JP6591224B2 - Processing apparatus and processing method - Google Patents

Description

  Embodiments described herein relate generally to a processing apparatus and a processing method.

Conventionally, as a means for purifying organic wastewater, a water treatment method using microorganisms has been mainstream, and in particular, an activated sludge method has been mainly employed. In recent years, from the point of further improving the quality of treated water, adoption of nitrogen and phosphorus removal methods such as anaerobic-aerobic activated sludge method, circulating nitrification denitrification method, anaerobic-anoxic-aerobic method (A2O method) has also been advanced It's getting on. Furthermore, a filtration membrane separation activated sludge method (MBR: Membrane Bio-Reactor), in which activated sludge is solid-liquid separated by a filtration membrane, has been introduced for the purpose of removing turbid components of treated water and saving space.
On the other hand, as means for enhancing the types of microorganisms, there is a method of enhancing the function of microorganisms and enhancing the degradability of the object to be treated by genetic manipulation. For microorganisms that have undergone genetic recombination, it is required to prevent them from flowing into environmental water by the Cartagena method, which is an international law. However, there have been cases where sufficient measures have not been taken to prevent the outflow of genetically modified microorganisms outside the processing plant system.

Cell surface engineering development and biotechnology development, Bioscience & Industry, 69 (1), 8-15, 2011

  The problem to be solved by the present invention is to provide a treatment apparatus and a treatment method capable of treating the water to be treated with high efficiency while preventing the genetically modified microorganisms from flowing out of the treatment plant system.

The processing apparatus according to the embodiment includes a biological reaction tank, a filtration membrane, a fluorescence measurement unit, a sterilization unit or blocking unit, and a control unit . The biological reaction tank holds a genetically modified microorganism in which a fluorescent protein gene and a function-enhanced gene that expresses a treatment function for a substance to be treated are incorporated by genetic recombination, and the treated water is biologically treated by the genetically modified microorganism. To do. The filtration membrane filters the treated water biologically treated in the biological reaction tank. The fluorescence measuring means measures the fluorescence of the treated water discharged from the biological reaction tank through the filtration membrane. Sterilization means is provided after the fluorescence measurement means and sterilizes the treated water. The blocking unit is provided at a subsequent stage of the fluorescence measuring unit and temporarily blocks the discharge of the treated water. The control unit controls the operation of the sterilization unit or the blocking unit based on the fluorescence measurement result obtained by the fluorescence measurement unit.

The figure which shows the structural example of the processing apparatus in 1st Embodiment. Explanatory drawing of the genetically modified microorganisms used suitably for embodiment. The figure which shows the structural example of the processing apparatus in 2nd Embodiment. The figure which shows the structural example of the processing apparatus in 3rd Embodiment. The figure which shows the structural example of the conventional processing apparatus.

  Hereinafter, a processing apparatus according to an embodiment will be described with reference to the drawings.

(First embodiment)
FIG. 1 is a diagram illustrating a configuration example of a processing device 1 according to the first embodiment. In the first embodiment, the processing apparatus is referred to as “processing apparatus 1a”. As an example, the processing apparatus 1a is installed in a sewage treatment plant.

  The processing apparatus 1a includes a biological reaction tank 10, a filtration membrane 20, a fluorescence measurement device (fluorescence measurement means) 40, and an ultraviolet irradiation device (sterilization means) 50. Moreover, the processing apparatus 1a includes a dehydrator 60 and a dryer 70.

The biological reaction tank 10 is a tank that holds a genetically modified microorganism and performs biological treatment of treated water with the genetically modified microorganism. Drainage, which is raw water for treatment water, is introduced into the biological reaction tank 10 through the pipe 2. Examples of the wastewater include sewage, agricultural settlement wastewater, and factory wastewater.
The pipe 2 is provided with a screen 2 a that removes relatively large dust in the waste water, and prevents introduction of dust into the biological reaction tank 10. For the screen 2a, for example, a filter of about 1 mm can be used.

