JPH044892A - Production of corrinoid using methane-forming bacterium and production device therefor - Google Patents

Abstract

PURPOSE:To continuously culture the title substance and to obtain the substance inexpensively and efficiently by anaerobically culturing a methane forming bacterium in a bioreactor while feeding a culture substance containing a substance of promoting production of corrinoid of the bacterium to the rector. CONSTITUTION:First, a methane forming bacterium is anaerobically cultured in a bioreactor having the methane forming bacterium in high concentration while supplying a culture solution containing a substance (e.g. L-glutamic acid) of promoting production of corrinoid of the bacterium to the bioreactor. Then a culture solution not containing cells is taken out from an outlet of the bioreactor and the objective substance in the culture solution is recovered by a method of adsorbing the substance on a columm packed with a hydrophobic resin.

Description

DETAILED DESCRIPTION OF THE INVENTION "Industrial Application Field" The present invention relates to a method for producing corrinoids (vitamin n, . . . ) using methane-producing bacteria and a production apparatus used therefor.

"Conventional technology" Vitamin B1. Since chemical synthesis of corrinoids (hereinafter referred to as corrinoids) is extremely difficult, their production is limited to anaerobic bacteria such as acetic acid bacteria and propionic acid bacteria, and Strebtomyces (S
treptmyees), Bacillus (Bacillus)
It is carried out by a fermentation method in which aerobic bacteria such as s.

To explain an example of a method for producing corrinoids using a conventional fermentation method, first, corrinoid-producing bacteria are inoculated into a culture tank filled with a culture solution suitable for the corrinoid-producing bacteria to be used.
Incubate while stirring as necessary. Next, the culture solution in the culture tank is centrifuged to collect the cultured bacteria, which is further dried and crushed (the cultured bacteria die at this point). Next, corrinoids are extracted from the crushed bacterial cells by heat extraction and solvent extraction, and the extract is further purified using purification means such as electrophoresis, ion exchange, and reversed phase chromatography to produce corrinoids.

[Problems to be Solved by the Invention] However, in the conventional fermentation method using acetic acid bacteria and propionic acid bacteria, the product is likely to be inhibited by the organic acids produced, and Streptomyces and Bacillus ,
Even in the method using j-aerobic bacteria, there were problems such as 2-produced corrinoids being denatured after an appropriate culture time.

Furthermore, in fermentation production methods using these bacteria, the produced corrinoids accumulate within the bacterial cells, so in order to obtain corrinoids, the cultured bacterial cells must be crushed to recover corrinoids and idos, making continuous production difficult. It is difficult and there are problems with poor production efficiency.

The present invention was developed in view of the above circumstances, and includes a production method that enables continuous culture of corrinoids and achieves high production efficiency in the production of corrinoids by fermentation, and a production apparatus suitable for the same. The purpose is to provide

[-Means for Solving the Problems] The present invention provides a bioreactor that maintains methane-producing bacteria at a high concentration, while supplying a culture solution containing a substance that promotes corrinoid production of methanogenic bacteria. The method for solving the above problem was to perform anaerobic culture of the producing bacteria, take out a culture solution containing no bacterial cells from the bioreactor outlet, and recover corrinoids in the culture solution.

Furthermore, after recovering the corrinoids in the culture solution taken out from the outlet of the bipedal bioreactor, it is desirable to use the corrinoid recovery residual liquid as the culture stock solution.

Furthermore, a corrinoid metabolism promoting substance that promotes the extracellular metabolism of corrinoids of methanogenic bacteria can be added to the above culture solution, and culture can be carried out.

Further, the production equipment used in the corrinoid production method according to the present invention includes a bioreactor that holds methane-producing bacteria at a high concentration, and a culture solution supply means for supplying a culture solution of methane-producing bacteria into the bioreactor. Preferably, there is a production device comprising a corrinoid recovery means for recovering corrinoids in a culture solution containing no bacterial cells taken out from the outlet of the bioreactor, and sending the corrinoid recovery residual solution to the culture solution supply means as a culture stock solution. used.

The present invention will be explained in detail below.

The methanogenic bacteria used in this invention are M e
methane bacteria such as Thanosarcina. Furthermore, both mesophilic methanogenic bacteria and high-temperature methanogenic bacteria can be used for corrinoid production according to the present invention.

