JPS63123373A - Cultivation apparatus for gaseous substrate - Google Patents

Abstract

PURPOSE:To obtain a cultivation apparatus for a gaseous substrate, containing a membrane filter having attached and immobilized microorganisms in the interior of a container having a hermetically sealable planar bottom, supply and discharge ports for a gas as well as supply port for a liquid and capable of accurately controlling cultivation. CONSTITUTION:A cultivation apparatus obtained by providing a flange 32 joining to a cover (B) at the top end part of a bottomed cylindrical body 31 having a horizontal bottom to form the body (A) of the apparatus, providing a discharge port 33 for a liquid at the end of the cylindrical body 31, supply port 37 for a gas, gas supply port 36 and gas discharged port 38 at the cover (B) and containing a membrane filter having attached and immobilized microorganisms in the bottom of the body (A).

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a container for culturing microorganisms, and more particularly to a container for culturing microorganisms using a gaseous substrate.

(Prior Art) As a means for culturing microorganisms that proliferate using a gaseous substrate, there is a solid-gas reaction type, that is, a method in which a gaseous substrate is directly or closely supplied to the microorganisms. Therefore, if you want to find the consumption rate of substrate gas or the production rate of product gas per area, you first need to calculate the area accurately and keep the amount of microorganisms constant. Also, in order to keep the pressure constant, you need to Needs to be supplied continuously. However, since microorganisms proliferate, it is not possible to accurately determine the area occupied by microorganisms, and it is also difficult to determine the amount of microorganisms.

In this way, conventionally, when culturing microorganisms using a gaseous substrate rather than a liquid or solid substrate, the cultivation was precisely controlled by supplying gas continuously or intermittently while taking out the produced gas. It was not possible to do so.

(Problems to be Solved by the Invention) As described above, the present invention was made for the purpose of developing a completely new culture vessel that enables gaseous substrate culture, which has not been possible in the past.

Another object of the present invention is to develop a culture vessel that can supply gas continuously or intermittently and sample the generated gas for analysis in the cultivation of microorganisms, particularly microorganisms that utilize gaseous substrates. It was made in

(Means for Solving the Problems) The present invention was made for the purpose of developing a novel culture vessel as described above.

For this purpose, various aspects were considered, but unlike liquid substrates, gaseous substrates have many problems unique to gases, such as contact with microorganisms, accurate usage amount, and volatilization into the air. I couldn't get it.

Therefore, we changed our thinking and came up with a new idea of organic bonding between immobilized microorganisms and a sealed container.As a result of further research, we succeeded in developing a new microbial membrane, and now the present invention has been completed. It is.

First, the microorganisms used are treated as follows to form a microbial film. That is, the culture solution obtained by culturing microorganisms is concentrated or directly filtered through a membrane filter without being concentrated, and the microorganisms are attached and immobilized on the filter, thereby producing a microbial film.

This microbial membrane is new, and while it firmly fixes the microbial cells of a certain density to the carrier with a certain thickness, it does not reduce the activity of the microorganisms in the slightest, making it extremely excellent. The film forming method will be described in detail below.

To do this, it is first necessary to culture the microorganisms and multiply the microbial cells, which can be done as appropriate using a stirring-type culture incubator or other conventional methods suitable for the growth of microorganisms. The culture fluid obtained can be directly subjected to the membrane treatment described below, but concentrating it to reduce the volume is very advantageous in terms of work efficiency, work cost, processing time, etc. be.

Examples of concentration methods include removing the supernatant by decantation after allowing the product to stand still, centrifugation, vacuum concentration, etc., but any other concentration method that does not harm microorganisms may be used. All can be used as appropriate.

After concentrating the culture solution in this manner, or without concentrating it, the culture solution is filtered through a membrane filter.

Examples of membrane filters include collodion membranes, formalin-hardened gelatin membranes, silicic acid membranes, cellophane membranes, and nitrocellulose type membranes, and commercially available membrane filters can also be used freely.

By holding the membrane filter on a flat support stand,
When the culture solution is filtered, a microbial film is formed on the membrane filter. In addition, at this time, an ultrafilter or a membrane filter is used, and instead of filtering under normal pressure as described above,
It is also possible to filter under reduced or increased pressure.

This filtration process can be performed using, for example, the apparatus shown in FIG. A membrane filter 2 is set below a glass filter holder 1, and connected to a suction bottle 3 using a rubber stopper. When the microorganism culture solution 4 is placed in the glass filter holder 1 and sucked, the microorganisms are attached and immobilized on the membrane filter, forming a microorganism film. Although suction is used in this example, it is also possible to filter by applying constant pressure from above the glass filter.

In addition to storing the culture solution of microorganisms in the glass filter holder 1, separately cultured microorganisms are suspended in water, buffer, culture solution, etc., and this suspension is placed in the glass filter holder 1. It may also be filtered.

In this way, various microorganisms can be uniformly adhered and fixed to the filter paper by extremely simple operations, and an extremely excellent microbial film with constant density and film thickness can be obtained. There are no particular restrictions on the microorganisms that can form films in this manner, and all microorganisms can form films, but methanogenic bacteria such as methane-producing bacteria can be advantageously used. : Methanogen HU strain (Hiroshima University Faculty of Engineering archived strain), Met
hanobactarium thermoautot
rophicum.

