JPH0889285A - Method for detecting anaerobic bacteria in fermentation tank or the like - Google Patents

Abstract

PURPOSE: To provide a method for dissolving problems concerning anaerobic bacteria in early stages by simply detecting existence of anaerobic bacteria in a aqueous system. CONSTITUTION: This method for detecting anaerobic bacteria is carried out by using an alloy of a metal selected from copper and titanium or a metal selected from iron, nickel, copper, aluminum and titanium as an indicate electrode 3 and measuring the potential difference between the indicate electrode 3 and the reference electrode 4.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting anaerobic bacteria which occurs in fermenters, industrial water systems and the like.

[0002]

2. Description of the Related Art In the production of microbially produced polymers,
Generally, an aeration stirring fermentation tank is used, and the fermentation tank is stirred by stirring blades so that the substrate and oxygen are made uniform in the culture solution. Examples of polymers produced using such equipment are xanthan gum, pullulan, dextran, curdlan, B-16 polymer [Alkagenes.
Litas B-16 strain (polysaccharide produced by FERM-BP-2015)] and the like. These polymers have a very high molecular weight, and the viscosity of the culture solution increases from the middle of the culturing, and the final viscosity is often 10,000 centipoise or more. When the viscosity becomes high as described above, the stirring becomes incomplete, and some areas become deficient in oxygen. In particular, polymers such as xanthan gum and B-16 polymer, which have non-Newtonian pseudoplastic viscosity characteristics, have a reduced viscosity only in the portion subjected to the shearing force of the stirring blade, and have high viscosity in other portions. Therefore, the stirring of the whole culture solution becomes very bad. as a result,
The oxygen concentration also becomes non-uniform, and an oxygen-deficient portion will occur. In the production of polymers by microorganisms, originally undesirable bacteria may be contaminated, but when the contaminated bacteria are aerobic bacteria, they grow from the initial stage of fermentation, and the growth of the desired bacteria is suppressed. Therefore, the determination can be made at a relatively early stage, and the culture can be stopped. However, if the contaminated bacteria are anaerobic bacteria, the growth is suppressed while the fermenter contains a lot of oxygen, but if the culture progresses and the viscosity of the culture solution rises to a certain extent, there will be oxygen deficiency. To breed. When the anaerobic bacteria grow, the nutrient source is consumed, so that the activity of the originally intended bacteria becomes dull and the yield of the culture product decreases. In this case, it is difficult to determine whether the failure of the culture is caused by the contamination of other species of bacteria or the external factors such as the medium components. Moreover, if the cause is an anaerobic bacterium, it will not be known until the latter half of the culture, so it is not only a trouble of redoing the culture,
Waste of time and energy will increase. Therefore, it becomes necessary to know the existence of anaerobic bacteria as soon as possible and to judge them.

When performing fermentative production of a polymer using a fermenter, it is difficult to obtain stable physical properties of a culture solution, particularly viscosity characteristics. The cause has been clarified as variation in activity of polymer-producing bacteria and variation among lots of medium components, but the present inventor found out that contamination of anaerobic bacteria is also one of the major causes. Even if the fermenter is thoroughly washed after use, stains are left on the concavo-convex parts such as the connection part in the device, and anaerobic bacteria are often attached to the stains. For example, a heat-resistant Clostodium genus, which is a spore-forming anaerobic bacterium, may not be completely sterilized by heat sterilization or steam sterilization. Conventionally, there is no method for easily detecting the presence of anaerobic bacteria, and it is common to actually sample the bacteria to identify the bacterial species.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method capable of easily detecting the presence of anaerobic bacteria in a water system and quickly solving the problems associated with anaerobic bacteria.

[0005]

As a result of systematically studying the properties of anaerobic bacteria, the present inventor has found that when the anaerobic bacteria act on an alloy of a specific metal, the potential of the alloy slightly decreases. Based on this finding, we have completed a method for detecting the presence of anaerobic bacteria.

That is, according to the present invention, a metal selected from copper and titanium or an alloy of a metal selected from iron, nickel, copper, aluminum and titanium is used as an indicator electrode, and a reference electrode that is present in the same aqueous system is used. The present invention relates to a method for detecting an anaerobic bacterium by measuring the potential difference between.