The culture medium used for culturing this methanogenic bacteria includes various components such as culture substrate (carbon source), nutrients (nitrogen source, vitamins, etc.), inorganic salts, and other substances that promote corrinoid production by methanogenic bacteria. A culture solution containing the following additives and having p I-1 adjusted as necessary is used. In addition, in order to carry out continuous culture, the residual solution of corrinoids collected after removing them from the bioreactor and collecting the corrinoids is used as a culture stock solution, and each of the above components lacking in this culture stock solution is added and mixed, and then added to the bioreactor. Use this as the culture solution to be supplied.

Suitable culture substrates for methanogenic bacteria include acetic acid,
These include methanol, medylamine, carbon dioxide and hydrogen, and formic acid, and one or more of these may be selected and used depending on the type of methanogenic bacteria used.

Moreover, organic industrial wastewater containing the above-mentioned culture substrate can also be used as the culture solution.

The corrinoid production promoting substances of methanogenic bacteria that are added to the culture solution include Co'' L-glutamic acid, glinone, 5-amylpurinic acid, vorphobilinogen, uroporphyrin, roinone, isoloinone, nosteine, nostin, hydroquinoproline, Examples include L-glutamine, histidine, α-ketoglutaric acid, tryptophan, etc., and one or more of these are selected and used.

The amount of the above-mentioned corrinoid production promoting substance added will be described in detail in the experimental examples described later, but generally it may be a small amount, and the appropriate concentration in the bioreactor is about 0.1 to 100 mM. In addition, some of these corrinoid production-promoting substances have an inhibitory effect on the growth of methane-producing bacteria when added at high concentrations. Therefore, the addition of corrinoid production-promoting substances should be started from a low concentration range to prevent the generation of methane gas. It is preferable to gradually increase the concentration while determining the growth status of bacteria in the bioreactor.

Furthermore, the organic industrial wastewater containing the above-mentioned corrinoid production-promoting substance can be used as a stock solution of the culture solution, and the corrinoid production-promoting substance in the solution can be utilized.

In the present invention, a bioreactor that retains methane-producing bacteria at a high concentration is used, or methods for retaining methane-producing bacteria at a high concentration in a bioreactor include: 1) A bioreactor that retains bacterial cells using a carrier; use

■Use an upflow anaerobic sludge bed bioreactor that retains bacteria by utilizing the self-granulation action of bacteria.

■Use a bioreactor that retains bacterial cells by entrapping and immobilizing them with a gel.

■A separation membrane for bacterial cell preparation is installed at the bioreactor outlet.

■By centrifugation.

■Install a natural (gravity) settling tank.

There are several methods, and these methods are appropriately selected and used depending on the type of bacteria used and culture conditions. By using these methods, it is possible to prevent cultured bacteria (methane-producing bacteria) from flowing out of the culture system.

Furthermore, a culture solution containing no cultured bacteria can be taken out from the bioreactor.

Methanogenic bacteria produce corrinoids by being cultured anaerobically in a bioreactor, and a portion of the produced corrinoids is metabolized outside the bacterial cells. A culture solution containing corrinoids produced by the cultured bacteria is then taken out from the bioreactor outlet.

Methods for recovering corrinoids in the culture solution taken out from the bioreactor outlet include: ■ a method of adsorbing and recovering corrinoids using a column filled with a hydrophobic resin; ■ a method of adsorbing corrinoids using an ion exchange resin; Methods such as ■solvent extraction method are used, but in particular, the method using a hydrophobic resin-filled column (■) is easy to maintain and regenerate the resin, and adsorbs ionic substances necessary for the growth of methane-producing bacteria. iii) Methanol, which is a culture substrate for methanogenic bacteria, can be used to recover corrinoids from the column. However, with the corrinoid recovery method using hydrophobic resin, it is not possible to selectively adsorb only the corrinoids in the culture solution, and instead adsorbs all the hydrophobic substances, so the recovery device requires multiple columns. It is.

The corrinoid thus recovered from the culture solution is purified using a purification method such as electrophoresis, ion exchange, or reversed phase chromatography to obtain purified corrinoid.

