WO1993013213A1

WO1993013213A1 - Fermentation process

Info

Publication number: WO1993013213A1
Application number: PCT/JP1987/000156
Authority: WO
Inventors: Shinko Kitaura; Yoshimasa Takahara; Shiro Nagai; Naomichi Nishio
Original assignee: Shinko Kitaura; Yoshimasa Takahara; Shiro Nagai; Naomichi Nishio
Priority date: 1986-03-14
Filing date: 1987-03-13
Publication date: 1993-07-08

Abstract

A fermentation process of producing a product by fermenting a gaseous substrate using microorganisms, which comprises retaining a microorganism immobilized on a carrier in a reaction vessel, feeding an aqueous solution so that at least part of the surface of the microorganism is moistened and passing the gaseous substrate through the void of the aggregate of the microorganism to thereby directly react the microorganism with the gaseous substrate. Thus, methane, formic acid, or the like can be effectively biosynthesized.

Description

Specification

Title of invention

Fermentation method

Industrial applications

The present invention relates to a method for producing a substance using a microorganism, and more particularly, to a novel method for biosynthesizing a target substance from a gaseous raw material using an immobilized microorganism.

Therefore, the present invention plays an important role in the technical field of biotechnology such as fermentation industry, microbial industry, enzyme industry, and food industry. The present invention can also be used extensively in the technical field of the chemical industry, because methane, hydrogen cyanide, acetylene and other various organic industrial chemicals can be produced using the methane and the like obtained by this method as a raw material. Is what is done.

Conventional technology

Conventionally, there is no system for industrially producing methane using microorganisms.By-products are produced by anaerobic digestion in human waste and sewage treatment or by spoilage of compost and other organic waste. It merely used methane, a by-product of the process, to decompose high-molecular-weight organic matter by microorganisms contained in sludge and the like to convert it into low-molecular-weight compounds, and finally produce It is a tan and does not biosynthesize methane from gaseous low molecular weight raw materials.

Recently, a system for producing methane using an apparatus as shown in Fig. 2 has been developed. It consists of methanotrophs in a liquid medium containing supplementary nutrients such as nitrogen sources and inorganic salts. z

In addition, carbon dioxide gas and hydrogen gas are forcibly supplied from the outside of the fermenter 13 into the liquid medium, and mechanically stirred by the stirring blades 14 (aeration-stirred fermenter 15) or draft. The air bubble 19 from the vent hole 18 is atomized by the air tube 16 (bubble column type fermenter 17) to increase the gas-liquid interface, and at the same time, the air bubble is retained in the culture medium for a long time, thereby increasing the culture medium. This system accelerates the rate of dissolution of carbon dioxide and hydrogen gas into the gas and causes a biochemical reaction by the methane-producing bacteria to obtain a product gas.This system also converts methane from the raw material gas. However, it cannot be biosynthesized by a direct gas phase reaction, and as described later, it cannot be used industrially because of its drawbacks such as low methane production rate.

Also, when producing a target substance by fermentation, an industrial system that directly produces the target substance by directly acting on microorganisms having a gaseous substrate immobilized as a raw material has been completely established. Not.

For example, a method has been proposed in which cells are suspended in a solution and gas is supplied to generate formic acid (SY * Eguchi et al., Appl. Microbiol. Biotechnol., 22, 148-151 (1985)). However, in this method, the generated formic acid was dissolved in the solution, and a high concentration of formic acid could not be obtained.

This method also does not biosynthesize the desired substance directly from the raw material gas by a gas phase reaction, and has the disadvantage that the production rate of the desired substance is low. I can not do such a thing.

As described above, there is no known technology for directly supplying low-molecular gas to microorganisms and directly biosynthesizing a target substance. At present, even the technology for biochemically synthesizing the target substance using immobilized microorganisms is not even known at all.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an example of an apparatus for carrying out the present invention.

Fig. 2 shows a conventional methane fermentation apparatus.

FIG. 3 illustrates an example of an apparatus for producing formic acid in the present invention.

Problems the invention is trying to solve

The present invention has been made for the purpose of developing an industrial system for directly producing a target product from a gaseous substrate, and firstly uses the above-described aeration type or bubble column type fermenter. We focused on the system.

However, this known system has the following disadvantages in order to achieve the above object.

1. Aeration-type fermenters require large power for mechanical stirring.

2.Because the dissolution rate of the substrate gas is limited, if the gas supply rate is increased further, most of the substrate gas will reach the liquid surface without being used by microorganisms, and contact with organisms will occur. The efficiency becomes extremely poor.

