JPS6352876B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for aerobically cultivating microorganisms, and particularly to a method for culturing microorganisms at high concentrations.

Microbial protein (Single Cell Protein)
Development is underway in various regions, and some have already reached the stage of practical use.

These methods involve inoculating pre-cultured inoculum into a sterilized culture solution at a rate of about 1% of the culture solution, but the bacterial cell concentration after culture is at most 20g.
drycell/. On the other hand, a method (described in Japanese Patent Publication No. 51-9833) has been developed in which oxygen-enriched gas is blown into the culture tank in order to increase the bacterial cell concentration during culture, but this method also increases the final bacterial cell concentration. is 20.5~23.1
The concentration is only about % by volume, and it has not yet reached a level where the concentration can be sufficiently increased.

As described above, when the concentration of bacterial cells in the culture solution is low, a step of separating the bacterial cells from the culture solution is required after completion of the culture, which complicates the process. In addition, since the bacterial cell concentration is low at the initial stage of culture, bacterial contamination sometimes occurs, causing a decrease in production in the culture tank. Furthermore, there is a problem in that the amount of culture waste fluid discharged is extremely large due to the low bacterial cell concentration.

The present invention was made in view of the above situation, and
The purpose is to provide a method that enables culture at a high bacterial cell concentration.

In order to achieve the purpose of the present invention, the high bacterial cell concentration culturing method of the present invention is carried out by washing the bacterial cells after the completion of culture with water or the like, or using a part of the bacterial cells after washing to reduce the moisture content by using a press filter or the like. The method is characterized in that a part of the bacterial cells concentrated to about 60 to 70% is returned to the culture tank as seed bacteria, and culture is carried out at a high concentration of initial bacterial cells.

In the present invention, a part of the bacterial cells is returned to the culture tank,
The reason for culturing at a high concentration of initial bacterial cells is as follows.

In microbial culture, if more bacteria can utilize a given amount of substrate, the productivity of the bacteria will improve. That is, if the initial bacterial cell volume is the same, by increasing the initial bacterial cell concentration and supplying a substrate in an amount commensurate with the bacterial cell amount, the bacterial cell growth amount per unit time can be increased. However, if the initial bacterial cell concentration is increased, the amount of oxygen respiration by the bacterial cells increases accordingly, but conventionally, air is used to aerate the culture tank, so a sufficient amount of oxygen cannot be supplied. From now on, in order to supply an amount of oxygen that can maintain a high bacterial cell concentration, pure oxygen or oxygen-enriched gas with an oxygen concentration of 21% or more must be aerated. In this way, by aerating oxygen-enriched gas or pure oxygen, the concentration of initial bacterial cells can be increased. In general, the final bacterial cell concentration is 20 to 50 g drycell/
Based on the above, in order to take advantage of the benefits of oxygen-enriched gas aeration, the initial bacterial mass must be 20g.
It is desirable to culture as a dry cell or more.
Naturally, the higher the initial bacterial concentration, the higher the bacterial productivity. It is necessary to decide based on concentration, etc.

For supplying oxygen-enriched gas or pure oxygen, an oxygen cylinder, liquid oxygen, a cryogenic air separation device, or an air separation device using molecular adsorption is used.

In order to increase the concentration of initial bacterial cells, a portion of the washed bacterial cells after cultivation is returned to the culture tank and used as seed bacteria for the following reasons. Since the bacterial cells are used as they are after cultivation, it is always possible to cultivate bacterial cells with constant properties, and it is also possible to cultivate bacteria in a highly active state, which prevents bacterial mutation and contamination with undesirable bacteria. It has the advantage of having less At this time, a part of the cultured at a high concentration can be used as a starter, but metabolites of the microorganisms are accumulated in the culture solution, which has a negative effect on the growth of the microorganisms. Therefore, in the washing process, a part of the bacterial cells is used after removing these metabolic substances with water.
It is also possible to use a part of the bacterial cells concentrated in the next concentration step.