Note that methane gas generated by culturing in the bioreactor can be stored in a gas holder and used as fuel gas.

As mentioned above, if methanogenic bacteria are used to produce corrinoids, there will be a large amount of corrinoids per dry weight of the cultured bacteria.Culture substrates and culture solutions are inexpensive.Product inhibition does not occur or is unlikely to occur. Since most of the corrinoids produced are metabolized outside the bacterial cells (in the culture solution), corrinoids can be easily recovered and are suitable for continuous production.In addition, methane-producing bacteria have traditionally been used mainly in water treatment of organic wastewater. Since it is used in bioreactors, there is the possibility of developing a process that combines wastewater treatment and corrinoid production.Furthermore, it has the advantage that methane gas produced by culturing in a bioreactor can be used for energy.

From the above points, if methane-producing bacteria are maintained at a high yield in a bioreactor and cultured continuously by supplying a culture solution containing a corrinoid production promoting substance, corrinoids can be produced in large quantities, and their recovery can be done using conventional processes. This makes it possible to construct a production system with extremely high production efficiency of corrinoids.

Next, another example of the corrinoid production method according to the present invention will be explained.

In one example of the corrinoid production method using the methanogenic bacteria described above, the methanogenic bacteria are cultured in a culture solution containing a corrinoid production promoting substance, and the corrinoid production ability of the bacteria can be increased. In the corrinoid production method described here,
A corrinoid metabolism promoting substance that promotes the extracellular metabolism of corrinoids of methanogenic bacteria is added to the culture solution, and culture is carried out. This method makes it possible to further improve the production efficiency of corrinoids.

Methanogenic bacteria originally produced about 60% of the corrinoids.
% is metabolized outside the bacterial body, but the remaining 40% remains within the bacterial body.

Methods for promoting corrinoid metabolism from within the bacterial cells of methanogenic bacteria include: ∎ Forced metabolism from within the bacterial cells using chemical and physical effects; ∎ Formation of a cytoplasmic membrane that facilitates permeation of substances to promote metabolism. There are ways to make it easier.

From these two viewpoints, it is important to use surfactants and antibiotics that cause cytoplasmic membrane synthesis disorders in bacterial cells at a concentration that does not inhibit the growth of methanogenic bacteria (if these substances are added in excess, they may inhibit the growth of methanogenic bacteria). By adding and culturing corrinoids (which have the effect of inhibiting corrinoids), it is possible to promote the metabolism of corrinoids within the bacterial cells.

Preferred examples of commercially available surfactants include Triton X-100 and SDS, and preferred antibiotics include colistin and polymyxin. It is sufficient that each of these substances has a purity that does not contain heavy metals that may affect microorganisms.

By adding appropriate amounts of these surfactants and antibiotics to the culture solution, it becomes possible to produce corrinoids with high efficiency using a bioreactor in which methanogenic bacteria are maintained at a high β degree.

In the corrinoid production method according to this example, the production efficiency of corrinoids is further improved by adding a corrinoid metabolism promoting substance that promotes the extracorporeal metabolism of methanogenic bacteria to the culture solution of methanogenic bacteria. can be done.

Next, a production apparatus according to the present invention will be described.

FIG. 1 shows, as an example of a corrinoid production apparatus according to the present invention, a continuous culture apparatus suitably used for continuous culture using methanogenic bacteria.

This continuous culture device is connected to a bioreactor 1 that holds methane-producing bacteria at a high concentration, a culture solution supply device 2 that supplies a culture solution into the bioreactor 1, and an outlet of the bioreactor 1. A solid-liquid separator 3 separates the solid content in the culture solution Lt taken out from the -1 outlet, and collects corrinoids in the culture solution L3 that do not contain bacterial cells separated by the solid-liquid separator 3, and The main component is a corrinoid recovery device 4 that sends the recovered residual liquid L4 as a culture stock solution to the culture solution supply device 2.

The format of the bioreactor 1 is not particularly limited, and various types of reactors such as fixed bed, fluidized bed, and tJAsB method can be used. Examples of suitable bioreactors include a bioreactor using a carrier, an upflow type anaerobic sludge bed bioreactor that uses the self-granulation effect of bacteria to retain bacteria, and a bioreactor that uses a carrier to retain bacteria. This includes a bioreactor that performs entrapping immobilization with gel, etc. In addition, when an ultrafiltration membrane is used in the solid-liquid separation device 3, it is also possible to culture without using the timely immobilization method.