The detector used in the method of the present invention comprises at least an indicator electrode, a reference electrode and an electrometer made of the alloy. The measurement is performed by immersing the indicator electrode and the reference electrode in the same water system as the water system to be measured so that both electrodes do not come into direct contact with each other. The distance between both electrodes is not particularly limited as long as it is in the same water system, but considering the electric resistance of the water system and the influence of noise, the distance between both electrodes is 10 cm, which is the distance where the electrodes do not come into direct contact with each other.
The following is preferably 5 cm or less. If the distance is larger than this, the sensitivity becomes dull and the noise becomes large, which is not preferable in practical use. The size of the indicator electrode is not particularly limited, but the closest point to the reference electrode acts as a counter electrode, so the closest point is 10 cm or less,
It is preferable to set it so that the distance is 5 cm or less, and connect it to the place. The indicator potential and the reference electrode are electrically connected, and an electrometer is placed between them to measure the potential difference between both electrodes. The potential in this case is +500 mV to -50
Since it is 0 mV, it is necessary to use an electrometer suitable for this.

The indicator electrode used in the present invention is made of a metal selected from copper and titanium or an alloy of a metal selected from iron, nickel, copper, aluminum and titanium.
As the alloy in this case, a corrosion-resistant alloy that forms a stable and uniform passivation film on the surface in water containing oxygen is suitable.
A typical example of the iron alloy is stainless steel, and SUS-201, SUS-302, SUS-30.
4, SUS-306, SUS-317, SUS-316
and so on. Examples of nickel alloys include cupronickel, monel, inconel, and the like. Examples of copper alloys include bronze, 70-30 copper nickel, silicon bronze, red brass, admiralty brass and the like. Examples of aluminum alloys include Type5005, Type50
52, Type 5154 and the like. Examples of titanium alloys include Ti-6Al-4V and Ti-5Al-2.5Sn. The reason why such a material having corrosion resistance is used is that a material having large corrosion cannot catch a minute current because the corrosion current is large. Practically, stainless steel is preferable in view of the measurement sensitivity of the present invention, workability of the material, and the like, and among these, SUS-316 and SUS-304 are particularly preferable.

The shape of the indicator electrode is not limited, but when the indicator electrode is immersed in water for use, a cylinder, a cylinder with a bottom, a flat plate or the like is suitable. On the other hand, it is also possible to separately provide a detector and extract water from the water system to be measured for measurement.

Examples of the reference electrode used in the present invention include a silver / silver chloride electrode, a hydrogen electrode, a sweet corn electrode, a mercuric sulfate electrode, and a mercury oxide electrode. Of these, the points that are easy to handle
The silver / silver chloride electrode is most suitable. A commercial item can be used for these.

The anaerobic bacterium includes a facultative anaerobic bacterium which can grow even in the presence of oxygen and an absolute anaerobic bacterium which cannot grow in the presence of oxygen, but the present invention is applicable to both. Examples of facultative anaerobes include Clostodium
Butyrican (ATCC 25779), Clostodium sporogenes (ATCC 7955), Actinomyces bovis (ATCC 13683) and Bifidobacterium bifidium (ATCC 2952) are available. As an example of an absolutely anaerobic bacterium, Shigella dysenteria (ATCC 13313). ), Entrobacter Sakazaki (ATCC 29544), Serratina Marcescens (ATCC 13880), Vibrio marinus (ATC)
C15381).

The generation of anaerobic bacteria is detected by the change in the potential difference between the indicator electrode and the reference electrode. As the standard,
Even if the false positive rate increases, if half-year detection is required, for example, 3
When it is required to reliably detect mV / hr, even if the detection is somewhat delayed, it can be considered that anaerobic bacteria have occurred when a change of, for example, about 10 mV / hr occurs. Since the above values differ depending on the accuracy of the detectors, it is basically assumed that anaerobic bacteria are generated when there is a change exceeding the noise of each detector. In the detection method of the present invention, basically there should be no change in the potential difference between the indicator electrode and the reference electrode, so it may be said that anaerobic bacteria have occurred when there was a change in the potential difference of a certain constant value. it can.

