JPS62215395A - Fermentation process - Google Patents

Abstract

PURPOSE:A gaseous substrate is brought into direct contact with an immobilized microorganism to effect the direct synthesis of an objective compound in a practical scale. CONSTITUTION:The objective product is formed, as a gaseous substrate is directly fed to the immobilized microorganism. In order to carry out the process according the present invention, for example, an immobilized microorganism is placed in the reactor 1 and the liquid containing nutrients such as nitrogen source, minerals and others is jetted from the nozzle 4 by the operation of the valve 6 or dripped or allowed to flow down to the carrier containing the immobilized microorganism. And, as the nutrient solution is dropped, a substrate gas of an appropriate nutrient composition is fed from the pipe 5 through valve 6 into the reactor 1 to bring the nutrient solution into contact with the microorganism immobilized on the carrier whereby the objective product is obtained. The objective substance formed is collected through the gas outlet 7 in a gas tank 10. The suitable microorganism is a methane-producing bacterium utilizing carbon dioxide, hydrogen gas as substrates.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing a substance using microorganisms, and more specifically, a method for directly synthesizing a target substance from a gaseous raw material using immobilized microorganisms. Concerning a novel method.

Therefore, the present invention plays an important role in the technical field of biotechnology such as fermentation industry, microorganism industry, enzyme industry, and food industry.

Moreover, since methanol, hydrogen cyanide, acetylene, and various other organic industrial chemicals can be produced using the target product obtained by honzuke as a raw material, the present invention is also of great use in the technical field of the chemical industry.

[Conventional technology]

Conventionally, when producing a target substance using the fermentation method, no industrial system has been established in which a gaseous substrate is used as a raw material to directly act on immobilized microorganisms to directly produce the target substance. Such a system is merely a slightly modified version of the usual aerated agitation culture using a liquid medium.

That is, using a device as shown in FIG. 2, the microorganisms 12 involved are suspended in a liquid medium 11 containing supplementary nutrients such as a nitrogen source and inorganic salts, and the gaseous substrate is introduced from the outside 13 of the fermenter. In addition to forcibly supplying air into the liquid medium, mechanical stirring by stirring X14 (aeration stirring type fermenter 1)
5) Alternatively, the air bubbles 19 from the vent hole 18 are atomized by the draft tube 16 (bubble column fermenter 17),
By enlarging the gas-liquid interface and at the same time allowing air bubbles to stay in the culture solution for a long time, the rate of dissolution of the gaseous substrate into the culture solution is accelerated, and the desired generated gas is generated through biochemical reactions by the microorganisms involved. This system also does not biosynthesize the target substance directly from the raw material gas through a gas phase reaction, and as will be described later, it has disadvantages such as a low production rate of the target substance, so it is not suitable for industrial use. cannot be used on a large scale.

In this way, there is no known technology for directly supplying low-molecular raw material gas to microorganisms to directly biosynthesize target substances.
Especially when it comes to technology for biochemically synthesizing target substances using immobilized microorganisms.

At present, even the technical issue itself is not known.

[Problem that the invention seeks to solve]

The present invention was made for the purpose of developing an industrial system for directly producing a target product from a gaseous substrate, and firstly, the present invention was developed for the purpose of developing an industrial system for directly producing a desired product from a gaseous substrate. I paid attention.

However, this known system has the following specific drawbacks in achieving the above objective.

1. Aeration-stirring fermenters require a large amount of power for mechanical stirring.

2. Since there is a limit to the dissolution rate of the substrate gas, if the gas supply rate is increased beyond that, most of the substrate gas will reach the liquid surface without being utilized by the microorganisms, and the efficiency of contact with the microorganisms will be extremely low. Deteriorate.

3. Continuation is difficult because it is not possible to supply a substrate gas that matches the substrate consumption rate of microorganisms.

4. A large amount of liquid and a large liquid depth are required.

The reactor has to be large, and the entire device cannot be downsized.

Moreover, fatally, these known systems cannot directly produce the target substance from the raw material gas.

[Structure of the invention]

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

To achieve this goal, we conducted extensive research and found that in order to increase the yield of the target substance, it is necessary to increase the concentration of the raw material gas and increase the contact rate with the bacteria that produce the substance. Ta. If the raw material gas is dissolved in the culture solution or supplied in the form of bubbles, as in conventional systems, the gas concentration cannot be sufficiently increased. Therefore, we completely changed the way we thought about a system for increasing the raw material gas concentration, and as a result, we were able to directly supply 1M raw material gas in a gaseous state, bring it into contact with microorganisms, and carry out biosynthesis, something that had never been dreamed of before. This led to the idea of a new technical idea.

As a result of intensive research from various angles on ways to concretely and industrially realize this new idea, we discovered that it was possible to do so by using immobilized microorganisms.
As a result of further research based on this useful new knowledge, the present invention was completed.