A gas holder 5 is provided at the top of the bioreactor 1 to collect methane gas produced by culture. It is also desirable to install a temperature control mechanism to control the temperature inside the bioreactor and maintain the culture temperature at the optimum temperature for the bacteria used.

The culture solution supply device 2 also includes a substrate storage tank 6 for adding a culture substrate, a nutrient factor storage tank 7 for adding a nutrient factor, and a corrinoid production promoting substance of methanogenic bacteria or a corrinoid production promoting substance and corrinoid metabolism. Additive storage tank 8 for adding both the promoting substance and the pH of the culture solution
Additives from each storage tank are added to the pH adjuster storage tank 9 for adding acid or alkali to adjust the pH, and to the corrinoid recovery residual liquid (culture stock solution) sent from the above-mentioned corrinoid recovery device 4. It is configured to include a mixing adjustment tank 10 that regenerates the liquid and supplies this culture liquid L1 to the bioreactor 1.

The substrate storage tank 6 is for supplying one or more types of substrates suitable for the bacteria to be used, such as acetic acid and methanol, and when supplying multiple types of substrates at different concentrations, each substrate is A supply tank is installed in the

The nutritional factor storage tank 7 is for adding various nutritional substances such as nitrogen sources, inorganic salts, vitamins, and growth promoting factors. When using a mixture of the culture substrate and the nutritional factor, one storage tank can supply the culture substrate and the nutritional factor.

Further, when it is necessary to control the concentration of two or more types of additives such as a corrinoid production promoting substance and a corrinoid metabolism promoting substance, the additive storage tank 8 is provided with an additional storage tank for each additive.

In addition, the pHl11 preparation storage tank 9 contains a predetermined amount of acidic solution so that the culture solution supplied to the bioreactor 1 falls within the optimum pH range of the bacteria used, based on the measurement result of p)I of the culture solution. Alternatively, it has a function to automatically add an alkaline solution. As the acidic solution, in addition to organic acids such as acetic acid, inorganic acids such as hydrochloric acid and sulfuric acid can also be used. Alkaline solutions include sodium hydroxide, calcium hydroxide,
Ammonia water and the like are suitable.

The mixing and preparation tank IO is for mixing the stock culture solution L4 sent from the corrinoid recovery device 4 and the additives in each storage tank 6, 7, 8, 9 to regenerate the culture solution Ll. In this mixing preparation tank 10, a sensor measures the concentration of the culture substrate, each nutritional factor, or a corrinoid production promoting substance in the culture stock solution L4, and automatically extracts the missing components from each storage tank according to the amount of the culture stock solution L4. Quantitatively added, together with pHEIM from the pH adjuster storage tank 9, a culture solution with a constant composition,
It is desirable to have a configuration that can supply the ion reactor l.

The solid-liquid separator 3 separates the solid content from the culture liquid taken out from the outlet of the bioreactor 1 using a centrifugal separator or a natural (gravity) settling tank, and separates the solid content S (
A concentrated liquid containing a large amount of solid content such as bacterial cells is returned to the mixing and preparation tank 10, and a culture liquid L3 from which the solid content S has been separated is sent to the corrinoid recovery device 4. In addition to the above-mentioned device, the solid-liquid separation device 3 includes a bacterial cell separation membrane such as an ultrafiltration membrane at the outlet of the bioreactor 1 to prevent bacteria from flowing out from the bioreactor 1. It can also be configured so that only the culture fluid 1-y that does not contain bacteria is taken out from the membrane. In this case, the step of returning the solid content S is omitted.

The corrinoid recovery device 4 preferably has a plurality of hydrophobic resin-packed columns connected together, but an ion exchange resin can also be used as the packed resin.

Further, the symbol +1 in this figure is a recovery solvent storage tank, and after the corrinoids in the culture solution L3 are absorbed by the corrinoid recovery device 4 for a certain period of time, the recovered solvent is supplied to the corrinoid recovery device @4, and the recovered solvent is absorbed by the resin. The corrinoids are eluted, and the eluted corrinoids are sent to a purification step 12.