The biological reaction tank 10 holds genetically modified microorganisms. The raw water of the treated water introduced into the biological reaction tank 10 is biologically processed with high efficiency by the genetically modified microorganism.
A genetically modified microorganism is a microorganism into which a fluorescent protein gene and a function-enhanced gene that expresses a treatment function for a substance to be treated are incorporated by genetic recombination. Since the genetically modified microorganism has a fluorescent protein gene incorporated, it expresses the fluorescent protein and exhibits fluorescence when irradiated with excitation light. In addition, since the genetically modified microorganism has a function-enhanced gene incorporated therein, it exhibits an excellent treatment function for the substance to be treated in the treated water. Genetically modified microorganisms proliferate using organic matter in wastewater or organic matter that is decomposed by its own wastewater treatment.

FIG. 2 is an explanatory diagram of a genetically modified microorganism used in the processing apparatus 1a.
It is possible to cause yeast to produce lipase by introducing a gene encoding lipase information into the yeast by genetic recombination. Lipase has a function of decomposing oils and fats.
Moreover, it is possible to make yeast produce fluorescent protein by introducing a fluorescent protein gene into yeast by gene recombination.
In the embodiment shown in FIG. 2, by incorporating the fluorescent protein gene EGFP and the lipase gene, the yeast cell wall exhibits fluorescence, and a lipase that is an oil-degrading enzyme can be produced on the surface layer. EGFP is one of green fluorescent proteins, and can detect a fluorescence wavelength of 509 nm when a wavelength of 488 nm is excited.

  In the embodiment, the treated water includes what is introduced into the biological reaction tank 10, what is stored in the biological reaction tank 10, and what is discharged from the biological reaction tank 10. At least a part of the liquid of the treated water passes through the filtration membrane 20 provided in the biological reaction tank 10 to remove the genetically modified microorganisms and is sent to the pipe 3.

The filtration membrane 20 is disposed in the biological reaction tank 10 and is provided in the treated water of the biological reaction tank 10. As the filtration membrane 20, for example, a membrane having pores smaller than that of the genetically modified microorganism can be used, and is usually used in a membrane separation activated sludge method (MBR) in which activated sludge is solid-liquid separated by a membrane. A film to be used may be used. For example, the diameter of the yeast is approximately 5 to 10 μm, and the filtration membrane may be any one as long as it has a smaller pore diameter. The pore size of MF membranes that are often on the market is about 0.1 μm to 0.5 μm.
The air supplied from the cleaning blower 10a is released into the treated water as bubbles with respect to the filtration membrane 20, thereby preventing the filtration membrane 20 from being clogged. The air supplied from the cleaning blower 10a increases the dissolved oxygen concentration of the treated water in the biological reaction tank 10 and contributes to the provision of the dissolved oxygen concentration suitable for the growth of the genetically modified microorganism. As shown in FIG. 1, in order to further increase the dissolved oxygen concentration in the biological reaction tank 10, an auxiliary aeration blower that supplies air to the treated water in the biological reaction tank 10 may be provided.

