JP2001212590A - Bioreactor having means for controlling decomposition of organic chlorine compound by microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a bioreactor which can appropriately control the decomposition speed of organic chlorine compounds by microorganisms. SOLUTION: The bioreactor is provided with a measuring means for measuring the decomposition speed of the organic chlorine compounds by microorganisms, an inducer storage tank in which an inducer for activating the microorganisms is stored, and an activated microorganism culture tank for culturing pre-activated microorganisms.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a bioreactor for decomposing organic chlorine compounds by microorganisms.

[0002]

2. Description of the Related Art Organochlorine compounds such as trichlorethylene (TCE) are widely used in precision machinery related industries, pharmaceutical and chemical industries, dry cleaning factories, etc. due to their nonflammability and excellent degreasing power. However, many of them show toxicity to the human body, and some of them show carcinogenicity. Furthermore, since organic chlorine compounds are hardly decomposable, they remain in the environment widely and cause serious soil / groundwater pollution, which is a major social problem.

[0003] As a method of treating an organic chlorine compound, there is a method of removing it by adsorbing it on activated carbon or the like, but this method only recovers pollutants and cannot make it harmless.
In addition, this method has not yet solved the essential problem, since secondary contamination during disposal of the breakthrough activated carbon can occur.

[0004] On the other hand, environmental remediation technology utilizing microorganisms (hereinafter referred to as bioremediation technology).
Is effective in decomposing and removing contaminants, and is low in cost and low in the risk of secondary pollution. Currently, attempts have been made to apply bioremediation technology to the decomposition of organic chlorine compounds.

For example, regarding the contamination by TCE, researches have been conducted using a purification technique using a methane-utilizing bacterium, a toluene-utilizing bacterium, or the like in the soil or on a ground bioreactor. However, the decomposition of TCE by these microorganisms is performed by co-metabolism with specific substances,
The E-degrading activity requires an inducer and is not stable, and furthermore, the decomposition reaction has a problem that the phenomenon of substrate competition inhibition between the inducer and TCE occurs.

As described above, at present, it is very difficult to control the processing capacity of a bioreactor that decomposes an organic chlorine compound, and it is necessary to establish a control technique for stably processing an organic chlorine compound. I have.

[0007]

SUMMARY OF THE INVENTION The present invention relates to a bioreactor for decomposing organic chlorinated compounds by the action of microorganisms, which comprises a means for measuring the rate of decomposition of the organic chlorinated compounds over time. By preparing
An object of the present invention is to provide a bioreactor capable of appropriately controlling a decomposition treatment speed.

[0008]

In order to solve the above-mentioned problems, the present invention provides a bioreactor for decomposing an organic chlorine compound in a fluid to be treated, wherein a microorganism capable of decomposing the organic chlorine compound is used. A contained reaction tank and a measurement connected to the reaction tank so that the fluid to be treated can be introduced from the reaction tank, and capable of measuring a decomposition rate of the organic chlorine compound in parallel with the decomposition processing of the organic chlorine compound. Means, an activated microorganism culture tank for culturing activated microorganisms to be supplied to the reaction vessel, and an organic chlorine of the microorganisms to be supplied to the reaction vessel, the microorganism being contained in the reaction vessel. Provided is a bioreactor provided with an inducer storage tank in which a substance capable of inducing compound decomposition activity is stored.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS [Experimental Example 1] In this experimental example, it was clarified that the rate of oxygen consumption by microorganisms is almost proportional to the rate of decomposition of TCE by microorganisms, and that the former can be used as an index for the latter.

The details of the experiment will be described below.

First, a phenol assimilating bacterium KO-30 strain (FERM P-17222) having TCE-degrading activity was inoculated into a medium A added to a phenol concentration of 500 mg / L, and cultured for 20 hours. . The composition in 1 L of medium A is as follows.