The invention will now be described in detail with reference to the apparatus shown in FIG. 1, which is illustrated as an example of an apparatus for carrying out the invention.

The reactor 1 contains immobilized microorganisms.

Immobilization of microorganisms is carried out by conventional methods, and any carrier binding method can be used.

Microorganisms can be immobilized in a spherical, cylindrical, granular or other appropriate shape and then filled into the reactor 1, directly immobilized on the wall of the reactor, or hollow fibers with microorganisms immobilized on the inner and/or outer surfaces can be used. After filling a large number of reactors, filling one or more microorganism-immobilized (porous) plates vertically or horizontally into a reactor, or filling a small column with the molded and immobilized microorganisms described above, A reactor is constructed by filling a large number of these into the reactor.

As the microorganism, any microorganism can be used as long as it can produce the desired product using a gaseous substrate.

For example, methane-producing microorganisms of the type that utilize carbon dioxide gas, hydrogen gas, etc. as substrates can be advantageously used, but the present invention is not limited to these microorganisms. All microorganisms can be used as long as they can biosynthesize.

Specifically, the methane-producing bacteria include Durham-negative methane-producing bacteria HU strain isolated from digested sludge at the Hiroshima heavy sewage treatment plant (strain preserved in the Mizuben Laboratory, Faculty of Engineering, Hiroshima University, freely available);
Methanobacteriumthern+oa
utotrophicum ATCC29183゜M,
formicicum, M. omelia
nskii.

M, propionicum, M, sohnge
niii.

Methanobacterium such as M, 5uboxydans; Methanococcus mazei,
M. vanielii and uN.Methanococcus;
Methanosaricina barkerii
ATCC29894, M, methanica,
Bacteria of the genus Methanomonas such as Methanomona
s methylovora ATCC21963゜M
, methylovara 5ubsp, thia
Methanomonas bacteria such as Minophila ATCC21370 can be used alone or in combination. Furthermore, without isolating the bacteria in this way, it is also possible to directly fix a source of bacteria, such as a culture solution, wet cake, activated sludge, or digested sludge, and use it in the present invention.

One drop of liquid 2 containing a supplementary nutrient source such as a nitrogen source and inorganic salts is sprayed or flowed down from a jet pipe 4 by means of a control valve 3 onto a carrier on which microorganisms or a group of microorganisms are immobilized. If necessary, these nutrient solutions may be held in a carrier in advance. In addition, if the bacteria used require specific substances in addition to gaseous raw materials such as CO2, H2, and CO when synthesizing the target compound, these liquid or solid raw materials may be preliminarily added to the nutrient solution 2. The present invention can be applied to all types of microorganisms and is very advantageous, since the intended purpose is sufficiently achieved by adding the microorganisms.

Simultaneously with the dropping of the nutrient solution 2, a substrate gas having an appropriate composition is supplied from the reactor lower pipe 5 through the control valve 6,
The microorganisms immobilized on the carrier are brought into contact with the nutrient solution to produce the desired product. The type and composition of the substrate gas used as raw materials differ depending on the bacteria used, so it is necessary to select the optimal one according to the bacteria used6.For example, in the case of the methane-producing bacterium HU strain, hydrogen gas and carbon dioxide gas are used as raw materials. is used, and the H2/CO2 ratio is preferably greater than 1.

For this purpose, a gas analyzer (not shown) is installed at the lower produced gas outlet.
A control valve 6 provided at the substrate gas inlet is operated to adjust the mixing ratio of the substrate gas and/or its supply amount and supply speed to the optimum value for methane production. It is preferable to do so. The same is true for other microorganisms, and the control valve 6 is controlled according to the data from the gas analyzer to supply substrates commensurate with the substrate consumption rate of the microorganisms.

Reactor 1 is surrounded by a jacket for heating or heat retention, and temperature-controlled water and temperature-controlled gas are flowed into the jacket, and heating wires are installed to promote the biosynthesis reaction. Alternatively, contrary to the above, it is also possible to supply the substrate gas from above the reactor and take out the product gas from the bottom of the reactor. Moreover, the nutrient solution that has fallen into the liquid collection tank 8 is not disposed of as is, but is returned to the nutrient solution tank 2 from the liquid outlet 9 via a pump and a pipe (not shown) for circulation use. Economic efficiency will further increase. Furthermore, if necessary, by pressurizing the inside of the reactor, the solubility of the gas can be increased and the reaction rate can be increased. By keeping the reactor airtight and adjusting the oxygen content within the reactor to the optimum value for the immobilized microorganisms, the production rate of the desired product can be maximized.

The target substance thus produced passes through the produced gas output lower and is collected in the gas storage 10.