The treated water discharged from the biological reaction tank 10 passes through the pipe 3 and is discharged out of the processing apparatus 1a.
A fluorescence measuring device 40 is installed in the pipe 3. Since the genetically modified microorganisms are separated by the filtration membrane 20, the genetically modified microorganisms are not contained in the treated water that has passed through the filtration membrane. However, the risk that the genetically modified microorganisms are mixed into the treated water that has passed through the filtration membrane 20 due to some abnormality such as the filtration membrane 20 breaking is not zero. However, according to the treatment apparatus 1a of the embodiment, the fluorescence measuring apparatus 40 can detect the fluorescence exhibited by the genetically modified microorganisms in the treated water that has passed through the filtration membrane, and the genetic recombination in the treated water discharged from the biological reaction tank. The presence of microorganisms can be detected. The fluorescence measuring device includes, for example, an excitation light source and a light receiving unit. When the genetically modified microorganism is irradiated with excitation light emitted from the excitation light source, fluorescence emitted from the fluorescent protein is detected by the light receiving unit. The excitation light source is, for example, a xenon lamp, a laser oscillator, an ultraviolet LED, or the like. The light receiving unit is, for example, a photodetector or a CCD image sensor.
For example, since the genetically modified microorganism of the embodiment expresses EGFP, the treated water is observed with a fluorescence measuring device 40 (excitation wavelength 488 nm, fluorescence wavelength 509 nm) corresponding to the wavelength of EGFP. The treated water may be observed all the time, for example, about once every 10 to 20 minutes. The measurement of fluorescence in the treated water does not have to target all the treated water that has passed through the filtration membrane and discharged from the biological reaction tank. May be performed.

The treatment apparatus 1a includes a sterilization unit that sterilizes the treated water after the fluorescence measurement unit.
In the pipe 3, an ultraviolet irradiation device 50 is installed downstream of the fluorescence measuring device 40. As described above, since the genetically modified microorganisms are separated by the filtration membrane 20, the treated water in the pipe 3 does not contain the genetically modified microorganisms. However, even if the genetically modified microorganism is mixed into the treated water due to some abnormality such as the filtration membrane 20 being broken, the genetically modified microorganism in the treated water is sterilized by the ultraviolet irradiation device 50 and killed. be able to. Since the treated water after passing through the ultraviolet irradiation device does not contain genetically modified microorganisms, it can be discharged into public water areas such as rivers.

The processing apparatus 1a includes a control unit that controls the operation of the sterilization unit based on the fluorescence measurement result obtained by the fluorescence measurement unit. That is, the processing apparatus 1a includes a control unit 80 that controls the ultraviolet irradiation of the ultraviolet irradiation device based on the fluorescence measurement value detected by the fluorescence measuring device 40.
For example, during normal operation, the ultraviolet irradiation device 50 does not operate, but when the fluorescence measurement device 40 measures even a slight amount of fluorescence, the ultraviolet irradiation device 50 is operated. The treated water may be sterilized by the ultraviolet irradiation device 50, and the genetically modified microorganisms in the treated water that has passed through the filtration membrane 20 may be sterilized.
Further, for example, the control unit 80 may include a calculation unit that calculates the ultraviolet irradiation amount of the ultraviolet irradiation device 50 based on the fluorescence intensity detected by the fluorescence measuring device 40. From the detected fluorescence intensity, the concentration of the genetically modified microorganism in the treated water in the pipe 3 that has passed through the filtration membrane 20 and the amount of ultraviolet irradiation that can sterilize the genetically modified microorganism are calculated. The control unit may control the ultraviolet irradiation amount of the ultraviolet irradiation device 50 so as to irradiate the calculated ultraviolet irradiation amount.

The processing apparatus 1a includes dehydrating means for dewatering sludge containing genetically modified microorganisms discharged from the biological reaction tank 10, and return means for returning the desorbed liquid obtained by the dehydrating means to the biological reaction tank. .
Since the genetically modified microorganisms and other microorganisms in the biological reaction tank 10 grow in accordance with the amount of wastewater treatment, it is preferable that the biological reaction tank 10 is periodically withdrawn from the biological reaction tank 10 as excess sludge.
A part of the extracted excess sludge is sent to the dehydrator 60 (dehydrating means) through the pipe 4 and dehydrated by the pump 4a. The dehydrator 60 is not particularly limited as long as it has means for dehydrating excess sludge, and examples thereof include a centrifugal dehydrator and a compression dehydrator. The desorbed liquid, which is a dehydrated liquid component, is returned to the biological reaction tank 10 via the return pipe 6. The return means is a return pipe 6 and a pump provided in the return pipe 6.
Part of the extracted excess sludge is returned to the biological reaction tank through the pipe 5 by the pump 5a as return sludge.

