered liquid in the fermenter. The method of producing an alcohol will now be described in more detail with reference to
In the method of producing an alcohol, the fermentation raw material is supplied to the fermenter (3) using the plurality of fermentation raw material supply systems (2a and 2b). The plurality of fermentation raw material supply systems (2a and 2b) each independently comprise a fermentation raw material tank (5) that stores the fermentation raw material, and a supply line (6) connecting the fermentation raw material tank (5) and the fermenter (3). Preferably, a pump (7) is disposed in the supply line (6) to deliver the fermentation raw material to the fermenter (3). The plurality of fermentation raw material supply systems (2a and 2b) supply the same fermentation raw material to the fermenter (3). The number of the fermentation raw material supply system is not particularly limited and may be two or more, for example, three to five, and may be set as appropriate by those skilled in the art in consideration of fermentation efficiency and the like.
In some examples, the production method comprises stopping one of the fermentation raw material supply systems (2a and 2b) from supplying the fermentation raw material during supply of the fermentation raw material using the plurality of fermentation raw material supply systems (2a and 2b). The fermentation raw material supply system to be allowed to stop supplying the fermentation raw material is selected from the plurality of fermentation raw material supply systems (2a and 2b) in rotation. For example, when the fermentation raw material supply system (2a) is stopped, the fermentation raw material supply system (2b) is operated to supply the fermentation raw material. On the other hand, when the fermentation raw material supply system (2b) is stopped, the fermentation raw material supply system (2a) is operated to supply the fermentation raw material. In continuous fermentation processes, the rate of supply of a fermentation raw material is preferably constant. Thus, regardless of the number of the fermentation raw material supply systems in operation, the total rate of supply of the fermentation raw material supplied from the fermentation raw material supply systems is preferably regulated to be constant. Thus, for easy regulation, one fermentation raw material supply system is preferably in operation. Preferably, the fermentation raw material tank (5) preferably contains a high concentration of the fermentation raw material. Thus, the fermentation raw material is preferably diluted into an appropriate sugar concentration with water just before being supplied to the fermenter. Thus, the supply lines (6) in the fermentation raw material supply systems (2a and 2b) are preferably provided with line mixers (17a and 17b), respectively. In addition, a water tank (14′) is also preferably disposed to supply water to the line mixers (17a and 17b). The water tank (14′) may be connected to the line mixers (17a and 17b), for example, through a line (18) having a T-branch as shown in
Either of the fermentation raw material supply systems (2a and 2b) is washed while stopping the supply of the fermentation raw material. Surprisingly, as described above, washing of fermentation raw material systems while stopping them in rotation at an appropriate interval described below enables a continuous fermentation to be continued for a long period of time without sterilization of a fermentation raw material.
For the fermentation raw material supply systems (2a and 2b), the interval at which the supply of the fermentation raw material is stopped is determined as appropriate by those skilled in the art depending on the type of bacteria, temperature, humidity, scale, and the like. For example, the interval is 1 to 10 days, preferably 1 to 6 days, more preferably 1 to 2 days.
As shown in
When the fermentation raw material supply system (2a) is stopped, the connecting valve (16a) can be opened to wash the fermentation raw material tank (5) and the line (6) in the fermentation raw material supply system (2a) with the washing liquid. When the fermentation raw material supply system (2b) is stopped, the connecting valve (16b) can be opened to wash the fermentation raw material tank (5) and the line (6) in the fermentation raw material supply system (2b) with the washing liquid. The number of the washing liquid tank connected to the fermenter is not particularly limited and may be one or two or more. From the viewpoint of equipment cost, one washing liquid tank is preferred as shown in
When the fermentation raw material supply system (2a or 2b) is washed, the liquid obtained by washing the fermentation raw material supply system (2a or 2b) is preferably supplied to the fermenter (3). The liquid obtained by the washing, which contains in solution the fermentation raw material remaining in the fermentation raw material tank (5) and the supply line (6), is preferably supplied to the fermenter from the viewpoint of efficient use of the fermentation raw material.