[0012] _{K 2 HPO 4 1.0g (NH 4} ) 2 SO 4 1.0g MgSO 4 · 7H 2 O 0.2g FeCl 3 · 6H 2 O 0.01g CaCl 2 · 2H 2 O 0.05g NaCl 0.05g bacteria solution centrifuged Collect, in 50 mM potassium phosphate buffer at pH 7.0, protein concentration 100, 200, and 300 mg protein /
The suspension was placed in a volume of L, and 20 mL of each was placed in a 100 mL vial, sealed with a Teflon-coated butyl rubber stopper, and sealed with an aluminum cap. TCE was added to these vials using a syringe so that the concentration would be 5 mg / L, assuming that all the components were dissolved in the bacterial cell solution, and the mixture was shaken at 27 ° C. and 250 rpm. The gas phase was sampled over time, and a decomposition test of TCE was performed. The results are shown in FIG.

As is clear from FIG. 1, the higher the cell concentration, the higher the TCE degradation rate (hereinafter abbreviated as _VTCE ).

[0014] Further, after aerating each of the bacterial cell solutions having the above-mentioned concentrations for 5 minutes, the mixture is placed in a 100 mL Erlenmeyer flask and 100 mg / L.
Phenol was added so as to have a concentration of, and the amount of dissolved oxygen was measured over time without air. The results are shown in FIG.

As shown in FIG. 2, it is clear that the higher the cell concentration, the higher the rate of reduction of dissolved oxygen (hereinafter abbreviated as V _{O 2} ).

The amount of dissolved oxygen when phenol was not added was also measured, but no apparent decrease in dissolved oxygen was found (not shown).

[0017] As described above, V _TCE and V _O2, since both showed similar trends of increasing cell concentration-dependent manner,
In addition, the initial velocity of both speed was measured and quantitatively examined the relationship of V _TCE and V _O2.

[0018]

[Table 1]

As shown in Table 1, the initial velocity of _VTCE was almost proportional to the initial velocity of _VO2 .

From the above results, it is clear that the TCE decomposition activity of the bacterial cell solution can be easily and quickly monitored by using the consumption rate of dissolved oxygen as an index.

[Experimental Example 2] In this experimental example, phenol assimilating bacterium KO-30 was added by adding phenol as an inducer.
It was demonstrated that TCE degradation activity of the strain could be induced.

A phenol-assimilating bacterium KO-30 strain (FERM P-17222) having a TCE-degrading activity was inoculated into a medium A supplemented with a phenol concentration of 500 mg / L, and subjected to aerobic conditions for one week. The culture was carried out under shaking. At this time, confirm that the cell solution has no TCE degrading ability, so that the concentration becomes 100 mg / L.
Phenol was added to the cell solution and cultured with shaking for 3 hours.

The bacterial cell solution was collected by centrifugation, and 50 mM of pH 7.0 was collected.
It was suspended in potassium phosphate buffer so that OD ₆₀₀ = 1.0. 5 mg / L assuming that all dissolved in the cell solution
Was added to the vial using a syringe, and the mixture was shaken at 27 ° C. and 250 rpm. The gas phase was sampled periodically to perform a TCE decomposition test.

FIG. 3 shows the results.

As shown in FIG. 3, cells that did not show the ability to degrade TCE before the addition of phenol were:
Addition of the inducer phenol induces TCE degradation activity.

From the results of the present experimental example and experimental example 1, when the dissolved oxygen consumption rate of the cell solution in the bioreactor is reduced, the activity of the microorganisms for decomposing organic compounds is reduced. It has been clarified that by adding an inducer, the ability of microorganisms to treat organic chlorine compounds can be restored and kept stable.

[Experimental Example 3] In this experimental example, the cells were cultured under three different conditions: no induction by phenol after shaking culture, induction by phenol after shaking culture, and continuous culture in the presence of a high concentration of phenol. The phenol-assimilating bacterium KO-30 strain (FERM P-17222) was used to compare TCE-degrading activities.

Under the conditions, a phenol-assimilating bacterium KO-30 strain (FERM P-17222) having TCE-degrading activity was inoculated into a medium A supplemented with a phenol concentration of 500 mg / L, and allowed to grow for one week. Shaking culture was performed under aerial conditions.