The processing apparatus 1a includes sludge sterilization means for sterilizing sludge containing the genetically modified microorganisms discharged from the biological reaction tank 10.
The solid content of the sludge remaining after dehydration is sent to a dryer 70 (dry sterilization apparatus) and dried by heat at 120 ° C. Since the genetically modified microorganisms contained in the solid content of the sludge are killed by the heat at 120 ° C., the dryer 70 also serves as a sludge sterilization means.
With such a configuration, the genetically modified microorganism is sterilized, and excess sludge is reduced. The surplus sludge that has been subjected to the drying treatment eventually becomes waste or can be used as fertilizer.

  According to the processing apparatus 1a of the first embodiment, as described above, since the fluorescence measuring apparatus 40 capable of detecting the fluorescence exhibited by the genetically modified microorganism into which the fluorescent protein gene is incorporated, the outflow of the genetically modified microorganism is provided. Can be detected.

  Furthermore, according to the treatment apparatus 1a of the first embodiment, since the ultraviolet irradiation device 50 for sterilizing the treated water is provided, the genetically modified microorganism can be sterilized, and the genetically modified microorganism flows out of the processing plant system. Can be prevented.

  Hereinafter, the processing method of the first embodiment will be described.

The processing method of the embodiment includes a processing step, a separation step, and a fluorescence measurement step. The treatment step is a step of biologically treating the treated water with a genetically modified microorganism into which a fluorescent protein gene and a function-enhanced gene that expresses a treatment function for a treatment target substance are incorporated by genetic recombination. The separation step is a step of passing at least a part of the biologically treated treated water through a filtration membrane that filters the treated water. The fluorescence measurement step is a step of measuring the fluorescence of the treated water that has passed through the filtration membrane.
An apparatus for carrying out the processing method of the embodiment is not particularly limited, but it is preferable to use the processing apparatus of the embodiment. Hereinafter, the processing method of the first embodiment using the processing apparatus 1a of the first embodiment will be described. In addition, description is abbreviate | omitted about the point which is common in the description with the processing apparatus of 1st Embodiment.

First, wastewater, which is raw water of treated water, is introduced into the biological reaction tank 10 holding the genetically modified microorganisms via the pipe 2, and the treated water is biologically treated with the genetically modified microorganisms (processing step). .
Biological treatment as used herein refers to, for example, a function that a genetically modified microorganism decomposes a target substance, a function that converts a target substance into another substance, a function that takes in a target substance, and adsorbs a target substance Function etc. can be mentioned. By these functions, the treated water can be purified.

  Next, at least a part of the biologically treated treated water is allowed to pass through the filtration membrane 20 that does not pass the genetically modified microorganisms in the treated water, but allows the liquid component in the treated water to pass through. Water is obtained (separation step). By passing the treated water through the filtration membrane 20, the genetically modified microorganisms are removed from the treated water.

  The treated water that has passed through the filtration membrane 20 is sent to the pipe 3, and the fluorescence of the treated water that has passed through the filtration membrane 20 and is discharged from the biological reaction tank 10 is measured by the fluorescence measurement device 40 (measurement step). Since the genetically modified microorganisms are separated by the filtration membrane 20, the genetically modified microorganisms are not contained in the treated water that has passed through the filtration membrane 20. However, when a genetically modified microorganism is mixed into the treated water that has passed through the filtration membrane 20 due to some abnormality such as the filtration membrane 20 breaking, the fluorescence emitted from the genetically modified microorganism is detected by the fluorescence measuring device 40. The presence of genetically modified microorganisms in the treated water can be detected.