3. Because it is not possible to supply the substrate gas that matches the substrate consumption rate of the microorganism, it is difficult to achieve continuous operation. 4. Since a large amount of liquid and a large liquid depth are required, the reactor must be large, and the entire device cannot be downsized.

And fatally, these known systems do not allow the production of the target substance directly from the source gas.

Invention

The present invention has been made to solve the above-mentioned drawbacks and to develop an industrial production method for producing the target substance in a large amount and economically.

As a result of extensive research to achieve this goal, it was found that in order to increase the yield of the target substance, it is necessary to increase the raw material gas concentration and increase the contact ratio with the substance producing bacteria. Obtained. As in the conventional system, the gas concentration cannot be sufficiently increased if the original gas is dissolved in the culture medium or supplied in the form of bubbles. Therefore, as a result of completely changing the idea of a system for increasing the concentration of the raw material gas and examining it, the conventional dream was to supply the raw material gas directly in gaseous form and contact it with microorganisms to perform biosynthesis. He came up with a new technical idea that was not taken into account.

As a result of diligent research from various angles on measures for realizing this new idea concretely and industrially, we obtained the finding that it would be possible to use immobilized persimmons. As a result of further research based on the new findings, the present invention was completed.

Hereinafter, the present invention will be described in detail with reference to the apparatus shown in FIG. 1 as an example of an apparatus for carrying out the present invention. Reactor l contains the surface-treated microorganisms. Immobilization of microorganisms is carried out by a conventional method, and any of the carrier binding methods can be used.

The microorganisms are immobilized in a spherical, cylindrical, granular or other suitable shape and then filled into the reactor 1, fixed directly to the reactor wall, or immobilized on the inner and / or outer surface. A large number of hollow fibers with immobilized microorganisms are filled into the reactor, and one or more (porous) plates with immobilized microorganisms are vertically or horizontally filled into the reactor. The above-mentioned molded and immobilized cells are filled into a small column and then filled into a large number of reactors to constitute a reactor.

As the microorganism, any microorganism can be used as long as it produces the target product using a gaseous substrate. For example, a methane-producing bacterium of a type utilizing carbon dioxide gas, hydrogen gas, or the like as a substrate can be advantageously used, but the present invention is not limited to only these microorganisms. All microorganisms can be used as long as the target product can be biosynthesized using the substrate.

Specifically, the methane-producing bacterium is a gram-negative methane-producing bacterium HU strain isolated from digested sludge at a sewage treatment plant in Hiroshima City (a strain preserved in the Nagai Laboratory, Faculty of Engineering, Hiroshima University; freely available for sale). ,

Met hanobact eri um t hermoaut ot rophicum, π. Methanosaliina used alone or in combination it can. In addition, without isolating the bacteria, it is also possible to directly fix bacteria sources such as culture solutions, wet cakes, activated sludge, and digested sludge and use them in the present invention. An aqueous solution 2 containing supplementary nutrients such as a nitrogen source and inorganic salts is sprayed, dropped or dropped from a squirt tube 4 onto a carrier on which organisms or microorganisms are fixed by a control valve 3. If necessary, these nutrient solutions may be stored in a carrier in advance. Also, when using bacteria of the objective compound synthesized, C0 _2, to request a specific substance in addition to the gaseous feedstock of H _2, CO, etc., these liquid or solid raw material in the said nutrient solution 2 Since the intended purpose can be sufficiently achieved by adding it in advance, the present invention can be applied to all types of microorganisms, which is extremely advantageous.

When the nutrient solution used in Examples 1 to 3 is used as the aqueous solution to be supplied to the microorganisms in the present invention, methane gas is generated, and when the solution in Example 4 is used, formic acid is generated. It is. At the same time when the aqueous solution 2 is dropped, a substrate gas having an appropriate composition is supplied from the lower pipe 5 of the reactor via the regulating valve 6 and brought into contact with the microorganisms and nutrient solution fixed on the carrier to produce the desired product. Is generated. Since the type and composition of the substrate gas used as a raw material differ depending on the bacterium used, it is necessary to select an optimum one according to the bacterium used. For example, in the case of the methane-producing HU strain, hydrogen gas and carbon dioxide gas are used as raw materials, and the H ₂ / CO ₂ ratio is preferably larger than 1. For this purpose, a gas analyzer (not shown) is installed at the product gas outlet 7 to analyze the product gas, It is preferable to operate the control valve 6 provided at the inlet so as to adjust the mixing ratio of the substrate gas and / or the supply amount and the supply speed thereof to the optimum values for the production of methane. The same applies to the case of other microorganisms, and the control valve 6 is controlled according to the data of the gas analyzer to supply the substrate in accordance with the substrate consumption rate of the microorganism.

