JPS6175256A - Fungus body density measuring apparatus - Google Patents

Abstract

PURPOSE:To elevate the response, by a method wherein the intensity of a transmission pulse is changed measuring the intensity of an ultrasonic signal received to obtain a fixed intensity of the received signal so that a mean processing can be done in a short time even at a shorter transmission pulse generation cycle. CONSTITUTION:A signal processing section of this measuring apparatus is made up of an ultrasonic wave transmitting/receiving element 300, an ultrasonic wave trans mission/reception controller 310, a waveform averaging processor 320, a propagation arithmetic unit 330, a temperature arithmetic unit 360, a fungus body arithmetic unit 370 and the like. The intensity of a transmission pulse is adjusted with the controller 310 so that the ultrasonic wave reaches a specified level when it transmitted from the transmitting/receiving element 300, reflected on the outer wall 240 propagating through a culture fluid 110 and received with the transmitting/receiving element 300 and then, the signal is fed to a processor 20 to under-go an averaging processing. Then, the ultrasonic wave propagation time is computed by the signal and based on the temperature obtained from the temperature arithmetic unit 360, the density of fungus is computed with the fungus density arithmetic unit 370. Thus, the response can be elevated in a wide density range.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to a device for measuring the concentration of microorganisms inside a culture tank or fermenter.

[Background of the invention]

Bacterial cell concentration meters for bioplants have strict functions such as (1) being able to measure high concentrations, (ii) being unaffected by air bubbles and agitation, and (i) having high-speed response. required.

Conventional bacterial cell densitometers for bioplants are known that utilize the fact that transmitted light and reflected waves of light (such as a laser) attenuate relative to concentration.

However, when focusing on the attenuation of light, air bubbles on the optical path cause errors, and it is difficult to measure up to a concentration of bacterial cells that is so high that it fills the optical path. Therefore, in the case of an optical type, functions (1) and (11) are not fully satisfied.

On the other hand, in Japanese Patent Laid-Open No. 57-50652, a method is known in which the number of bacteria is measured using the received voltage of ultrasonic waves (attenuation of ultrasonic waves) as an indicator. However, JP-A-57-5065
In Publication No. 2, when the amount of air bubbles entering the ultrasonic propagation path changes, even if the number of bacteria is constant, the received voltage changes, making it impossible to accurately measure the number of bacteria. Also, JP-A-57-506
In Publication No. 52, since the measuring device is directly immersed in the culture solution, consideration must be given to the sterilization operation, maintenance is complicated, and (I) and (II) are not satisfied.

On the other hand, a method using the sound velocity of ultrasonic waves as a solution concentration meter is known (Japanese Patent Application Laid-open No. 77656/1983, etc.)
. However, the method of determining concentration using the speed of sound was considered inapplicable to non-aqueous solutions such as suspensions. In particular, if there are bubbles in the suspension, the bubbles will scatter the ultrasonic waves.
Concentration cannot be measured.

In this way, the functions (1) to (11) were not satisfied with conventional bacterial cell concentration meters, so physical measurements such as the oxygen concentration in the supply gas, the oxygen concentration in the exhaust gas, and the charcoal gas concentration were used. An indirect method was used in which chemical quantities were measured and the microbial cell concentration was calculated from these. In this way, bacterial cell concentration and bacterial cells! Therefore, accurate values such as the growth rate of the bacterial cells and the yield of the product were not known, making efficient production difficult.

[Purpose of the invention]

The purpose of the present invention is to measure the microbial cell concentration inside a culture tank or fermentation tank in a wide range from low to high concentrations, even in agitated microbial suspensions containing air bubbles, with a fast response time and on-line measurement. The goal is to provide equipment.

[Summary of the invention]

The present invention measures the microbial cell concentration by focusing on the relationship between the microbial cell concentration in a microbial suspension and the sound speed (or time for propagation of a predetermined distance) of a sound wave propagating in this microbial suspension. In the device, ultrasonic waves are generated in a microbial suspension that is stirred and contains air bubbles at a constant repetition period, the ultrasonic waves are propagated through the gaps between the air bubbles, and the propagation waveform is received, and further, If you average the captured waveforms multiple times,
It was discovered that it was possible to measure the bacterial cell concentration, and when performing the averaging process, it was necessary to average multiple pulses in a short period of time in order to improve the responsiveness of the measuring device. Although the return cycle must be shortened, the concentration meter a
When the t range is wide, for example, the transmitting pulse intensity is set so that a received signal can be obtained even at high concentrations, and when measuring low concentrations, the ultrasonic received signal due to the ultrasonic transmitting pulse generated at a certain time T and the time T Among the ultrasonic reception signals generated by the ultrasonic transmission pulses generated earlier, signals that have not yet been completely attenuated (hereinafter referred to as reverberant echoes) interfere with each other, making accurate measurement difficult. Therefore, this device measures the strength of the ultrasound reception signal and changes the ultrasound transmission pulse strength to obtain a reception signal of a constant strength and shorten the ultrasound transmission pulse generation cycle. However, the present invention is characterized by making it possible to perform averaging processing over a wide concentration measurement range in a short time without receiving interference from reverberant echoes, thereby improving the responsiveness of the measuring device.