Preferably, the supply lines (6) in the fermentation raw material supply systems (2a and 2b) are each provided with a sugar concentration meter (9) to check whether the fermentation raw material is diluted by the line mixer according to the setting or to measure the concentration of the remaining fermentation raw material in the washing liquid. The sugar concentration meter (9) may be placed at any particular position in the supply line (6), preferably near the junction between the supply line (6) and the fermenter (3) from the viewpoint of accurate measurement of the sugar concentration in the fermentation raw material.
The method of producing an alcohol comprises culturing a microorganism in the fermentation raw material in the fermenter using the fermentation raw material supplied from the plurality of fermentation raw material supply systems as described above to obtain a fermented liquid; filtering the obtained fermented liquid through a separation membrane module to obtain an alcohol-containing filtered liquid and an unfiltered liquid; and retaining or refluxing the obtained unfiltered liquid in the fermenter.
As shown in
Any number of fermenters may be employed in the production method without impairing the desired effects. In the production method, the continuous culture operation is usually and preferably performed in a single fermenter as shown in
The concentration of sugars in the culture is preferably maintained at 5 g/L or less. The concentration of sugars in the culture is preferably maintained at 5 g/L or less to minimize the loss of sugars by removing of the culture. The concentration of sugars in the culture is adjusted as appropriate according to the composition or the supply rate of the fermentation raw material supplied from the fermentation raw material supply system.
The microorganism can usually be cultured at a pH of 4 to 8 and at a temperature of 20 to 40° C. The pH of the culture can be adjusted to a predetermined value usually at a pH of 4 to 8 with an inorganic or organic acid, or an alkaline substance, or even urea, calcium carbonate, ammonia gas, or the like. Further, aeration may be performed as needed.
A batch culture or fed-batch culture may be performed early in the cultivation to increase the concentration of the microorganism before the start of the continuous culture. The concentration of the microorganism may be increased, then the increased concentration of the microorganism may be seeded, and thereafter the continuous culture may be performed at the start of the cultivation. The supply of the fermentation raw material and continuous culture of the culture can be started at appropriate times. The supply of the fermentation raw material and the continuous culture are not necessarily started at the same time.
To achieve efficient productivity, the concentration of the microorganism (including cultured cell) in the culture in the fermenter is preferably maintained in a high level, at which the environment of the culture is suitable for the growth of the microorganism and the ratio of death does not increase. As an example, the concentration of the microorganism in the culture can be maintained at 5 g/L or more by dry weight to obtain good production efficiency.
The microorganism can be removed from the fermenter as needed. For example, since the separation membrane tends to be clogged when the concentration of the microorganism in the fermenter is too high, the clogging can be avoided by removing the microorganism.
Next, microorganisms that can be used in the production method are described below. The microorganism used in the production method is not limited as long as it can be used for the alcohol production. Examples of the microorganism that can be used for the alcohol production include yeasts belonging to the Genus Saccharomyces, Genus Kluyveromyces, and Genus Schizosaccharomyces. Among them, Saccharomyces cerevisiae, Kluyveromyces lactis, and Schizosaccharomyces pombe can be suitably used.
The microorganism that can be used for production of the alcohol production may be a microorganism or a cultured cell having an artificially enhanced capability for producing an alcohol. Specifically, the microorganism that can be used for production of the alcohol production may be a microorganism having characteristics partially modified by mutation or gene modification. One example of the microorganism having partially modified characteristics include a yeast that obtains an ability to metabolize raw starch by introduction of a glucoamylase gene of a mold belonging to the Genus Rhizopus (Microorganisms, 3: 555-564(1987)).
In some examples, the fermented liquid obtained as described above is filtered through a separation membrane module to obtain an alcohol-containing filtered liquid and an unfiltered liquid, and then the obtained unfiltered liquid is retained or refluxed in the fermenter. The microorganism and the like contained in the unfiltered liquid can be retained or refluxed in the fermenter as described above to perform an efficient alcohol fermentation.
As shown in
The fermenter (3) is also connected to the separation membrane module (4) via the second line (11), and an unfiltered liquid (for example, larger matters than the pore size of the porous membrane (12) such as microorganism) obtained by filtering the fermented liquid through the separation membrane module (4) is delivered to the fermenter (3) through the second line (11). The second line (11) may be provided with a pump (7) to deliver a fermented liquid obtained in the fermenter (3) to the separation membrane module (4).