Reactor 1 is surrounded by a jacket for heating or keeping the temperature warm, in which hot water or temperature-adjusted gas is flown, and a heating wire is provided to conduct biosynthesis reaction. May be promoted. Conversely, it is also possible to supply the substrate gas from above the reactor and take out the generated gas from below the reactor. Also, the aqueous solution that has fallen into the liquid collecting tank 8 is not discarded as it is but is passed from the liquid outlet 9 to the aqueous solution tank 2 via a pump and a pipe (not shown). Recycling further increases its economic efficiency. If the product is a water-soluble substance such as formic acid, it is dissolved in the aqueous solution and falls into the liquid collecting tank 8, so that the aqueous solution is withdrawn from the liquid outlet 9 to recover the water-soluble substance. Can be. If necessary, pressurizing the inside of the reactor can increase the gas solubility and increase the reaction rate. If the reactor is kept airtight and the base gas content in the reactor is adjusted to the optimal value for the immobilized microorganisms, the production rate of the target product can be maximized.

When the target substance thus generated is a gas, it is collected in the gas storage 10 through the generation gas outlet 7. Also, the target substance If is a water-soluble substance, it is recovered from the liquid collection tank 8.

Examples 1-3

Zeolite, foam brick, and inorganic foam (particle size: 7.1 to 12.6 mm) were used as carriers, and the HU strain, which had been isolated in the laboratory of Hiroshima University's Faculty of Engineering, was used as the carrier. It was fixed by the adsorption method.

The methane-producing bacteria immobilized on the carrier in this manner are filled in the reactor shown in Fig. 1 (reactor capacity: 75 mJi), and the gaseous substrate is obtained using the equipment shown in Fig. 1 under the following specifications. Methane fermentation was performed. That is, in the reactor 1 filled with the carrier on which the microorganisms are immobilized, the nutrient solution 2 shown in Table 1 is dropped from the upper part of the carrier from the ejection pipe 4 by the control valve 3 onto the surface of the carrier, and the lower part of the reactor is An appropriate flow of the substrate gas was supplied from the pipe 5 by the control valve 6, and the gas generated by the microorganisms on the carrier was obtained from the upper outlet 7. A jacket was provided around the reactor, and the temperature was adjusted to the optimal temperature (37 ° C) for microbial reaction by passing temperature-controlled water.

Reactor actual capacity: 75πυί

Fermentation temperature: 37

Immobilized bacterial mass: per reactor

Example 1 Zeolite 0.675g-dry cell

Example 2 Foam brick 0.643g-dry cell

Example 3 Inorganic foam 0.604g-dry cell

Supply rate of nutrient solution: 25-30mfi / day per reactor

Substrate gas supply rate: 4760mfi / day Substrate gas composition _{(%): H 2 81.5%} , C0 2 18.5%

table 1

Composition of nutrient solution

NH C1 o.9gje 1) trace metal solution

NaH ₂ P0 ₄ * 2H ₂ 0 3 EDTA 1 £

K2HPO4 7 "Fe3 (Ρθ4) 2.8Η ₂ 0 1.02

MgCl · 6Η 0 0.36 "HnCl2.4H20 0.1

Na S-9H 0 0.5 0ο0ΐ2.6 · 0 0.17 trace metal solution 1) 9 mfi / £ ZnCl ₂ 0.1 vitaniin soluion 2) 5 "CaCl2 0.02

H ₃ B0 ₃ 0.019 a ₂ Mo0 -2H ₂ 0 0.01

2) vitamin solution

biotixi 2 mg / £ pyridoxine-HCl 10 folic acid 2 riboflavin 5 thiamine 5 nicotinic acid 5

Ca-pantothenate 5 vitamin Bi ₂ 0.1 α-lipoic acid 5

P-amiJiobenzoic acid 5 As a result, the following results were obtained.

Generated gas composition (%) H ₂ co ₂ CH ₄ Example 1 Zeolite 45.2 0 54.8 Example 2 Foaming media 46.5 5 0.8 52.7 Example 3 Inorganic foam 43.5 0 06.5 0

As is apparent from the above results, according to the present invention, it can be biosynthesized directly methane gas from H ₂及beauty C0 _2, yet little C0 ₂ of the roller and the raw material base poor from the product gas Only a small number is detected, indicating that methane gas, a huge product, can be obtained very efficiently at high dullness.