Since the method of the present invention detects the presence of anaerobic bacteria, its application is not limited to fermenters. For example,
Occurrence of slime and fouling due to anaerobic bacteria in industrial cooling water systems, wastewater treatment systems, papermaking processes such as papermaking processes, etc., can be similarly used for detection of anaerobic bacteria in these fields.

In the industrial cooling water system, the circulating water is reused by evaporating a part of the circulating water in the cold water tower to dissipate the heat and cooling. The water in the system is concentrated and the concentration of nutrients and pollutants rises, and at the same time the number of microorganisms such as bacteria, algae and fungi also increases. Some of these bacteria produce sticky substances, and the adsorption of suspended matter is added to the sticky substances to form slime. Also, in paper mills, process water is circulated and reused, and microorganisms in the water increase, and suspended matters such as pulp, starch, and talc are accumulated to form slime. In the case of dirt accompanied by microbial activity, the lower layer of the adhered portion becomes anaerobic, and anaerobic bacteria grow unless special consideration is given.
Thus, the method of the present invention can detect when the activity of the anaerobic bacterium becomes active. However, it is not possible to detect it because the electric potential does not change just by creating an anaerobic state by depositing suspended matter. When the effect of slime control is exerted by adding a microbicide or the like, the activity of microorganisms including anaerobic bacteria is suppressed.
In such cases, the method does not detect slime.
Therefore, the quality of slime control can be evaluated by using this method. The present invention also includes the detection of anaerobic bacteria in such industrial water systems and the accompanying slime.

[0015]

The alloy used in the present invention forms a passive oxide film on the surface in water and is less likely to corrode. However, if a defect occurs in a very small portion of the passive film due to some reason, the action of compensating for the defective portion of the passive oxide film works. Electrically, it is the oxidation of the surface by oxygen,
Cathode reaction occurs.

However, when dirt components such as microorganisms and pulp adhere to a certain place, the aerobic state is maintained at the initial stage, but when the slime grows, the oxygen gradually becomes insufficient. When it becomes anaerobic in this way, anaerobic bacteria grow. In the growth process, organic acid is generated to increase the hydrogen ion concentration and promote the cathode reaction, so that the balance between the anode reaction and the cathode reaction is lost. Metals that easily corrode in water, such as mild steel, zinc metal, and tin metal, have large corrosion currents, so minute currents cannot be captured, but the alloys of the present invention have extremely weak corrosion currents, so even slight corrosion can cause potential Changes. The method of the present invention measures this change in potential as the potential with respect to the reference electrode.

Therefore, the potential does not change even if the state is simply anaerobic. The activity of microorganisms is added thereto, and the potential changes only when there is a slight corrosive action by the microorganisms.

[0018]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 Test Using Fermenter Two MBF-500MPA type fermenters 1 (schematic diagram is shown in FIG. 1) manufactured by Tokyo Rika Co., Ltd. were used, and stainless steel was used as an indicator electrode 3 therein. A cylindrical cup (diameter 20 mm, height 30 mm) made of SUS-316, and a silver / silver chloride standard electrode (HA-10 manufactured by Towa Denpa Co., Ltd.) as a reference electrode 4.
The detector 2 consisting of 1) was installed.

In addition to the detector 2, the fermenter 1 is equipped with a stirrer 5 with a rotating blade, a condenser 6, and a dissolved oxygen meter 7. A ventilation pipe 8 is connected near the base end of the stirrer 5, and the air dedusted by the filter 9 passes through the inside of the rotary shaft and is aerated from the tip thereof into the culture solution (not shown). The indicator electrode 3 and the reference electrode 4 of the detector 2 are connected to a potentiometer 10, and the potentiometer 10 is further connected to a recorder (not shown). A warm water jacket 11 is provided around the fermentation tank 1 so that the temperature can be adjusted.

According to the method disclosed in Japanese Patent Application Laid-Open No. 4-200389, the following medium composition was used, and one of the fermenters had Alcaligenes laetus B-16 (FERM BP-2015).
(Experiment A), the other was Alcaligenes Laetus B-16 and Clostridium sporogenes A
Inoculated with TCC7955 (anaerobic bacterium) (experiment B).
(Alkaline generatus B-16 strain polymer production medium)

[0022] glucose; 2% yeast extract; 0.05% urea; 0.05 wt% K ₂ HPO _4; 0.84 wt% KH ₂ PO _4; 0.44 wt% MgSO ₄ · 7H ₂ O 0.02 wt% NaC; 0.01 wt% distilled water; balance

2.7 l of the above medium was sterilized and put into a fermenter having a volume of 5 l, inoculated with the above-mentioned bacteria, and cultured at 30 ° C for 5 days. The aeration and stirring conditions were an air flow rate of 3 l / min and a rotation speed of 500 rpm.