After shaking and culturing under the same conditions as above for one week, phenol was added to a concentration of 100 mg / L, followed by shaking and culturing for 3 hours. After the bacterial cell solution was collected by centrifugation, it was diluted with 50 mM potassium phosphate buffer to the volume before centrifugation.

In each separate bioreactor (3L volume)
After injecting 1 L of the cells cultured under the conditions and
Air containing pm (5 mg / L) TCE was supplied at 10 mL / min. Samples were taken at the inlet (black diamond) and outlet (black square) of the bioreactor to test for TCE degradation in the reactor.

Under the conditions, a concentration of 10% was placed in a 1.5-L incubator.
The above strain was continuously cultured by continuously injecting the medium A containing 00 mg / L phenol.

Under a condition that the culture solution was appropriately passed through a bioreactor having a volume of 3 L, air containing TCE having a concentration of about 4 ppm (4 mg / L) was supplied at a flow rate of 10 mL / min. Sampled similarly, and tested for TCE degradation.

The results are shown in FIGS.

FIG. 4 shows the results of TCE degradation when using cells cultured under the conditions. When the cells were used, the TCE-degrading activity was already low at the start of the operation of the bioreactor, and the TCE-degrading activity disappeared in 20 hours.

FIG. 5 shows the results of TCE degradation using the cells cultured under the conditions. The cells had a higher TCE decomposition activity at the start of the bioreactor operation and maintained the TCE decomposition activity until the end of the operation (about 60 hours) as compared to the cells under the conditions, but the TCE decomposition activity gradually decreased with time. did.

FIG. 6 shows the results of TCE degradation using the cells cultured under the conditions. The cells have a higher TCE degrading activity at the start of the bioreactor operation than the cells under the conditions, and unlike the conditions, at the end of the operation (24
T), there was no change in TCE degradation activity.

From the above results, by appropriately introducing activated microorganisms into a bioreactor for decomposing TCE, the TCE decomposition activity in the reaction tank is controlled, and the processing capacity of the bioreactor is stabilized. It became clear that it could be kept.

As described above, from the above three experimental examples, when the activated microorganisms and / or inducers are added to the reaction vessel of the bioreactor while measuring the oxygen consumption rate by the microorganisms, the organic chlorine compound using the microorganisms can be reduced. It became clear that the decomposition process could be adjusted appropriately.

Hereinafter, a bioreactor designed based on this experimental example will be described. However, the following example is not intended to limit the scope of the present invention in any way.

[Example 1] In this example, a measuring means capable of monitoring the decomposition rate of the organic compound in real time is provided in parallel with the decomposition processing of the organic chlorine compound by the microorganism, so that the processing speed of the organic chlorine compound is reduced. The following describes a bioreactor capable of appropriately controlling the bioreactor.

As shown in FIG. 7, the bioreactor of the present embodiment includes a reaction tank 1 containing a microorganism capable of decomposing an organic chlorine compound and a fluid to be treated connected to the reaction tank 1 and containing the organic compound. A pump 2 for sending or feeding air to the reaction tank 1, a measuring means 3 for sampling the microorganism solution contained in the reaction tank 1 with time and measuring the decomposition rate of the organic compound in real time, Activated microorganism culture tank 4 for culturing activated microorganisms to be supplied to tank 1
And a control valve 5 disposed between the reaction tank 1 and the activated microorganism culturing tank 4, and a substance to be supplied to the reaction tank 1,
An inducer storage tank 6 in which a substance capable of inducing the activity of decomposing the organochlorine compound of the microorganisms accommodated in the reaction tank 1 is stored, and a control valve 7 disposed between the reaction tank 1 and the inducer storage tank 6. Have.

The fluid to be treated by the bioreactor of this embodiment is a liquid or gas containing TCE and an organochlorine compound such as dichloroethylene, or a mixture thereof.
The fluid to be processed is sent to the reaction tank 1 by the action of the pump 2. The fluid to be treated is typically wastewater from a precision machine factory, a pharmaceutical and chemical factory.

