JPH0449995B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to a method for aerobically cultivating microorganisms, and particularly to a method and apparatus for controlling the amount of substrate added in yeast culture.

[Background of the invention]

In the production of yeast cells such as food/feed yeast and baker's yeast, a fed-batch culture method is used in which a substrate is supplied little by little during culture. In order to carry out this culture efficiently, the fed substrate must be completely consumed by the yeast.
In addition, it is essential that by-products such as ethanol be small.

Conventionally, since there is no means to rapidly measure the concentration of bacterial cells or the activity of bacterial cells during culture, a method has been used in which substrates are added to a culture tank according to a predetermined program. In this method, it was not possible to feed the substrate in accordance with the bacterial activity in the tank, and the productivity was not high.

[Objective of the Invention] The present invention was made in view of the above-mentioned current situation, and its purpose is to improve the efficiency of yeast cell production by appropriately controlling the amount of substrate fed during culture. The present invention provides a method and apparatus for doing so.

[Summary of the invention]

The substrate fed-batch method for achieving the purpose of the present invention detects the production of by-products such as ethanol by increasing RQ, reduces or eliminates the amount of substrate fed, and then the generated by-products are transferred to yeast. This method is characterized by detecting the assimilation by the increase in dissolved oxygen concentration and increasing the amount of substrate fed.

Next, the method of the present invention will be explained in detail. To implement this control method, it is necessary to quickly know the production and consumption of ethanol, but at present there is no effective means to directly and quickly measure the ethanol concentration in the culture solution. Therefore, the present inventors detected the production of ethanol using the respiratory quotient RQ, and also discovered a phenomenon in which dissolved oxygen concentration rapidly increases due to consumption of ethanol by yeast, leading to the present invention.

In yeast culture using sugar as a substrate, RQ = 1.0 indicates good cell growth, and RQ > 1.0 indicates ethanol production because a large amount of carbon dioxide gas is produced. Therefore, if RQ > 1.0, it is assumed that ethanol has been produced, and the substrate feeding is reduced or stopped. On the other hand, if RQ<1.0, it may be due to insufficient substrate.
Either the yeast is assimilating the produced ethanol, and it cannot be determined whether the yeast has sufficiently consumed the ethanol. However, when the ethanol in the culture solution is sufficiently assimilated by the yeast with substrate feeding reduced or stopped, the amount of oxygen consumed by the yeast decreases, and the dissolved oxygen concentration rapidly increases. By detecting this rapid rise in dissolved oxygen concentration, it is possible to infer sufficient consumption of ethanol.
This increases the amount of substrate fed.

An experimental example is shown in FIG. 2.5 hours after the start of culture,
The respiratory quotient RQ exceeded 1.0, and ethanol production was detected. At this time, the ethanol concentration reached 9 g/or more, and the yield relative to sugar tended to decrease. Therefore, starting from the third hour, the substrate flow acceleration per bacterial cell amount (referred to as the α value) was set to zero, and the substrate feeding was stopped, and the produced ethanol was assimilated by the yeast. By the 5th hour after the start of culture, approximately 4 to 6
Dissolved oxygen (DO) controlled in the range of mg/
Substrate feeding was resumed as the concentration rose rapidly and a decrease due to ethanol assimilation was detected. During the suspension time of quality fed batch, the sugar yield tends to increase,
The maximum value reached 0.53 when substrate feeding was restarted.

Substrates used in the present invention include glucose, fructose, sucrose, and industrial raw material molasses. In addition, ammonium sulfate, urea, aqueous ammonia, monopotassium phosphate, yeast extract, magnesium sulfate, monosulfuric acid sulfate, which are used in normal culture as auxiliary raw materials.
It contains iron and various vitamins and minerals.

An example of a culture apparatus for the method of the present invention is shown in FIG. Inoculum is placed in a culture tank 1, and a substrate is supplied from a substrate tank 7 by a substrate supply pump 8. At this time, an inlet oxygen partial pressure measuring device 5, an inlet gas amount measuring device 6, an outlet gas oxygen partial pressure measuring device 11, a carbon dioxide gas partial pressure measuring device 1
2. The data from the exhaust gas amount measuring device 13 is input into the control electronic computer 4 to calculate the respiratory quotient RQ, from which the α value is changed to control the substrate flow acceleration, and the substrate supply pump 8 is activated according to the speed. put it into operation. When the production of by-products is detected and the substrate feeding amount is very small or zero, the signal from the dissolved oxygen sensor 9 is detected by the dissolved oxygen meter 10 and sent to the electronic computer 4, and the α value is determined from the change. The substrate flow acceleration is controlled by changing the Also, during cultivation, it is necessary to maintain the oxygen level at 2 mg/min or more so that the bacterial cells are sufficiently supplied with oxygen. By controlling the inlet gas oxygen concentration and the inlet gas amount using the oxygen separator 3, the dissolved oxygen concentration is maintained at a constant value.