[Embodiments of the invention]

FIG. 1 is an embodiment of the present invention, FIG. 2 is a sectional view taken along the line ■-■ in FIG. 1, FIG. 3 is a detailed explanatory diagram of an embodiment of the signal processing circuit section of the present invention, and FIG. , FIG. 5 is an explanatory diagram of the operation of the signal processing circuit of the present invention.

In these figures 1 to 3, 10o is a culture tank (
(shown in a cross-sectional view), culture cells such as individual microorganisms, animal and plant cells, and tissues. The culture tank 100 is usually cylindrical, and contains a culture solution 110 in which the bacterial cells are cultured.

The ultrasonic transceiver 300 is an ultrasonic transceiver controller 310
The ultrasonic wave is transmitted in response to an electric signal, propagated through the culture medium 110, reflected by the ultrasonic wave reflecting section outer wall 240, propagated in the reverse direction along this path, and the propagated ultrasonic wave is received by the ultrasonic transmitter/receiver 300. The transmission pulse intensity is adjusted so that the propagating ultrasound received by the ultrasound reception control device e310 reaches a predetermined level, and after adjustment, the signal is sent to the waveform averaging processing device 320)
, where the waveform signal is subjected to averaging processing. Next, the propagation time calculation device 330 calculates the ultrasonic propagation time of the culture solution 110 from the electrical signal obtained by the waveform averaging processing device 320. On the other hand, based on the signal obtained by the temperature detector 350, the temperature of the culture solution 110 is measured by the temperature calculation device 36θ. The bacterial cell concentration calculating device 370 calculates the bacterial cell strength from each electrical signal obtained from the propagation time calculating device 330 and the temperature calculating device 360. 340 is a control device that controls these.

120 is a compressor that supplies oxygen-containing gas such as air to the culture tank 10 from the aeration device 140 via the gas supply pipe 130.
0 to the culture solution 110 inside.

The motor 150 rotates agitators 1170A, B fixed to the shaft 160 to promote gas-liquid contact. The rotational speed may range from several rpm to several hundred rpm.The vane 180 is an exhaust pipe for eliminating foam. The substrate 210 stored inside the substrate tank 200 is
The substrate is supplied into the culture tank 100 by a substrate supply pump 220 through a substrate supply pipe 230. Bacterial cells proliferate in such a system, and the bacterial cell concentration increases over time.

Furthermore, as shown in FIG. 2, the ultrasonic transmitter/receiver 300 and the outer wall 240 of the ultrasonic reflector are arranged offset from the center so that the propagating ultrasonic waves are not obstructed by the shaft 160 in the culture medium 110. .

The details of the operation of the ultrasonic cell concentration measuring device having these characteristics will be described below.

In FIG. 3, the setting value F of the repetition period setting device 342 is
The control circuit 341 connects the variable intensity pulse transmitter 311 along
Issue a pulse transmission command to In response to this command, a transmission pulse emitted from the variable intensity pulse transmitter 311 is applied to the ultrasonic transceiver 300. The ultrasonic waves generated by the ultrasonic transmitter/receiver 300 propagate through the culture medium 110 and reach the ultrasonic reflector 24.
0 and returns to the ultrasonic transceiver 300, where it is converted into an electrical signal and sent to the amplifier 312 where it is amplified.

Here, the setting of the repetition period will be described.

In the present invention, in order to deal with disturbances in the received ultrasonic waveform due to the aeration state (gas supply amount) and stirring state (stirring rotation speed) of the culture solution 110, the received waveform is averaged multiple times (N times). I am doing it. Therefore, the time T required to obtain the received waveform as information for calculating the propagation time is as shown in FIG. 4(a), where N: the number of averaging processes F: the transmission pulse repetition frequency. In other words, it can be seen that in order to speed up the responsiveness of the measuring device, it is sufficient to reduce the number of times N of averaging processing and increase the transmission pulse (return frequency F). However, the number of averaging processes N
It is desirable to do this 10 times or more, and it cannot be reduced unnecessarily. Therefore, the increase in the transmission pulse repetition frequency will be highlighted.