Since the reaction tank 1 contains a microorganism capable of decomposing the organic chlorine compound, the organic chlorine compound in the fluid to be treated sent to the reaction tank 1 is decomposed in the tank. The reaction tank 1 is a general one used for a normal bioreactor.

The microorganisms to be contained in the reaction tank 1 include Acinet
obactor sp strain G4 (Appl.Environ.Microbiol., 5
2, 383 (1986), USP4925802, ATCC53617), Pseudomonas
putida F1 (Appl.Environ.Microbiol., 54, 1703 (198
8)), Pseudomonas fuluoresence PFL12 (Appl.Environ.
Microbiol., 54, 2578 (1988)), Pseudomonas mendocin
a KR-1 (Bio.Tecnol., 7, 282 (1989)), Mycobacterium
vaccae JOB5 (Appl.Environ.Microbiol., 54, 2960 (19
89), ATCC 29678), Welchia alkenophila sero 5 (USP 48
77736, ATCC 53570), Welchia alkenophila sero 33
(USP 4877736, ATCC 53571), Methylocystis sp. Strain
M (Agric. Biol. Chem., 53,2903 (1989), Methylosinu
a trichosporium OB3b (Appl.Environ.Microbiol., 2
9, 365 (1989), JP-A-02-92274, JP-A-03-292970), Met
hylomonus sp MM2 (Appl.Environ.Microbiol., 57, 2
36 (1991), Alcaligenes denitrificans ssp.Xylosoxid
ans JE75 (Arch, microbiol., 154,410 (1990)), Alcali
genes eutrophus JMP134 (Appl.Environ.Microbiol.,
56, 1179 (1990)), Pseudomonas putida BH (Journal of the Japan Sewerage Association, 24, 27 (1987)), Pseudomonas sp.
9), Pseudomonas mendocina KWI-9 (JP 06-70753A),
Pseudomonas cepacia KK01 (JP 06-22776), Alcalig
enes eutrophus KS01 (JP 07-123976), Pseudomonas
Aeruginosa JI104 (JP 07-236895A), Corynebacterium
sp J1 (JP 08-66182A), Pseudomonas Alcaligenes KB
2 (Japanese Patent Application Laid-Open No. 08-154668), Alcaligenes sp TL2 (Japanese Patent Application Laid-Open
54669), Pseudomonas sp.TL1 (JP 08-187077), Pseu
domonas Aeruginosa JI104 (JP-A-07-236895), and Pse
Organochlorine-degrading microorganisms such as, but not limited to, udomonasu cepacia strain KO-30 (FERM P-17222).

These microorganisms are preferably immobilized on a carrier dispersed and contained in a tank. Carriers for immobilizing microorganisms include, but are not limited to, using porous cellulose, collagen and the like.

A measuring means 3 is connected to the reaction tank 1 so that a part of the microorganism solution in the tank can be introduced. The measuring means 3 is a means capable of quickly and easily measuring the activity of decomposing the organochlorine compound of the microorganism solution introduced from the reaction tank 1.

As shown in the above experimental example, since the oxygen consumption rate of the microorganism solution is almost proportional to the decomposition rate of the organochlorine compound, the means quickly and simply measures the oxygen consumption rate of the microorganism solution. It is preferably an oxygen concentration measuring means. Specific examples include, but are not limited to, an oxygen electrode and an oximeter.

The rate of decomposition of the organochlorine compound by the microorganisms measured by the measuring means 3 is an index for controlling the state of the pump 2 for sending or supplying air and the opening and closing states of the control valves 5 and 7. The control signal based on the index is transmitted to the control valve 5, and the control valve 7 (broken line in FIG. 7).

The control signal indicates that when the rate of decomposition of the organochlorine compound by the microorganisms in the reaction tank 1 decreases, the liquid supply or gas supply amount of the pump 2 decreases, the degree of opening of the control valve 5 increases, and This signal is a signal that causes at least one of an increase in the degree of opening of the control valve 7.