[Embodiments of the invention]

Hereinafter, one embodiment of the present invention will be described in detail, but the present invention is not limited thereto.

Bacterial body: Saccharomyces cerevisiae (baker's yeast) Medium: glucose 500g, urea 53.75g,
_{Na2HPO4 ・ 2H2O25g} , _MgSO4・_7H2O9.5g ,
KC5.5g, sodium citrate 62.5g, yeast extract 12.2g, vitamin liquid 25ml and mineral liquid 25ml
was added to tap water 1, dissolved, and adjusted to pH 5.0.

However, the vitamin liquid contains 0.04g of biotin and vitamin
B ₁ 0.08g, vitamin B ₆ 2.0g, calcium pantothenate 1.0g and inositol 20g in distilled water 1
Mineral liquid is prepared by dissolving _CuSO4 .
_5H2O0.05 , _ZnSO4・_7H2O0.8g and _FeSO4
It was prepared by dissolving 0.3 g of (NH ₄ ) ₂ ·6H ₂ O in 1 part of distilled water.

Culture conditions: Using a 50° fermenter, temperature: 30°C, pH: 5.0, dissolved oxygen concentration: 4 to 4 depending on stirrer rotation speed, oxygen partial pressure of aeration gas, and aeration amount
The amount was controlled within the range of 6 mg/. Note that the oxygen partial pressure of the ventilation gas was changed using an air compressor and an oxygen cylinder. The tank internal pressure was controlled at 0.5 Kg/cm ₂ G, and the exhaust gas carbon dioxide concentration was controlled within 20%. The initial liquid volume is
15, the initial bacterial cell concentration was 50 g/h, and the substrate flow acceleration per bacterial cell was 0.3 g/g·h.

Result: At the end of 6 hours of culture, ethanol concentration was 0.
g/, and the final sugar yield was 0.44. At this time, 99.8% of the fed glucose was consumed, and the bacterial cell concentration reached 66.3 g/g/.

〔Effect of the invention〕

The present invention enables substrate fed-batch control that ultimately reduces by-products, which has the effect of achieving bacterial cell culture with an improved final sugar yield, and improves the productivity of the culture tank. .

[Brief explanation of the drawing]

FIG. 1 is a diagram showing an experimental example of the present invention, and FIG. 2 is a schematic diagram of an example of a culture apparatus of the present invention. 1... Culture tank, 2... Stirrer, 3... Oxygen separator, 4... Control electronic computer, 5... Oxygen partial pressure measuring device, 6... Inlet gas amount measuring device, 7... Substrate tank , 8... Substrate supply pump, 9... Dissolved oxygen sensor, 10... Dissolved oxygen meter, 11... Oxygen partial pressure measuring device, 12... Carbon dioxide gas partial pressure measuring device, 13... Exhaust gas amount measuring device, 14 , 15, 16, 17... Conduit.

Claims (1)

[Claims] 1. During aerobic cultivation of yeast, when by-products are produced when feeding substrates into a culture tank, the amount of substrate feeding is reduced, and by-products are assimilated by yeast. If it decreases, the amount of substrate fed is increased.In the culture method, if the respiratory quotient exceeds a predetermined value, it is determined that byproducts are excessively produced, and the amount of substrate fed is lowered. 1. A substrate fed-batch control method, characterized in that when the dissolved oxygen concentration increases to a predetermined value or more under the conditions, it is determined that the generated by-products have been sufficiently utilized, and the amount of substrate fed is increased. 2. A yeast culture tank placed in an aerobic environment, a device for supplying gas to the culture tank, a device for discharging gas from the culture tank, a device for calculating the respiratory quotient of yeast from each gas, and the above. It is equipped with a device that detects the dissolved oxygen concentration of the culture solution in the culture tank, and a device that supplies substrate to the culture tank, and when the respiratory quotient exceeds a predetermined value, it is determined that byproducts have been excessively produced, and the substrate flow is stopped. When the dissolved oxygen concentration increases to a predetermined value or more with the amount of substrate added and the amount of substrate fed reduced, it is determined that the generated by-products have been sufficiently utilized, and the amount of substrate fed is increased. Substrate fed-batch control device.