M. formicicum: Mstanoco
ccus vannielii; etc.

The culture container according to the present invention houses the microbial membrane prepared in this manner, and an example thereof will be described below with reference to FIG. 2.

The culture container consists of a container body A and a cover B. Both are airtightly joined to prevent gas from leaking. These materials are glass.

Transparent plastics and other commonly used culture containers can be used as appropriate. When glass is used, it can be autoclaved, so it is convenient for repeated use, and when plastic is used, it is suitable for use as a disposable type.

The main body A consists of a cylindrical body 31 with a horizontal bottom, and a donut-shaped flange 32 is provided on the upper edge thereof so as to fit with the cover B.

If necessary, a liquid outlet 3 is provided on the bottom side of the cylindrical body 31.
3, which is provided with suitable means 1 such as a pinch cock 3.
It can be opened and closed freely using the 4th grade.

Since the microbial membrane 35 is arranged on the bottom surface of the cylindrical body 31, the size of the bottom surface is preferably made slightly thicker than that of the microbial membrane. The liquid discharge port 33 may be provided on the side of the cylindrical body as shown in Figure 1, or may be opened at an appropriate position on the bottom surface of the pond at the center.゛Cover the entire body A with cover B. A and B are
After rubbing, screwing, and inserting an O-ring, etc.,
If necessary, it is further tightened with a clip or clamp to maintain airtightness, but other airtightness maintaining means can also be used as appropriate. Cover B has one through hole 36, 37, and 38 each.
Or drill more holes and use it as a gas supply port, liquid supply port, or gas discharge port. A lid may be attached to these holes to maintain airtightness, or a variety of rubber plugs may be attached to these holes, and gas or liquid may be supplied or discharged using a syringe needle. .

Above, the container of the present invention was described as being circular.
Since the size and shape of the microbial membrane can be changed freely, the culture container can also be freely designed accordingly.

In the culture container shown in FIG. 2, the bottom surface of the main body A is a flat surface as shown in FIG. 2-2, but it can also be made into a bottom surface as shown in FIG. 3.

That is, one or more grooves 51 are carved in the flat bottom surface 50 of the container body A (for example, width 1 to 3 cm and depth 1 cm), and a liquid containing nutrient salts etc. is poured into the grooves 51. The supply is mounted on it. Nutrient salts can also be supplied to the microbial film adhered and immobilized on the filter by osmosis or other actions. In this case, it is more convenient to provide the liquid supply port 37 in the bottom portion 50 without providing it in the cover B.

The groove 51 may be carved up to the inner wall surface of the container A;
As shown in Figure 1, some space may be left. The cross-sectional shape of the groove 51 may be triangular as shown in FIG. 3-2, or may be semicircular, quadrangular, or any other suitable shape. Further, the planar shape of the groove may be not only a straight line but also a circular shape, a spiral shape, and other shapes.

When the culture container according to the present invention is actually used, it is necessary to control the temperature, and therefore it must be used in a constant temperature room or a constant temperature bath.However, as shown in FIG. If a jacket C is provided integrally or removably around the jacket, and temperature-controlled water, air, or other liquid is allowed to flow through it, or if a heater and/or cooler is installed, it can be used not only under constant temperature conditions but also under normal atmosphere. Culture can be performed freely and is very convenient.

When using this culture device, after attaching the microbial membrane 35 and sealing it with cover B, with or without adding a culture solution, the gas supply port 35 is closed while controlling the temperature.
A gaseous substrate is supplied from 6 to react with microorganisms, and the generated gas is taken out from the gas outlet 38 and used as a target product as it is, or it can be sampled, measured and analyzed, and used for various microbiological studies. It is used for research.

Since the subject of the invention is the use of a gaseous substrate, the container must be kept tightly closed, in fundamental contrast to conventional culture vessels.

Not only when using a gaseous substrate to produce a target gas or other target product, but also when using a gaseous substrate to measure the consumption rate of the gas or the generation rate of the gas produced.

It is necessary to determine the flow rate and composition of gas entering and exiting this container. In other words, it is impossible to measure the flow rate if gas leaks from the container, and conversely, if outside air etc. enters the container, the reaction conditions will change, so it must be sealed tightly.

Below, an actual example of the method for forming a microbial membrane used in the present invention and an application example of advantageously fermenting and producing methane gas using a culture vessel incorporating the method will be described.

(Example of microbial film formation) A Durham-negative methane-producing bacterium HU strain isolated from digested sludge at the Hiroshima heavy sewage treatment plant (strain kept in Mizuben Laboratory, Faculty of Engineering, Hiroshima University, freely available) was cultured in advance and suspended. A liquid (bacteria cell concentration 0.250 g/Q) was obtained.

Take a certain amount (0.5~1Q) of this culture solution and use a Toyo Roshi membrane filter (pore size 0.45 μm, size diameter 90+S layer, nitrocellulose type) and a glass filter holder (filtration area 38.5 cm). 1st
Using the apparatus shown in the figure, suction filtration was performed to form a microbial film on the filter.