More specifically, when the decomposition rate is higher than the set value, the control signal indicates that both valves are closed to keep the pump 2 pumping or supplying air constant, and the decomposition rate decreases below the set value. If so, at least one valve is partially opened in accordance with the degree of decrease in the decomposition rate, and / or the amount of liquid or air supplied by the pump 2 is reduced in proportion to the degree of decrease in the decomposition rate. As such, it is preferable to control the pumps and valves.

The control valve 5 is provided between the activated microorganism culturing tank 4 and the reaction tank 1 and connects both tanks. When the control valve 5 is opened, the reaction from the activated microorganism culturing tank 4 is started. Activated microorganisms are added into the tank 1.

The term "activated microorganism" as used herein means that the activity of decomposing the organochlorine compound is previously induced by culturing in the presence of a substance that induces the activity of decomposing the organochlorine compound.
Means activated microorganisms. Therefore, when the activated microorganisms are added to the reaction tank 1 when the decomposition rate is reduced, the decomposition rate is increased and the processing capacity of the bioreactor can be kept constant. Further, according to the increase in the decomposition rate, it is preferable to control the amount of liquid or air supplied from the pump 2 in proportion to the increase in the decomposition rate.

The microorganisms to be cultured in the activated microorganism culturing tank 4 may be the above-mentioned microorganisms having an activity of decomposing organochlorine compounds. The activated microorganism and the microorganism in the reaction vessel 1 may be different microorganisms, but are preferably the same.

The type of the inducer to be added to the activated microorganism culturing tank 4 varies depending on the type of the microorganism. For example, phenol is used for phenol-utilizing bacteria and toluene is used for toluene-utilizing bacteria. Various substances for other microorganisms,
In particular, since an organic compound can be used as the inducer, an appropriate inducer may be selected according to the type of microorganism used. If the amount of the inducer in the activated microorganism culture tank 4 is increased, the activity of the activated microorganism is also increased. Therefore, the activity of the activated microorganism can be adjusted by the amount of the inducer. The preferred amount depends on the type of inducer, but 1 mg to 10 g / L,
Preferably 10mg-1g / L, more preferably 100mg-500mg / L
Will.

The amount of the activated microorganism to be added to the culture tank 1 may be determined according to the organic compound decomposing activity of the activated microorganism and the organic compound decomposing activity in the reaction tank 1. The activity of the activated microorganism for decomposing an organic compound in the presence of a predetermined amount of an inducer may be measured in advance, and it is not necessary to measure the activity sequentially when the bioreactor is operated.

As described above, the control valve 5 controls the amount of the activated microorganisms added to the activated microorganism tank 4 to the reaction tank 1, thereby controlling the activity of decomposing the organochlorine compound in the reaction tank 1. Can be appropriately adjusted.

On the other hand, the control valve 7 is connected to the induction substance storage tank 6.
When the control valve 7 is opened, the inducer is added from the inducer storage tank 6 into the reaction tank 1 because it exists between the reaction tank 1 and the two tanks.

The inducer to be added to the reaction tank 1 is an inducer for the organochlorine compound decomposing microorganisms contained in the reaction tank 1. Therefore, the type of the inducer is determined according to the type of microorganism contained in the reaction tank 1. The amount to be added varies depending on the type of the inducer, but is such an amount that the concentration in the reaction tank 1 becomes 1 mg to 10 g / L, preferably 10 mg to 1 g / L, more preferably 100 mg to 500 mg / L. possible.

Since the inducing substance increases the activity of decomposing the microorganisms contained in the reaction tank 1 to decompose the organochlorine compound, if the control valve 7 is opened when the activity decreases, the processing capacity of the bioreactor can be controlled. It becomes possible. Further, according to the increase in the decomposition rate, it is preferable to control the amount of liquid or air supplied from the pump 2 in proportion to the increase in the decomposition rate.