On the other hand, the time T2 during which the ultrasound waves propagate through the culture
As shown in FIG. 2, if the distance between the ultrasonic transmitter/receiver 300 and the ultrasonic reflector 240 is 61, and the propagation velocity of ultrasonic waves in water is V, then t T, = □ .・・・・・・・・・(2)
■ It becomes.

In this way, it can be seen that it is desirable to set the transmission pulse repetition frequency F as high as possible at a frequency of 1/T or less.

Now, the received signal amplified by the amplifier 312 is sent to the gate 3
Sent to 13th. The gate 313 is a gate control circuit 314
The signal reflected from the ultrasonic reflector 24.0 among the received signals (Fig. 4 (C)) is controlled as shown in Fig. 4).
)) only pass through.

The reflected signal that has passed through the gate 313 is first judged by a reflected signal strength judgment circuit 315.

Here, the operation of the reflected signal strength determination circuit 315, which is the key point of the present invention, and the variable strength pulse transmitter that operates in conjunction with the circuit 315 will be described in detail.

First, in order to speed up the response of the measuring device, the transmission pulse repetition frequency F is set to t/'rt as shown in equation (3).
It is necessary to set the frequency as high as possible at a frequency equal to or lower than t'r, (propagation time). However, in this case, the measuring device can be adjusted from low to high concentrations, e.g. Og/t~200g
In order to enable continuous measurement over a wide concentration range of /l, the following problems occur.

As the bacterial cell concentration increases, the attenuation of the ultrasonic waves propagating there increases, so the transmission pulse is large enough to obtain at least the first reflected signal at the ultrasonic transmitter/receiver 300, taking into account the effects of agitation and bubbles. It is necessary to apply P, . The received signal obtained at that time is shown in FIG. 5(a). Next, as the concentration of J gradually decreases, the attenuation of the ultrasonic wave decreases, the reflected signal gradually increases, and the propagation time T becomes longer. At this time, in order to correspond to a wide concentration measurement range, high concentration C
With the transmission pulse strength P set to correspond to 1 o'clock, as shown in FIG. 5(b), the low concentration C1 (< C+
) Sometimes the reflected signal remains as a reverberation for the first time, second time, third time, etc. (-time, second time... means that the ultrasonic wave generated from the ultrasonic transceiver 300 travels the propagation distance t shown in FIG. 2 by 2t, 4t (2tX2),
This is a signal that is received by propagating multiple times while attenuating as 6/, (2tX3)... Hereinafter, this will be referred to as a reverberant signal. Therefore, a situation occurs in which the first reflected signal and the reverberation signal necessary for measuring the propagation time interfere with each other, making measurement difficult or impossible.

In order to avoid this situation 2, as shown in Figure 5(c), by decreasing the transmission pulse repetition frequency (increasing V), we can secure enough time for the reverberant signal to decay even at a low concentration of C8, and Although a method of avoiding interference between reflected signals and reverberant signals may be considered, this method impairs the commercial responsiveness of the measuring device. On the other hand, if the transmission pulse P is set to coincide with the low concentration C7, the first reflected signal will not be detected at the high concentration C6.

In order to solve this problem and realize a measuring device with high-speed response over a wide concentration measurement range, the present invention employs the following means.

In advance, at a certain propagation distance t, only the -th reflected signal is received and the second and subsequent reflected signals are approximately 0, and the first reflected signal is set as L, (Fig. 5(d)),
This is set in the reflected signal strength discrimination level setter 316 in FIG. This reflected signal level L is 313t
- Compare the reflected signal that has passed through the reflected signal intensity determination circuit 315, and if the reflected signal that has passed through the gate 313 is >L, send a transmission pulse intensity increase command to the variable intensity pulse transmitter 311 via the control circuit 341. , increase the transmit pulse strength. Also, if the reflected signal passing through the gate 313 is <L, then
A transmission pulse intensity reduction command is sent to the variable intensity pulse transmitter 311 via the control circuit 341 to increase the transmission pulse intensity. In this way, when the reflected signal passing through the gate 313 and the reflected signal strength discrimination level Ll become almost equal,
That is, when the second and subsequent reflected signals no longer appear, the waveform averaging processing device 320 is activated.

By adopting this method, it becomes possible to measure the bacterial cell concentration over a wide concentration range without lowering the transmission pulse repetition frequency F and without impairing the responsiveness of the measuring device.

Now, the reflected signal sent from gate 313 is the logical number)
. This is shown in FIG. 4(d). The output of the divider 321 is
The A/D converter 322 performs analog-to-digital conversion to convert the reflected signal waveform into a digital signal.
/F time is stored in the memory 324. This is designated as mu 1 as shown in FIG. 4(d). Next, the same operation is performed on the reflected signal of the flat panel 2, and when storing it in the memory 324, the previously stored reflected waveform of &1 is read out to the buffer 325, and the output of this buffer 325 and the A/D converter 322 are The adder 323 adds the outputs of the . By repeating the same operation N times, data obtained by averaging the reflected signal waveform N times is stored in the memory 324 for 1/F time. Fourth
Figure (e). These series of operations are performed based on commands from the control circuit 341.