The processing method of the first embodiment may have a control step of controlling the operation of the sterilization means based on the fluorescence measurement result obtained in the measurement step. For example, during normal operation, the ultraviolet irradiation device 50 does not operate. However, in the measurement process, when even a slight amount of fluorescence is measured by the fluorescence measuring device 40, the ultraviolet irradiation device 50 is operated to detect the genetic recombination detected. Microorganisms may be sterilized by ultraviolet irradiation.
In the processing method of the first embodiment, the control step may include a calculation step of calculating the ultraviolet irradiation amount of the ultraviolet irradiation device based on the fluorescence measurement result obtained in the measurement step. For example, from the fluorescence intensity detected in the measurement step, the concentration of the genetically modified microorganism in the treated water that has passed through the filtration membrane 20 and the amount of ultraviolet irradiation that can sterilize the genetically modified microorganism are calculated. The control unit may control the ultraviolet irradiation amount of the ultraviolet irradiation device 50 so as to irradiate the calculated ultraviolet irradiation amount based on the calculated ultraviolet irradiation amount.

  According to the treatment method of the first embodiment, by using a genetically modified microorganism to which a lipase functional gene is added, it is possible to treat wastewater containing oils and fats that are usually difficult to biodegrade.

  According to the treatment method of the first embodiment, solid-liquid separation on a filtration membrane is easy by using yeast, which is a relatively large microorganism of 5 to 10 nm, as a recombinant.

  According to the treatment method of the first embodiment, a genetically modified microorganism can be detected with high sensitivity by using a fluorescent protein. Further, by using a fluorescent protein having a known excitation wavelength / fluorescence wavelength, a genetically modified microorganism can be detected easily and accurately.

(Second Embodiment)
In 2nd Embodiment, it replaces with the biological reaction tank 10 in the processing apparatus 1, the point provided with the biological reaction tank 11, the storage tank 30, and the pipes 7, 8, and 9 are provided, ultraviolet irradiation It is different from 1st Embodiment that it replaces with the apparatus 50 and the cutoff valve 100 (blocking means) which interrupts | blocks discharge of treated water temporarily is provided. The biological reaction tank 11 is a reaction tank that holds an anaerobic genetically modified microorganism and biologically treats treated water with the anaerobic genetically modified microorganism. In the second embodiment, only differences from the first embodiment will be described.

  FIG. 3 is a diagram illustrating a configuration example of the processing device 1 according to the second embodiment. In the second embodiment, the processing apparatus is referred to as “processing apparatus 1b”.

The biological reaction tank 11 is a hollow cylindrical container that stores a carrier. A treatment water inlet 11d is provided at the lower end of the biological reaction tank 11, and a treatment water discharge port 11c is provided at the top. In addition, an exhaust port 11e for gas generated with anaerobic treatment is provided at the upper end of the biological reaction tank 11. The biological reaction tank 11 stores a carrier to which microorganisms are attached. The treated water is configured to flow from the lower part to the upper part of the biological reaction tank 11. The treated water introduced from the inflow port 11d is subjected to anaerobic treatment by anaerobic microorganisms when passing through the carrier. The treated water that has passed through the carrier is discharged to the outside of the biological reaction tank 11 through the discharge port 11c. The gas generated during the anaerobic treatment is discharged from the exhaust port 11e.
In the processing apparatus 1a of the first embodiment, since the microorganisms in the biological reaction tank use aerobic microorganisms including genetically modified microorganisms, the biological reaction tank 10 includes the auxiliary aeration blower 10b and the cleaning blower 10a. It was configured to send air into it. In the processing apparatus 1 b shown in FIG. 3, air is not introduced from the outside of the tank, but the gas discharged from the exhaust port 11 e is sent to the cleaning blower 11 a through the pipe 7. The gas supplied from the cleaning blower 11a to the filtration membrane 20 becomes bubbles and is released into the water in the biological reaction tank 11, so that the filtration membrane 20 is prevented from being clogged.
One end of the pipe 8 is connected to the discharge port 11 c of the biological reaction tank 11. The treated water in the biological reaction tank 11 is discharged through the pipe 8 and stored in the storage tank 30. The pipe 9 is connected to the inlet 11 d of the biological reaction tank 11. The treated water in the storage tank 30 is introduced into the biological reaction tank from the inlet 11d through the pipe 9.