As described above, the bioreactor of the present embodiment has a measuring means for measuring the activity of decomposing organochlorine compounds in the reaction tank in real time, and an activity capable of increasing the activity in the reaction tank according to the measurement result. Since a separate tank is provided for each of the activated microorganism and the inducer, precise control of this type of bioreactor, which has been difficult in the past, can be achieved.

In the present embodiment, the most preferred bioreactor having all the measuring means, the activated microorganism culturing tank and the inducer storage tank has been described. However, the bioreactor having only the measuring means and any one of the tanks has been described. Also belong to the scope of the present invention.

Further, a bioreactor provided with only the measuring means also belongs to the scope of the present invention. In such a bioreactor, if necessary, a human may add an appropriate amount and type of activated microorganisms and / or inducers based on the measurement results.

[0063]

According to the bioreactor of the present invention, the decomposition treatment can be performed while monitoring the ability of the microorganism to decompose the organic chlorine compound over time.

Further, the bioreactor of the present invention can appropriately control the ability to decompose organic chlorine compounds based on the results of the monitoring.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing the change over time in the residual amount of trichloroethylene when trichlorethylene is decomposed by different concentrations of cells.

FIG. 2 is a graph showing the change over time of the dissolved oxygen concentration when trichlorethylene is decomposed by cells having different concentrations.

FIG. 3 is a graph showing the change over time in the residual amount of trichloroethylene when trichloroethylene is decomposed after adding phenol to cells in which trichloroethylene decomposition activity has not been induced.

FIG. 4 is a graph showing a time-dependent change in trichloroethylene degrading activity when cells that were not induced by phenol were used.

FIG. 5 is a graph showing a time-dependent change in trichloroethylene degrading activity when using cells that have not been induced by phenol.

FIG. 6 is a graph showing a time-dependent change in trichlorethylene degrading activity when using cells continuously cultured in the presence of phenol.

FIG. 7 is a schematic diagram of the bioreactor of Example 1.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12M 1/36 C12N 1/20 D 1/40 F C12N 1/20 A 1/38 11/02 1/38 11/12 11/02 B01D 53/34 134F 11/12 (72) Inventor Tsuyoshi Nakamura 1-8-1 Koura, Kanazawa-ku, Yokohama-shi, Kanagawa F-term in Basic Technology Research Laboratory, Mitsubishi Heavy Industries, Ltd. 4B029 AA02 AA21 BB02 CC04 CC10 DA03 DF04 DF05 DG06 4B033 NA12 NB12 NB45 NB58 NB68 NC04 ND04 NE05 NF01 NF04 NF06 4B065 AA01X AA04X AA12X AA24X AA31X AA36X AA41X AA43X AA44X AC20 BB04 A02 GB06 BC06 BC06 BC04 BC06 BC06 BC06 DD11 DD31

Claims (4)

[Claims] 1. A bioreactor for decomposing an organochlorine compound in a fluid to be treated, comprising: a reaction vessel containing a microorganism capable of decomposing the organochlorine compound; and containing the microorganism from the reaction vessel. Measuring means connected to the reaction tank so as to be able to introduce the fluid to be treated, the measuring means being capable of measuring the decomposition rate of the organic chlorine compound in parallel with the decomposition processing of the organic chlorine compound. Bioreactor. 2. The bioreactor according to claim 1, wherein said measuring means is an oxygen concentration measuring means. 3. The bioreactor according to claim 1, wherein an activated microorganism culture tank for culturing activated microorganisms to be supplied to the reaction tank and / or an activated microorganism culture tank to be supplied to the reaction tank. A bioreactor, further comprising an inducer storage tank that stores a substance that is capable of inducing an activity of decomposing the microorganism in the organochlorine compound contained in the reaction tank. 4. Based on the measurement of the decomposition rate of the organochlorine compound by the microorganism, both the inducer for activating the microorganism and the injection amount of the previously activated microorganism,
Or a method of controlling the processing capacity of a bioreactor using the microorganism by controlling any one of the methods.