The separation membrane module (4) has a substantially cylindrical shape and a porous membrane (12) inside. The porous membrane (12) is arranged so that the interior space of the separation membrane module (4) is divided to form two regions, R1 and R2. Thus, the separation membrane module (4) is in a state where the interior space is divided by the porous membrane (12) and the two divided spaces are communicated with each other only through the pores. In the separation membrane module (4), the first line (10) and the second line (11) are joined to communicate with the region R1, while a third line (13) is joined to communicate with the region R2.
In separation membrane module (4), the fermented liquid delivered from the fermenter (3) is filtered through the porous membrane (12) and separated into an alcohol-containing filtered liquid and an unfiltered liquid. Specifically, the alcohol-containing filtered liquid that has passed through the pores of the porous membrane (12) enters the region R2 and is delivered outside through the third line (13). On the other hand, the unfiltered liquid that has not passed through the pores of the porous membrane (12) remains in the region R1, refluxed to the fermenter (3) through the second line (11), and held in the fermenter (3). As described above, the fermenter (3) is subjected to supply of the fermentation raw material from the raw material supply tank (5) and the reflux of the microorganism and the like from the separation membrane module (4) repeatedly, which enables an efficient continuous fermentation.
The separation membrane module (4) may comprise the porous membrane (12) described above to divide the interior space, or comprise a hollow fiber separation membrane module comprising a plurality of hollow fiber membrane. When hollow fiber separation membrane modules are used, for example, when an external pressure hollow fiber membrane module is used, the first line (10) and the second line (11) are joined to the module to communicate with a space formed on the side of the outer surface of the hollow fiber membrane (corresponding to the region R1), while the third line (13) is joined to the module to communicate with a space formed on the side of the inner surface of the hollow fiber membrane (corresponding to the region R2). When an internal pressure hollow fiber membrane module is used, the first line (10) and the second line (12) are joined to the module to communicate with a space formed on the side of the inner surface of the hollow fiber membrane, while the third line (13) is joined to the module to communicate with a space formed on the side of outer surface of the hollow fiber membrane.
From the alcohol-containing filtered liquid obtained by the production method, an alcohol can be suitably separated and purified by, for example, purification via distillation, or purification via concentration using an NF membrane, an RO membrane, or a zeolite separation membrane.
The concentration of an alcohol in the alcohol-containing filtered liquid is not particularly limited, and can be adjusted according to fermentation conditions, the rate of filtration, and the like, and can be, for example, 30 to 120 g/L.
Preferred examples of the alcohol obtained by the production method include methanol, ethanol, propanol, butanol and combinations thereof. Preferably, the alcohol is ethanol.
The production method can also prevent any contamination to advantageously reduce the pH decrease in the culture in the fermenter. The pH of the culture in the fermenter during the continuous fermentation is, for example, about 3 to 8, preferably about 4 to 6.
The production method enables a long-term continuous fermentation even using a non-sterile fermentation raw material. The period of performing the continuous fermentation is not particularly limited. The suitable lower limit can be selected from, for example, 100 hours, 200 hours, 300 hours, 400 hours, 900 hours and 1,000 hours. The suitable upper limit of the period of performing the continuous fermentation can be selected from, for example, 1,000 hours, 1,500 hours, and 2,000 hours. Particularly suitable period of performing the continuous fermentation can be, for example, 100 to 2,000 hours.
The continuous fermentation apparatus as illustrated in
Preferably, the continuous fermentation apparatus comprises a washing liquid tank and/or a line mixer as shown in
The washing liquid tank, the lines (such as supply line, first line, and second line) and the fermenter can be a known tank, pipe and fermenter, respectively, depending on the desired size and characteristics of the fermentation raw material.
The separation membrane module has a substantially cylindrical shape as illustrated in
The porous membrane or the hollow fiber membrane may be composed of organic materials and/or inorganic materials. Preferably, the organic materials are organic polymers, including polyethylene resins, polypropylene resins, polyvinyl chloride resins, polyvinylidene difluoride resins, polysulfone resins, polyether sulfone resins, polyacrylonitrile resins, cellulose resins and cellulose triacetate resins. Among them, from the viewpoint of ease of film formation with the solution and excellent physical durability and chemical resistance, polyvinyl chloride resins, polyvinylidene difluoride resins, polysulfone resins, polyether sulfone resins and polyacrylonitrile resins are preferred. Particularly preferably, a polyvinylidene difluoride resin or a resin mainly containing it is used. Preferred inorganic membranes include ceramics.