The processing apparatus 1b includes a control unit that controls the operation of the blocking unit based on the fluorescence measurement result obtained by the fluorescence measurement unit. That is, the processing apparatus 1b includes a control unit 80 that controls the opening / closing of the shutoff valve 100 based on the fluorescence measurement value detected by the fluorescence measurement apparatus 40.
For example, during normal operation, the shutoff valve 100 is open, but when the fluorescence measuring device 40 measures even a slight amount of fluorescence, the shutoff valve 100 is closed and the detected genetically modified microorganism flows out of the system. You may make it prevent.

  According to the processing apparatus 1b of 2nd Embodiment, since the cutoff valve 100 is provided in the piping 3, the outflow of the genetically modified microorganisms outside a system can be prevented.

  According to the treatment apparatus 1b of the second embodiment, anaerobic microorganisms can be used to perform wastewater biological treatment, and the anaerobic microorganisms are used as genetically modified microorganisms with enhanced wastewater treatment functions. be able to.

  Hereinafter, the processing method of the second embodiment using the processing apparatus 1b of the second embodiment will be described. In the second embodiment, only differences from the first embodiment will be described.

  The processing method of the second embodiment may have a control step of controlling the operation of the blocking means based on the fluorescence measurement result obtained in the measurement step. That is, the processing method according to the second embodiment may include a control step of controlling the opening / closing of the shutoff valve 100 based on the fluorescence measurement value detected by the fluorescence measurement device 40. For example, during normal operation, the shut-off valve 100 is kept open, and the shut-off valve 100 is closed when fluorescence is measured even slightly by the fluorescence measuring device 40 in the measurement process. In this way, the detected genetically modified microorganism may be prevented from flowing out of the system.

  According to the treatment method of the second embodiment, by controlling the operation of the shutoff valve 100, the treated water containing the genetically modified microorganism can be blocked, and the genetically modified microorganism can be prevented from flowing out of the system.

(Third embodiment)
The third embodiment is different from the first embodiment in that the treatment apparatus 1 is provided with a microorganism culture tank 90 for culturing genetically modified microorganisms. In the third embodiment, only differences from the first embodiment will be described.

  FIG. 4 is a diagram illustrating a configuration example of the processing apparatus 1 according to the third embodiment. In the third embodiment, the processing device is referred to as a “processing device 1c”.

  The microorganism culture tank 90 is a tank for culturing genetically modified microorganisms. For example, genetically modified microorganisms may be periodically (for example, once a week) added from the microorganism culture tank 90 to the biological reaction tank 10, and when the biological treatment performance in the biological reaction tank 10 decreases, You may comprise so that it may add in addition. Alternatively, the genetically modified microorganisms may be constantly fed in a constant amount from the microorganism culture tank 90 to the biological reaction tank 10.

The microorganism culture tank 90 includes means for suitably growing the genetically modified microorganism. Examples of such means include temperature control means, pH control means, substrate concentration control means such as a turbidimeter, pressure control means, stirring means and the like.
As an example, when yeast is used as the genetically modified microorganism, the microorganism culture tank 90 is controlled so that the environment in the tank becomes 20 to 30 ° C. and pH 4 to 7.

A substrate tank for storing a medium to be supplied to the microorganism culture tank 90 may be connected to the microorganism culture tank 90. The medium is not particularly limited as long as it can cultivate microorganisms or the like in the microorganism culture tank 90, and a medium containing a carbon source, a nitrogen source, inorganic salts and the like is preferable.
Examples of the carbon source include saccharides such as glucose, fructose, and sucrose; and carbohydrates such as starch or starch hydrolyzate.
Nitrogen sources include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, and ammonium acetate, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal, soybean meal hydrolyzate And various fermented bacterial cell digests.
Examples of inorganic salts include magnesium phosphate, magnesium sulfate, sodium chloride, monopotassium phosphate, dipotassium phosphate, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like.