Methanol is particularly preferable as the recovery solvent when a hydrophobic resin is used as the resin of the corrinoid recovery device 4.

- The continuous culture device configured as described above maintains a high concentration of methanogenic bacteria in the bioreactor, and supplies a culture solution suitable for culturing this bacteria from the bioreactor population. , perform anaerobic culture in the bioreactor 1, take out the culture solution L3 that does not contain bacteria from the bioreactor outlet through the solid-liquid separation device 3, and send it to the corrinoid recovery device 4 to collect the culture solution and the corrinoids inside. Then, the remaining culture solution is sent to the culture solution supply device 2 as a culture stock solution L4, and various additives are mixed in this culture solution supply device 2 to regenerate the culture solution, and this culture solution L1 is transferred to the bioreactor L. By supplying it, methane-producing bacteria are continuously cultured. The corrinoids recovered by the corrinoid recovery device 4 are eluted with a recovery solvent at an appropriate time, sent to a purification process, and purified using methods such as electrophoresis, ion exchange, and reversed phase chromatography to obtain purified corrinoid products.

Note that the corrinoid-producing bacteria used in this continuous culture device include methane-producing bacteria (Methanogen
ic bacteria), but corrinoid-producing anaerobes other than methanogenic bacteria (e.g. Sultate-reducLing) are present in the bioreactor.
bacteria and Acetogenic bacteria
It is also possible to culture in a mixed state.

According to this continuous culture device, corrinoids can be continuously produced and recovered by continuously culturing methane-producing bacteria without draining the cultured bacteria outside the culture system.
− can be produced with high efficiency.

In addition, there is almost no liquid flowing out of the system, which reduces heat loss when culturing methane-producing bacteria at high temperatures, and is advantageous in terms of thermal efficiency.

In addition, since almost no liquid flows out of the system and the residual liquid from corrinoid recovery is recycled for culture, additives such as nutritional factors and corrinoid production promoting substances can be saved significantly. In particular, some of the nutritional factors, corrinoid production promoting substances, corrinoid metabolism promoting substances, etc. are often expensive, and even additives that could not be used in the past because they could not be recovered and reused can be used with this continuous culture device. It can be used if

Moreover, the recovery efficiency of corrinoids is good, and purification becomes easy.

Furthermore, there is no need to crush the bacterial cells, and the corrinoid production process can be significantly omitted.

Experimental examples related to the present invention will be described below.

(Experiment example) ■About the effects of corrinoid production promoting substances Vitamin B1
．． (corinoid), yeast extract, and polypeptone-free mineral medium (carbon source: methanol).
M92906) was used to inoculate the above medium with various substances shown in Table 1 added thereto, and cultured at a culture temperature of 53 degrees Celsius for 4 days. As an evaluation method, the amount of extracellular corrinoids in the system cultured in the basic medium and the amount of extracellular corrinoids in the system to which the substance was added were compared by TLC. The results are shown in Table 1.

Table 1 Furthermore, among the substances shown in Table 1, the relationship between the amount of Co'+ added and the amount of corrinoid produced is shown in FIG.

As is clear from Table 1 and Figure 2, by adding a corrinoid production promoting substance to the basic medium and culturing,
It was confirmed that corrinoid production ability was improved.

■ Regarding the effect of corrinoid metabolism promoting substances, using the above-mentioned bacteria, surfactant (Triton X-100, 5
DS), antibiotics (colistin, polymyxin), and cultured to investigate the effect of each metabolism promoting substance on extracellular corrinoid metabolism. The results are shown in Figures 3 to 6.

As can be seen from these figures, growth inhibition occurs in Triton , the additive-free system (plot of concentration O in each of the figures 3 to 6 above: 55%)
It can be seen that the corrinoid metabolic rate is extremely high compared to .

Next, we examined the influence of the experimental concentrations of the above substances on the growth of methanogenic bacteria, and the results are shown in Table 2.

(Margin below) Table 2 From the results shown in Table 2, Triton x-to.

Growth inhibition was observed as the amount of addition increased in the addition system, but growth below this level showed growth of 0.8 or more compared to the non-addition system.