Now, by referring to this memory 324, the propagation time measurement gate signal generator 331 generates a propagation time measurement gate signal (FIG. 4(f)). This signal controls the gate 332 to input the clock generated by the clock generator 334 (FIG. 4) to the counter 333 for a propagation time of 15 minutes (FIG. 4(h)). By counting this clock with the counter 333, the propagation time T,
The number of clocks M is obtained according to (FIG. 4(i)).

As described above, a method has been described in which the propagation time can be measured stably by averaging even in a stirred microbial suspension containing bubbles, and at high speed by using the above-mentioned means.

Next, a method for measuring temperature will be explained.

The temperature detector 350 can be a resistance temperature sensor such as a thermistor. The electrical signal generated by the temperature detector 350 is sent to the temperature calculation device 360, where the temperature is calculated.

Now, we will explain how to obtain two pieces of information: propagation time and temperature. Here, we will briefly describe a method for determining the bacterial cell concentration based on these two pieces of information. Generally, the bacterial cell concentration Cw is expressed as a function of propagation time Tt and temperature θ as follows: Cw=f(Tt, θ) (4).

Also, the function f in equation (4) differs depending on the target microorganism, so memo the function depending on the microorganism! Store it in J380. Therefore, based on the information on propagation time and temperature,
A function is called from the function memory 380, and the bacterial cell concentration is determined by the bacterial cell concentration calculating device 370.

In this example, the propagation time was measured from the time when the transmitted pulse was generated to the first reflected signal, but from the -th time to the second reflected signal, or from the time when the transmitted pulse was generated to the third reflected signal. It is obvious that the combination is arbitrary. Similarly, although the set level of the reflected signal strength discrimination level setter 316 was set to a level at which the second and subsequent reflected signals do not appear, the present invention is not particularly limited to this.

〔Effect of the invention〕

According to the present invention, when measuring the bacterial cell concentration of an agitated microbial suspension containing air bubbles by applying ultrasound, waveform averaging processing is unavoidable (to reduce the deterioration of measurement response). It is possible to realize a microbial cell concentration measuring device that is kept to a minimum and has high-speed responsiveness over a wide concentration measurement range.

[Brief explanation of the drawing]

1 and 2 are system diagrams of an embodiment of the present invention, FIG. 3 is a detailed explanatory diagram of a signal processing section, and FIGS. 4 and 5 are operational diagrams. 100...Culture tank, 110...Culture solution, 120...
・Compressor, 130... Gas supply pipe, 140...
- Aeration device, 150... Motor, 160... Shaft, 170A, B... Stirring train, 180... Defoamer, 190... Exhaust pipe, 200... Substrate tank, 21
0... Substrate, 220... Substrate supply port, 230...
・・Substrate supply pipe, 240 ・・Ultrasonic reflector, 300・
...Ultrasonic transmitter/receiver, 310... Ultrasonic transmitter/receiver control device, 311... Variable intensity pulse transmitter, 312...
Amplifier, 313...Gate, 314...Gate control circuit, 315...Reflected signal strength determination circuit, 316...
・Reflected signal strength discrimination level setter, 320... Waveform averaging processing device, 321... Division! , 322...A/
D converter, 323... Adder, 324... Memory, 325... Buffer, 330... Propagation time calculation device, 331... Propagation time measurement gate signal generator,
332... Gate, 333... Counter, 334...
・・Clock generator, 340 ・・Control device, 341・
... Control circuit, 342 ... Repeat cycle setting device, 35
0...Temperature detector, 360...Temperature calculation device, 37
0... Bacterial cell concentration calculation device, 380... Function memory.

Claims (1)

[Claims] 1. In a stirring tank containing a microbial suspension, a temperature detection sensor, a temperature calculation device, an ultrasonic transmitter/receiver, an ultrasonic receiver, an ultrasonic received signal strength measuring device, It consists of an ultrasonic transmission pulse intensity variable device, an ultrasonic reflection unit, a received waveform average processing device, a propagation time calculation device, and a microbial cell concentration calculation device, and the microorganism suspension is performed without being affected by stirring or air bubbles. A bacterial cell concentration measuring device that measures the concentration of a liquid at high speed. 2. The microbial cell concentration measuring device according to claim 1, characterized in that a plurality of the ultrasonic transmitter/receivers are provided independently for transmission and reception.