As a result of measuring the amount of microorganisms per unit area on the filtration surface thus obtained, the results shown in the following table were obtained. The amount of bacterial cells was constant in all parts of the filter surface.

Table Application Example The microbial membrane prepared in the previous example was mounted on the bottom of a flat culture vessel as shown in FIG. This culture vessel is used as a reactor, and a raw material gas having the following composition is supplied, and a nutrient solution having the following composition is supplied from the liquid supply port to carry out methane fermentation under the following conditions, and the produced methane is discharged from the gas. I took it out from the exit.

Fermentation temperature: 37℃ Nutrient solution supply rate: 0.2mQ/hr per reactor
Substrate gas composition and gas supply rate 2 40.12 18.20 41.68 1
274.63 20.22 1g, 69 61.
09 1225. INaH,PO,・2H,03#
EDTA 1 1Jn,) IP
o, 7 # Fe, (PO
4), -11ul, 0 1.02MgCl, -island 0
0.361 MnC1z"4H-0'0-I
Na, S-9H20o, s II CoC1,・6
H200,17NaHC0,3//ZnCl20
．． 1trace metal 5solution 1)
9 ml/Q CaC1,0,02vital
ILn 5solution 2) 5
7F) et BO30,019Nae o04-2
H,00,01 2) Vitamin 5 solution biotin
2 y@/Qpyridoxine
-HCl 10folic acid
2 riboflavin 5 thialline5 nicotinic acid 5Ca-pan
tothenate 5vitamine B,,
0.1α−1 ipoic acid
5p-aminobenzoic acid 5 As a result, the following results were obtained.

Composition of produced gas and gas production rate 1 629.480.311? , 69 0 2.00
12.621243.140.1218.204
2.740.61 7.63 1206.419.
0018.5962.040.37 4.5 As is clear from the above results, according to the present invention, methane gas can be directly biosynthesized from H2 and CO2, and in particular, as is clear from the application examples, The methane production rate changes depending on the H2 concentration (partial pressure) in the substrate gas. In other words, if the H2 concentration (partial pressure) decreases, the methane production rate also decreases. Therefore, by increasing the H2 concentration (partial pressure),
It can increase the rate of methane production.

(Effects of the Invention) As described above, the present invention provides a novel microbial membrane obtained by adhering and immobilizing microorganisms on a membrane filter, and a culture vessel having a unique configuration particularly suitable for using a gaseous substrate. , and by adopting such a novel structure, the present invention is capable of cultivating microorganisms, fermentative production of specific substances, and other methods using gaseous substrates. In addition to the remarkable effect that biochemical conversion of substances into other substances can be carried out very advantageously, it also has the following remarkable effects.

(1) In order to use a membrane filter, whether or not a culture solution with a constant bacterial cell concentration is used, by changing the amount of culture solution to be filtered, only the thickness and area of the membrane can be adjusted. The density of microorganisms can also be controlled.

(2) Since the area and shape of the membrane filter can be changed freely, any type of microbial membrane can be created1, and therefore, it can be freely manufactured into various types of culture vessels accordingly. .

(3) As long as you have a culture solution or suspension, you can easily prepare a microbial membrane anytime and anywhere in a very short time, and as soon as you replace this membrane, you can set up a culture container, which is extremely simple. The culture container is assembled.

(4) Since the structure of the container is simple and dead space can be eliminated, operations such as cleaning are easy and the container can be used repeatedly without the risk of contamination.

(5) By using a sealed structure and a microbial membrane, the pressure inside the container can be kept constant, and sampling can be performed while continuously supplying gas.

The gas consumption rate of the microbial film can be easily determined.

(6) Since a certain concentration of microorganisms is immobilized by microbial membranes, it is not only convenient for studying microbial metabolism, physiology, etc., but also serves as a basis for analyzing and researching various biological reactions using microorganisms. Very good.

(7) The present invention is excellent in the field of basic research on microorganisms, such as the immobilization of microorganisms themselves, as well as the analysis and measurement of substances using microorganisms such as bioassays.

[Brief explanation of the drawing]

FIG. 1 illustrates an example of an apparatus for forming a microbial film used in the present invention. FIG. 2 illustrates an example of the culture container according to the present invention, with FIG. 2-1 being a plan view and FIG. 2-2 being a sectional view. FIG. 3 shows another embodiment of the bottomless culture container, in which FIG. 3-1 is a plan view and FIG. 3-2 is an enlarged sectional view. FIG. 4 is a diagram illustrating another embodiment of the culture container. Agent Patent attorney Door 1) Parent Male No. 1 Figure 2-1 Figure 2-2 η臥′Xπμ＾ Figure 3-1 Figure 3-2

Claims (1)

[Claims] It consists of a sealable container with a flat bottom that houses a membrane filter on which microorganisms are attached and immobilized, and one or more gas supply ports, gas discharge ports, and liquid supply ports are penetrated through the container. 1. A culture container for microorganisms using a gaseous substrate, characterized by having a perforation.