Example 4 Formation of formic acid from H ₂ and C 0 ₂

Using the apparatus shown in Fig. 3, a suspension of HU strain (dry cell concentration of 10.86 g / β) was placed in a reactor 111 filled with sintered glass beads (diameter 7.6 to 10.5 mm) 112 as a carrier. Was placed anaerobically and aseptically, allowed to stand for 24 hours to allow the cells to adhere to the beads, and then the remaining liquid was gently drained from below. Then it causes please droplets of a solution as shown in the solution inlet 113 good Ri Table 2, the Moto賓gas H ₂ / C0 _2, and Kyo耠under downcomer from the gas inlet is 4, the bottom by Li biochemical reactions The resulting solution containing formic acid and the unreacted gas were recovered.

115 is a formic acid-containing solution outlet, 116 is a gas outlet, 117 is a temperature control water inlet at 32, 118 is a temperature control water outlet, and 119 is a jacket.

Table 2 Composition of solution

Phosphoric acid loose mouth liquid (0.1M) pH 8.0

Na HC0 ₃ 40 g / fi

_{N a S · 9H 2 0 0.1} "

Tr i t on X-l 00 2 "

Methyl viologen 7.5 mMol / fi Table 3 shows the fermentation conditions for formic acid. Under these conditions 32 to 2 weeks anti The results are shown in Table 3.

The formic acid was quantified by the method of Lang et al. (Lang E, Lang H., Z. Anal. Chem., 260, 8-10 (1972)), and the gas composition was gas chromatograph, gas chromatograph. The flow rate was determined by the stone membrane method.

Table 3

Reactor inner diameter 68mm

Sintered glass ball capacity 250ιηώ (apparent capacity) Length about 70mm

Inlet gas supply speed

C O, 1345.6 mil / day Solution supply rate 18.0 mfi / day Outlet gas discharge rate

H ₂ 6013.0 mfi / day CO: 1315.5 mfi / day Formic acid concentration in solution 104 mmol / £

From Table 3, the conversion of H ₂ to formic acid is

H ₂ content in formic acid

Conversion rate: X 100

Consumption 11 ₂ quantity

When asked by

(18.0 / 1000) (104/1000)

Conversion rate: X 100

(6056.0-6013.0) / (22400), and an extremely high conversion rate could be obtained. However, the amount of H ₂ dissolved in the solution was calculated because it was extremely small. Ignoring

Claims

/ The scope of the claims

1. For the production of substances by fermentation of gaseous substrates by microorganisms

In the meantime, the microorganisms immobilized on the carrier are held in the reaction vessel, and an aqueous solution is supplied into the reaction vessel so that at least a part of the surface of the microorganisms is moistened. A fermentation method characterized by directly reacting a microorganism with a gaseous substrate by passing the gaseous substrate through the above to biosynthesize the substance.

2. The fermentation method according to claim 1, wherein a gaseous substrate having low solubility in an aqueous solution is supplied to the microorganism.

3. A gaseous substrate containing one or more gases selected from hydrogen gas, carbon dioxide gas, carbon monoxide gas, oxygen gas, and nitrogen gas is supplied to the microorganism. 3. The fermentation method according to claim 2, wherein the request is:

4. The mixing ratio of hydrogen gas and carbon dioxide gas is 4: 1 or more

4. The fermentation method according to claim 3, wherein the mixed gas is supplied to the methane-producing bacteria fixed in the reaction vessel to biosynthesize methane gas.

5. Hydrogen gas, carbon dioxide gas, Trit on X-100 and methyl

5. The fermentation method according to claim 4, wherein an aqueous solution containing rubiologen is supplied to a methane-producing bacterium to biosynthesize formic acid.

6. Microorganisms immobilized on a carrier, gaseous substrate supply means, water-soluble

A fermentation apparatus comprising a liquid supply means, a biosynthetic substance collection means, and a reaction vessel, wherein the microorganism is installed in the reaction vessel. ί Ί

And a means for supplying a poorly gaseous group, a means for supplying an aqueous solution, and a means for collecting biosynthetic materials, respectively, which are connected to the inside of the reaction vessel.

7. The fermentation apparatus according to claim 6, wherein a means for adjusting the temperature in the reaction vessel is provided.