As a result, in Experiment A, 9.2 g / l of polysaccharide was accumulated and the final viscosity reached 4800 cps (No. 3 rotor, 30 rpm). Experiment B is 4.6 g / l
Polysaccharides accumulate and the final viscosity is 3600 cps (No. 3
Rotor, 30 rpm). The potential change in this culture is shown in FIG. In Experiment B, an increase in the electric potential was observed from the 3rd day of culture, and the growth of anaerobic bacteria could be detected.

The method for detecting anaerobic bacteria was performed as follows. That is, cooked meat medium [E-M108 (trade name) manufactured by Eiken Kagaku Co., Ltd.] was put in a petri dish, 0.1 ml of the fermentation solution was inoculated, spread with a conradi stick, and this was deoxidized material [Mitsubishi Gas Chemical Co., Ltd. [Ageless (trade name) manufactured by Co., Ltd.] was placed in a coexisting desiccator. After thoroughly replacing the air in the desiccator with carbon dioxide, at 37 ° C under reduced pressure.
The culture was allowed to stand for 2 days at 37 ° C., and the presence of C. sporogenes was confirmed by the growth of colonies.

Example 2 Test Using Fermenter The same apparatus as in Example 1 was used. However, for the indicator electrode 3, a cylindrical cup (diameter 20 mm, made of Inconel,
A height of 30 mm) was used. Xanthomonas campestris ATCC1 in one fermenter with the following medium composition
3951 was inoculated (Experiment C), and the other was inoculated with Xanthomonas campestris ATCC 13951 and Clostridium sporogenes ATCC 7955 (Experiment D). The culture conditions were the same as in Example 1. Experiment C
Has 15.3 g / l of polysaccharides accumulated and has a final viscosity of 2,0
00 cps (No. 3 rotor, 30 rpm) was reached. In Experiment D, 4.6 g / l of polysaccharide was accumulated and the final viscosity reached 1400 cps (No. 3 rotor, 30 rpm). The change in the potential in this culture is shown in FIG. 4. In Experiment D, the potential was increased from the 3rd day of culture, and the growth of anaerobic bacteria could be detected.

(Xanthomonas campestris polymer producing medium) Glucose; 3.5 wt% yeast extract; 0.2 wt% glutamic acid; 0.1 wt% CaCl ₃ ; 0.001 wt% K ₂ HPO ₄ ; 0 0.1% by weight KH ₂ PO ₄ ; 0.1% by weight MgSO ₄ .7H ₂ O; 0.02% by weight NaC; 0.01% by weight distilled water; balance

[Embodiment 3] A glass tube 11 and a stainless SUS-304 tube 3 are assembled as shown in FIG. 6, a silver / silver chloride electrode 4 and a stainless tube 3 are connected as shown in FIG. 7, and a potentiometer 10 is provided in the middle. It was set so that the potential between the two could be measured. This device was placed beside a paper machine (newspaper) at a paper mill to evaluate slime 12 generation and a bactericide. White water of the process was passed through the shell side of the double pipe at a flow rate of 300 ml / min by a perita pump. The stainless steel tube 3 is obtained by screwing up to the front of the silver / silver chloride electrode 4. 8 and 9 show the measurement results of the change in potential difference with time. Fig. 8 shows the time course of the potential when a bactericide was added to the white water system once every 24 hours, and Fig. 9 shows the time course of the white water system once every 8 hours.
The changes over time in the potential when the bactericidal agent is added are shown. In this step, the bactericidal agent was added once a day, but the potential started to rise about 6 hours after the device of the present invention was installed. Remove slime from the stainless tube of the device of the present invention, wash, re-install and restart the test,
The fungicide was added three times a day. As shown in FIG. 9, the potential increased, but the potential decreased when the bactericide was added. When the sterilizing agent was added when the potential was increased as described above, the potential did not change as a whole, and slime could be eliminated even in this paper machine.