According to the processing apparatus 1c of the third embodiment, the same effects as those of the first embodiment can be obtained, and the following effects can also be obtained.
According to the processing apparatus 1c of the third embodiment, since the genetically modified microorganisms cultured in the microorganism culture tank 90, which is a culture tank different from the biological reaction tank 10, can be introduced into the biological reaction tank 10, the biological reaction The processing performance of the tank 10 can be stabilized.

  In each of the above embodiments, the ultraviolet irradiation device is exemplified as the sterilizing means for sterilizing the genetically modified microorganisms in the treated water. However, the sterilizing apparatus is provided with means for killing the genetically modified microorganisms in the treated water. If there is no particular limitation. Examples of the sterilization apparatus include those provided with ozone treatment means, heating means, pH adjustment means, pressure control means, and the like. It may be provided with a sterilization means using saturated steam at high temperature and high pressure like an autoclave.

  In each of the above embodiments, the filtration membrane is exemplified in an embodiment arranged in a biological reaction tank. However, if the filtration membrane is capable of filtering the treated water biologically treated in the biological reaction tank, the biological reaction is performed. You may arrange | position outside the tank.

  In each of the above embodiments, the genetically modified microorganism is a microorganism into which the fluorescent protein gene EGFP and the lipase gene are introduced by genetic recombination, but the following microorganism may be used.

Hereinafter, the genetically modified microorganism will be described in detail.
Microorganisms can synthesize various substances such as enzymes based on the genetic information (DNA) they possess. Therefore, for example, a specific microorganism can be artificially introduced (genetic recombination) into a gene that encodes information on a substance that has the function of treating the target substance in wastewater, thereby causing the microorganism to produce the target substance. Is possible.

In the embodiment, the “fluorescent protein gene” is a gene encoding a fluorescent protein. When the fluorescent protein gene is introduced into the microorganism, the fluorescent protein is expressed in the microorganism.
The fluorescent protein may be any fluorescent protein having a known excitation wavelength / fluorescent wavelength such as GFP, YFP, CFP, mOrange, and mBana, in addition to EGFP.
A microorganism can be marked by introducing a fluorescent protein gene into the microorganism.

  A fluorescent protein is introduced into a microorganism as a fluorescent protein gene and synthesized by the microorganism based on information on the fluorescent protein gene. If a method that does not inherit a progeny, such as fluorescent staining of microorganisms, is adopted as a fluorescent marker, the microorganisms that have grown are not marked. On the other hand, in the present embodiment, since the fluorescent protein gene is inherited to a progeny, even when the microorganism grows, the microorganism is marked even in the grown microorganism.

In addition, in FIG. 2, although the microorganisms whose cell wall exhibited fluorescence were illustrated, fluorescent protein should just be located in the site | part which can detect a microorganism, and may be located inside the microorganism. Further, the fluorescent protein may be localized at a specific site of the microorganism, or may be uniformly arranged throughout the microorganism.
As shown in FIG. 2, the fluorescent protein is preferably disposed on the cell wall or cell membrane of the microorganism. By arranging the fluorescent protein on the cell wall or cell membrane of the microorganism, the detection sensitivity of the microorganism is increased. Moreover, as shown in FIG. 2, it is preferable that fluorescent protein is arrange | positioned uniformly so that the shape of microorganisms can be specified. By arranging the fluorescent proteins uniformly, the shape of the microorganism can be detected in the measurement process of the embodiment, and the detection accuracy of the microorganism is increased.

In the present embodiment, the “function-enhanced gene” has a function of imparting or promoting a treatment function for a substance to be treated by being introduced into a microorganism and expressing its function.
The function-enhanced gene includes not only a gene that encodes a protein having a processing function for a substance to be processed, but also includes all inheritable functional factors such as gene regulatory sequences that enhance the original processing function of microorganisms and RNAi-derived nucleic acids. .
By introducing the function-enhanced gene into the microorganism, the function of the wastewater treatment of the microorganism can be enhanced.