On the other hand, JP-A-54-32687, JP-A-53-
Publication No. 118584, Japanese Patent Publication No. 53-47583, Japanese Patent Publication No. Sho
No. 53-29985, etc., disclose a method in which a part of the culture solution is extracted during microbial culture, bacterial cells are recovered from the culture solution containing growth-inhibiting substances by centrifugation, and all of the recovered bacterial cells are returned to the culture tank. I'm going.
This prevents growth-inhibiting substances from accumulating in the culture tank liquid, making it possible to achieve high bacterial cell concentration culture. This method has the advantage of being able to culture from a low initial bacterial cell concentration to a high bacterial cell concentration, but the bacterial cells are diluted in order to return the concentrated bacterial cells to the culture tank by centrifugation. Since this means that concentration and dilution are performed continuously or continuously, energy loss is large. Furthermore, since the operation of repeatedly withdrawing a portion of the culture solution and returning it to the tank during culturing is repeated, there is a high risk of bacterial contamination.

In the present invention, a part of the washed bacterial cells after the completion of culture is used as a seed culture to increase the concentration of initial bacterial cells, so it is possible to culture with low energy loss and high bacterial cell productivity. Become.

When culturing is performed at a high initial bacterial cell concentration according to the present invention, the final bacterial cell concentration will be 100 g drycell/or more. At such a high concentration, it is possible to omit the bacterial cell separation step that is essential for normal fermentation processes.
As a result, the microbial cells after completion of culture are immediately sent to the microbial cell washing step. Centrifugation is usually performed in the bacterial cell separation process, and being able to save on the cost required for this is a huge advantage.

When culturing microbial cells, when the growth of microorganisms is inhibited by a highly concentrated substrate, such as when methanol or ethanol is used as a substrate, or when the amount of alcohol produced is suppressed, such as when cultivating baker's yeast using molasses. For example, it is necessary to perform fed-batch culture in which substrates are gradually added during culture. However, in other cases, a method is used in which the entire culture medium is charged into a culture tank in advance and culture is started.

The cleaning process for bacterial cells is carried out using undiluted solution 2 as in the usual method.
This is carried out once or twice by adding ~ several times as much washing water, and a separating plate type continuous centrifuge is mainly used for this purpose. This makes it possible to remove growth-inhibiting substances, which are metabolites of microorganisms, and to improve the quality of the product.

Microorganisms used in the present invention include the genus Saccharomyces, the genus Hansenula, and Torulopsis.
Genus Pichia, Candide and Mycotorula
Yeast belonging to the genera Methylomonas, Pseudomonas, Alcaligenes, Bacillus, and Corynebacterium, Nocardia
Genus and Streptomyces
Actinomycetes belonging to the genus Penicillium, molds belonging to the genus Aspergillus, and Trichoderma are used.

As culture substrates, carbohydrates such as molasses, n-paraffin, methanol, ethanol, acetic acid, fatty acids, etc. are used. Ammonium sulfate, urea, aqueous ammonia, potassium phosphate, yeast extract, magnesium sulfate, ferrous sulfate, and various vitamins and minerals used in normal culture are used as auxiliary materials other than the substrate.

The figure shows a schematic diagram of the process for carrying out the method of the invention. First, a culture medium is supplied to the culture tank 1 from the culture medium supply pipe 3, a seed culture is supplied from the seed culture return pipe 10 or 14, and while the stirrer 2 is rotated, an oxygen-enriched gas is supplied from the oxygen-enriched gas supply pipe 4. Culture the bacterial cells. After the cultivation is completed, the highly concentrated cultured cells are sent to a centrifugal separator 6 through a conduit 5, and centrifuged while the cells are washed with washing water from a washing water supply pipe 7. Some of the washed bacterial cells are returned to the culture tank 1, while other bacterial cells are sent to the filter press 11 through the conduit 9 and concentrated. A part of the concentrated microbial cells is returned to the culture tank 1, while other concentrated microbial cells become product microorganisms as they are, or are sent to the dryer 16 and dried and then become product microorganisms.

Next, examples of the present invention will be specifically described, but the present invention is not limited thereto.

Example 1 Bacterial cells: Saccharomyces cerevisiae (baker's yeast) Medium: Glyose was adjusted to a concentration of 30%, and urea, monosodium phosphate, magnesium sulfate, sodium citrate, yeast extract, and vitamin liquid were added and dissolved in tap water.

Culture conditions: Using a 1-volume mini-jar fermenter, the above medium was fed at a temperature of 30°C and a pH of 5.0, and the oxygen concentration in the aeration gas and the rotation of the stirrer were adjusted to maintain the dissolved oxygen concentration at 2 to 5 mg/. changed the number. The initial volume of culture solution was 350 ml, and the initial concentration was 50 g drycell/inoculation using washed bacterial cells.