Examples 1 to 3 Zeolite, foam brick, and inorganic foam (particle size 7.1 to 12.6 mm) were used as carriers, and the preserved bacteria HU strain purified and isolated at Mizuben Laboratory, Faculty of Engineering, Hiroshima University, was used as a carrier. It was fixed by adsorption method.

The methane-producing bacteria fixed on the carrier in this way are
Fill the illustrated reactor (reactor capacity 75nQ),
Using the apparatus shown in FIG. 1, methane fermentation from a gaseous substrate was carried out under the following specifications.

That is, the nutrient solution 2 is dripped onto the surface of the carrier from the upper part of the actor 1 through the jet pipe 4 by means of the control valve 3, which is filled with a carrier having immobilized microorganisms;
Substrate gas was supplied at an appropriate flow rate through a control valve 6 from the lower pipe 5 of the reactor, and the gas produced by the microorganisms on the carrier was obtained from the upper outlet lower. A jacket was provided around the reactor and temperature-controlled water was passed through it to maintain the optimum temperature (37°C) for microbial reactions.

Actual reactor capacity: 75 mQ Fermentation temperature: 37°C Amount of immobilized bacterial cells: per reactor Example 1 Zeolite 0.675 g-dry c
ell Example 2 Foam brick 0.643g-dry
Cell nutrient solution feed rate: 25-3 per reactor
0raQ Daily substrate gas supply rate: 4760 m12/day Substrate gas composition (%): O281.5%, co218
．． 5%NaH2PO4”21120 3 I
I EDTA 1 g/j! 2)
IPO47II Fe2(PO4)24H201,
02MgC12・6H200,36# MnCl2
-4H200, lNa2S・91120
' 0.5 If CoCl2・61120
0.17trace +1etal 5olution
n 1) 9 ml/Q ZnCl2
0.1vita@in 5solution 2)
5 # CaCl2 0.02
H3BO30,O19 Na2M0O4”2H200,01 2) vitamin 5 solution biotin
ZffI&/Qpyridotin
e-Fl 10folic acid
2riboflavin 5 tMaaine 5 nicotinic acid 5Ca-pan
tothenate 5vita11irIB12
0.1 α-1ipoic acid 5p-amino
benzoic acid 5As a result, the following results were obtained.

Produced gas composition (%) 82C02CH.

Example 1 Zeolite 45.2 0 54.
8 Example 2 Foam brick 46.5 0.8 52.7 Example 3 Inorganic foam 43.5 0 56.5 As is clear from the above results, according to the present invention, H2
It is possible to directly biosynthesize methane gas from CO2 and CO2, which is the raw material substrate, from the produced gas.
Almost or only slightly detected, it can be seen that the target product, methane gas, can be obtained with high purity and very efficiently.

(Effects of the Invention) As detailed above, according to the present invention, not only the completely novel effect of being able to directly biosynthesize a target compound from a raw material substrate gas, but also the following outstanding advantages are achieved. Therefore, the present invention can be realized on a large scale as a large-scale industrial synthesis method, and is particularly excellent as an industrial method: 1. The gas-liquid contact area is large and the gas dissolution rate is increased. Therefore, no power is required for stirring the liquid or aerating the substrate gas, and no power costs are required for this purpose.

2. The efficiency of contact between substrate gas and microorganisms can be increased, and as a result, the size of the reactor can be reduced.

The flow of the substrate gas in the reactor follows the gas supply rate, so that the gas does not form bubbles and reach the liquid surface.

3. It becomes possible to supply a substrate gas commensurate with the substrate consumption rate of microorganisms, making it possible to achieve continuity.

4. Appropriate porosity exists in the reactor, so the generated gas can freely pass through the reactor.

Furthermore, according to the present invention, when the substrate gas is supplied from the bottom, the product gas rises together with the remaining substrate gas, but as it rises, the substrate gas is consumed and the product gas becomes rich. Conversely, if the substrate gas is supplied from the top, the produced gas will descend together with the remaining substrate gas, but as it descends, the substrate and gas will be consumed and the produced gas will become rich. Therefore, by collecting gas from a part in the middle of the reactor, it is possible to obtain a mixed gas containing not only the target product gas but also a predetermined amount of raw material gas as needed.
It is very advantageous.

[Brief explanation of drawings]

FIG. 1 illustrates an example of an apparatus for carrying out the present invention. FIG. 2 illustrates a conventionally used methane fermentation device. Agent Patent Attorney Door 1) Parent Male Figure 1 Figure 2 Figure 11, *Den Jl Determination 12, Hanyo Offer 1 Mono 13, Kapopusu Entrance 20.1 Wide R Suero Procedure Amendment Belt January 7, 1985

Claims (1)

[Claims] A fermentation method characterized by producing a target product while directly supplying a gaseous substrate to immobilized microorganisms.