Further, when anaerobic bacteria including facultative anaerobic bacteria were detected from the recovered slime, it was 2 × 10 ⁸ c
ell / g (per 1 g of dried slime) was detected, and when one of the bacterial species was identified, it was an anaerobic bacterium of the genus Escherichia coli. The method for detecting anaerobic bacteria is based on “Classification and Identification of Microorganisms (Bottom)” by Takeharu Hasegawa (Society Publishing Center, pages 150-151), and those that anaerobically decompose sugars to produce acids are anaerobic bacteria. It was measured.
The composition of the medium used is as follows.

Peptone; 2.0 wt% NaC; 5.0 wt% K ₂ HPO ₄ ; 0.3 wt% agar; 2.0 wt% bromothymol blue; 0.08 wt% distilled water; balance pH: 7 .1

[Embodiment 4] Using the apparatus described in Embodiment 3, the effects of an apparatus for filtering industrial cooling water circulating water were compared. The circulating water was pumped from the pit of the cold water tower with a peristaltic pump, and the shell of the double tube composed of the glass tube 11 and the stainless steel tube 3 was used for both cases where the circulating water was passed through and where it was not passed. On the side,
Water was passed at a flow rate of 300 ml / min. The results are shown in Fig. 10. In the cold water tower in which the device of the present invention was installed and the filtration device was not installed, the spontaneous potential began to rise after 30 hours, while the potential of the cold water tower in which the filtration device was installed did not change. There wasn't. Further, in the case where the filtration device was not installed, when the stainless tube was replaced with the tube whose surface was not threaded (FIG. 5) and the test was conducted again, there was almost no change in the potential as shown in FIG. . Therefore,
It was found that slime may be generated at a place where suspended solids in the circulating water are easily accumulated, but slime is not generated on a smooth surface such as the surface of the heat exchanger tube.

Slime adhering to the stainless steel tube was collected, and anaerobic bacteria were detected in the same manner as in Example 3. As a result, 7 × 10 ⁵ cells / g (per 1 g of dry slime) was detected, and one of the bacteria was detected. When the species was identified, it was an anaerobic bacterium belonging to the genus Serratina marcescens.

[0033]

According to the method of the present invention, anaerobic bacteria that proliferate in a production facility can be easily and quickly detected and can be traced over time, so that the harmful effects of anaerobic bacteria can be prevented.

[Brief description of drawings]

FIG. 1 is a vertical sectional view of an apparatus used in an embodiment of the present invention.

FIG. 2 is an enlarged perspective view of a detector portion of the device.

FIG. 3 is a graph showing the results of investigating changes with time in potential by culturing using the device of FIG. 1 in the case where anaerobic bacteria are present and in the case where anaerobic bacteria are not present.

FIG. 4 is a graph showing the results of investigating changes with time in potential by culturing using the apparatus of FIG. 1 in the presence and absence of anaerobic bacteria, respectively.

FIG. 5 is a sectional view showing a structure of a detector used in one embodiment of the present invention.

FIG. 6 is a sectional view showing a structure of a detector used in one embodiment of the present invention.

FIG. 7 is a block diagram showing a configuration of a measuring device using the detector.

FIG. 8 is a graph showing a change with time in the electric potential obtained by measurement in an implementation system using the apparatus of FIG.

FIG. 9 is a graph showing the change over time in the electric potential obtained by measurement in an implementation system using the apparatus of FIG.

FIG. 10 is a graph showing the change over time in the electric potential obtained by measuring in the implementation system using the apparatus of FIG.

FIG. 11 is a graph showing the change over time in the electric potential obtained by measuring in the implementation system using the apparatus of FIG.

[Explanation of symbols]

 2 Detector 3 Indicator electrode 4 Reference electrode

Claims (3)

[Claims] 1. An anaerobic property by measuring a potential difference between a reference electrode and a metal selected from copper and titanium or an alloy of metals selected from iron, nickel, copper, aluminum and titanium. Bacteria detection method. 2. The indicator electrode is made of stainless steel.
The method for detecting an anaerobic bacterium described. 3. The method for detecting anaerobic bacteria according to claim 1, wherein a silver / silver chloride electrode is used as a reference electrode.