Result: After 12 hours of culture time, the culture solution volume was 750ml.
The bacterial cell concentration reached a high concentration of 102 g drycell/. The yield based on sugar at this time was 49%.

Therefore, when the cells obtained here were washed and cultured again under the above conditions, almost the same results were obtained, and the cell concentration reached a high concentration of 105 g drycell/.

Example 2 Bacterial strain: Saccharomyces cerevisiae (baker's yeast) Medium: Blackstrap molasses was prepared into a 40% sugar solution, and urea,
Sodium phosphate was dissolved.

Culture conditions: Using a 1-volume mini-jar fermenter, the above medium was fed at a temperature of 30°C and a pH of 5.0, and the oxygen concentration in the aeration gas and the rotation of the stirrer were adjusted to maintain the dissolved oxygen concentration at 2 to 5 mg/. changed the number. The initial volume of culture solution was 350 ml, and washed bacterial cells were used as the inoculum, and the initial concentration was 25 g drycell/.

Result: After 15 hours of culture time, the culture solution volume was 750ml.
The bacterial cell concentration reached a high concentration of 110 g drycell/.

Example 3 Bacterial strain: Saccharomyces cerevisiae (baker's yeast) Medium: Blackstrap molasses was prepared into a 40% sugar solution, and urea,
Sodium phosphate was dissolved.

Culture conditions: Using a 1-volume mini-jar fermenter, the above medium was fed at a temperature of 30°C and a pH of 5.0, and the oxygen concentration in the aeration gas and the rotation of the stirrer were adjusted to maintain the dissolved oxygen concentration at 2 to 5 mg/. changed the number. The initial culture volume was 350 ml, and the initial concentration was 70 g drycell/inoculation using washed bacterial cells.

Result: After 15 hours of culture time, the culture solution volume was 750ml.
The bacterial cell concentration reached a high concentration of 131 g drycell/. The yield based on sugar at this time was 46%.

Therefore, when the bacterial cells obtained here were washed and cultured again under the above conditions, almost the same results were obtained, and the bacterial cell concentration reached a high concentration of 130 g dry cell/.

Example 4 Bacterial strain: One strain belonging to Hansenula Medium: Ethanol as substrate, ammonium sulfate, monopotassium phosphate, disodium phosphate as auxiliary raw materials,
Magnesium sulfate, ferrous sulfate, and thiamine were used.

Culture conditions: Using a 1-volume mini-jar fermenter, ethanol was fed at a temperature of 35°C and a pH of 3.5, and the oxygen concentration in the aeration gas and the rotation speed of the stirrer were adjusted to maintain the dissolved oxygen concentration at 2 to 5 mg/. changed. The volume of culture solution was 500 ml, and the initial concentration was 50 g drycell/inoculation using washed bacterial cells.

Result: Bacterial cell concentration was 110g after 15 hours of culture time.
A high concentration of drycell/ was reached. The yield at this time was 70%.

Therefore, when the cells obtained here were washed and cultured again under the above conditions, almost the same results were obtained, and the cell concentration reached a high concentration of 106 g drycell/.

As described above, the present invention enables high bacterial cell concentration culture, so the bacterial cell isolation step can be omitted.
This has the effect of preventing bacterial contamination, improving the productivity of the culture tank, and reducing the amount of waste water.

[Brief explanation of drawings]

The figure is a schematic diagram of a culture process that is an example of the method of the present invention. 1... Culture tank, 6... Centrifugal separator, 11... Filter press, 10, 14... Inoculum return tube.

Claims (1)

[Claims] 1. A step of aerobically cultivating microorganisms using pure oxygen or oxygen-enriched gas, a step of extracting cultured microbial fluid and washing the microbial cells after completion of the culture, and concentrating the washed microbial cells. A method for culturing microorganisms at a high bacterial cell concentration, the method comprising a step of returning a part of the bacterial cells obtained from the washing step of the bacterial cells as seed bacteria for the culturing step. 2. The method for culturing a microorganism at a high bacterial cell concentration according to claim 1, wherein in the microorganism culturing step, the initial bacterial cell concentration is 20 g dry cell/or more.