From these facts, three substances other than Triton (S D
It was confirmed that if methane-producing bacteria (S, colistin, polymyxin) were used in the range of this experiment, the effect of efficiently metabolizing corrinoids out of the bacterial cells was obtained without inhibiting bacterial growth.

"Effects of the Invention" As explained above, according to the present invention, the following effects can be obtained.

Methanogenic bacteria are used to produce corrinoids, and a corrinoid production-promoting substance that increases the corrinoid productivity of the bacteria is added to the culture, resulting in a large amount of corrinoids produced per bacterial weight of the cultured bacteria. In addition, the culture substrate and culture solution are inexpensive. In addition, product inhibition is less likely to occur. Furthermore, since most of the produced corrinoids are metabolized outside the bacterial cells (in the culture solution), corrinoids can be easily recovered and are suitable for continuous production.

Furthermore, since methanogenic bacteria have traditionally been used primarily for water treatment of organic wastewater, there is a possibility of developing a process that combines wastewater treatment and corrinoid production. Another advantage is that methane gas produced during cultivation in a bioreactor can be used for energy.

From the above points, if methane-producing bacteria are kept at a high concentration in a bioreactor and cultured continuously by supplying a culture solution containing a corrinoid production promoting substance, corrinoids can be produced in large quantities and their recovery is also faster than in conventional processes. This makes it possible to construct a production system with extremely high corrinoid production efficiency.

Furthermore, by culturing with the addition of a corrinoid metabolism promoting substance that promotes the extracorporeal metabolism of methanogenic bacteria, the recovery rate of corrinoids can be improved and the production efficiency can be further increased.

Furthermore, in the corrinoid production apparatus according to the present invention, it is possible to continuously produce and collect corrinoids by continuously culturing methane-producing bacteria without leaking the cultured bacteria outside the culture system, and the production of corrinoids is possible. High efficiency. Almost no liquid flows out of the system, and when culturing methane-producing bacteria at high temperatures, heat loss is reduced, which is advantageous in terms of thermal efficiency.

Almost no liquid flows out of the system, and the residual liquid from corrinoid recovery is reused for cultivation, so additives such as nutritional factors and corrinoid production promoting substances can be greatly saved. Many of the promoters and corrinoid metabolism promoters are expensive, and even additives that could not be used in the past because they could not be recovered and reused can be used with this continuous culture device. You can get the same effect.

[Brief explanation of drawings]

FIG. 1 is for explaining an example of a corrinoid production device according to the present invention, and is a schematic diagram of a continuous culture device for methane-producing bacteria, and FIGS. 2 to 6 are detailed explanatory diagrams of the present invention. Figure 2 is a graph showing the relationship between Co''' concentration and corrinoid production amount, and Figures 3 to 6 are graphs showing the relationship between the concentration of various corrinoid metabolism promoting substances and the extracellular metabolic rate. This is a graph. 2... 4... L 4... Bioreactor culture solution supply device Corrinoid recovery device Lx, Ls... Culture solution culture stock solution Figure 1

Claims (4)

[Claims] (1) Methane-producing bacteria are cultured anaerobically in the bioreactor while supplying a culture solution containing a substance that promotes corrinoid production of methane-producing bacteria into a bioreactor that maintains a high concentration of methane-producing bacteria. A method for producing corrinoids using methane-producing bacteria, characterized in that a culture solution containing no bacterial cells is taken out from an outlet and corrinoids in the culture solution are recovered. (2) Corrinoid production using the methane-producing bacteria according to claim 1, characterized in that after recovering corrinoids in the culture solution taken out from the bioreactor outlet, this corrinoid recovery residual liquid is used as a culture stock solution. Method. (3) The method for producing corrinoids using methanogenic bacteria according to claim 1, characterized in that a corrinoid metabolism promoting substance that promotes extracellular metabolism of corrinoids of methanogenic bacteria is added to the culture solution. (4) A bioreactor that holds methane-producing bacteria at a high concentration, a culture solution supply means for supplying a culture solution of the methane-producing bacteria into the bioreactor, and a culture solution that does not contain bacterial cells and is taken out from the outlet of the bioreactor. A corrinoid production apparatus comprising a corrinoid recovery means for recovering the corrinoid therein and sending the corrinoid recovery residual solution as a culture stock solution to the culture solution supply means.