The continuous fermentation apparatus may also be provided with sugar concentration meters, thermometers, and pH meters at desired positions in, for example, the fermenter and the lines, from the viewpoint of monitoring the progress of the fermentation and washing. The lines may also be provided with a valve to control the direction of liquid delivery or washing at a desired position.
The continuous fermentation apparatus may also be provided with a control unit to control the stop duration of the fermentation raw material supply system, the washing duration, the rates of delivering liquids and the like. For example, the control unit can store information from the sugar concentration meters, the thermometers, and the pH meters deposited in the continuous fermentation apparatus, and based on the information, can control the stop duration of the fermentation raw material supply system, the washing duration, the rates of delivering liquids and the like. The control unit can be provided by using a semiconductor memory such as a flash memory or a DRAM (Dynamic Random Access Memory).
In some examples, a fermentation raw material supply system that does not comprise a fermentation raw material sterilizer can be used. The term “a fermentation raw material supply system that does not comprise a fermentation raw material sterilizer” refers to a fermentation raw material supply system that does not comprise any device that sterilizes the above-described fermentation raw material. Examples of the fermentation raw material sterilizer include tanks capable of retaining the fermentation raw material at a high temperature or at a high temperature and high pressure; continuous sterilizers capable of continuously performing a high-temperature treatment or a high-pressure treatment; filter devices that remove the microorganism from the fermentation raw material; and devices that irradiate the fermentation raw material with UV or gamma-ray. The fermentation raw material supply system that does not comprise a fermentation raw material sterilizer not only is more convenient and low-cost than the fermentation raw material supply system that comprises a fermentation raw material sterilizer, but also eliminates the need for sterilization management of the fermentation raw material supply system, and therefore is advantageous in reducing the management burden.
Our method will now be described in more detail with reference to the Examples. Unless otherwise stated, the unit and the measuring method are in accordance with the provisions of the Japanese Industrial Standard (JIS).
To determine the possible duration of continuous fermentation when a continuous fermentation apparatus comprising a separation membrane module is used to produce an alcohol without sterilization of a fermentation raw material and the equipment, the continuous fermentation apparatus (1) shown in
The continuous fermentation apparatus (1) shown in
The conditions of continuous fermentation using the continuous fermentation apparatus (1) were as follows:
As a result of the continuous fermentation, the concentration of ethanol in the filtrate was as shown in
On the other hand, the concentration of glucose, the concentration of fructose, and the concentration of lactic acid in the filtrate, and the pH value of the culture in the fermenter were as shown in
From the above results, we found that the possible duration of continuous fermentation without sterilization of the fermentation raw material and the equipment using the continuous fermentation apparatus (1) shown in
As described in WO '260, it is known that there is a risk of contamination in continuous fermentation at the fermenter and the like. The region including the fermenter (3) enclosed in the dotted line (21) in the continuous fermentation apparatus (1) shown in
The results were as shown in
Fermentation was carried out using the continuous fermentation apparatus (1) shown in
As a result, the concentration of ethanol was as shown in
The concentration of glucose (fermentation raw material), the concentration of fructose (fermentation raw material), the concentration of lactic acid and the pH value of the culture in the fermenter were as shown in
From the above results, we found that the supply line is the site that causes contamination and interferes continuous fermentation in the continuous fermentation apparatus comprising a separation membrane module. From these facts, we found that a long-term continuous fermentation without contamination can be achieved by preparing a plurality of fermentation raw material supply systems comprising a fermentation raw material tank and a supply line, and using the plurality of fermentation raw material supply systems in rotation at a determined interval capable of preventing contamination that interferes continuous fermentation while washing the fermentation raw material supply system out of use repeatedly. We found that a long-term continuous fermentation set in the same test as in Example 3 can be carried out by the continuous fermentation apparatus shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2017-020394 | Feb 2017 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2018/004087 | 2/6/2018 | WO | 00 |