The fluorescent protein gene and the function-enhanced gene are incorporated into a microorganism by genetic recombination so that both can be inherited by progeny. For example, the fluorescent protein gene is introduced in the immediate vicinity of the portion into which the function-enhancing gene is introduced. By inheriting both the fluorescent protein gene and the function-enhanced gene, it can be confirmed by fluorescence whether it is a genetically modified microorganism.
Only one kind of each of the fluorescent protein gene and the function enhancement gene may be introduced, or a plurality of kinds of each may be introduced.

  The substance to be treated is a substance contained in the treated water and may be an organic substance or an inorganic substance. Examples of the processing target substance include various organic substances, phosphorus, phosphorus compounds, metals, and heavy metals.

  Function-enhanced gene processing functions include, for example, a function in which a microorganism expressing a function-enhanced gene decomposes a target substance, a function that converts a target substance into another substance, a function that takes in a target substance, and a target to be processed The function of adsorbing substances can be mentioned. By these functions, the treated water can be purified.

Whether or not the function-enhanced gene has a function of degrading the substance to be treated can be determined by comparing a recombinant microorganism into which the function-enhanced gene is incorporated and a control microorganism into which the function-enhanced gene is not incorporated.
For example, in the comparison of the same conditions, the concentration of the target substance in the treated water is lower in the treated water biologically treated with the recombinant microorganism than in the treated water biologically treated with the control microorganism. If it has increased, it can be determined that the function-enhanced gene has a decomposition function for the substance to be treated.

Whether or not a functionally enhanced gene has a conversion function for a substance to be treated can be determined by comparing a recombinant microorganism incorporating the enhanced gene with a control microorganism not incorporating the enhanced gene. is there.
For example, in the comparison of the same conditions, the treated water biologically treated with the recombinant microorganism has a lower concentration of the substance to be treated in the treated water and the conversion product increases than the treated water biologically treated with the control microorganism. If so, it can be determined that the function-enhanced gene has a conversion function for the substance to be treated.

Whether or not a functionally enhanced gene has an uptake function for a substance to be treated can be determined by comparing a recombinant microorganism incorporating the functionally enhanced gene with a control microorganism that does not incorporate the functionally enhanced gene. is there.
For example, in the comparison of the same conditions, the concentration of the substance to be treated in the treated water is lower in the treated water biologically treated with the recombinant microorganism than in the treated water biologically treated with the control microorganism. If the target substance has increased, it can be determined that the function-enhanced gene has a function of taking up the target substance.

Whether or not the enhanced gene has an adsorption function for the substance to be treated can be determined by comparing a recombinant microorganism incorporating the enhanced gene with a control microorganism not incorporating the enhanced gene. is there.
For example, in the comparison of the same conditions, the concentration of the target substance in the treated water is lower in the treated water biologically treated with the recombinant microorganism than in the treated water biologically treated with the control microorganism. If the target substance has increased, it can be determined that the function-enhanced gene has an adsorption function for the target substance.

The function-enhancing gene is preferably a gene encoding an enzyme having a processing function for the substance to be processed.
As the enzyme, hydrolase, oxidoreductase, transferase, eliminase, isomerase, and synthetic enzyme are preferable, and hydrolase is more preferable.
Examples of hydrolases include aminoacylase, α-amylase, glucoamylase, cellulase, invertase, carboxypeptidase, leucine aminopeptidase, penicillin amidase, AMP deaminase, protease, lysozyme, papain, chitinase, cholesterol ester hydrolase, alkaline phosphatase, β- Glucosidase, β-D-galactosidase, asparaginase, glutaminase, urease, penicillinase, creatininase, creatinase, creatine deiminase, pectinase, catalase, lipase, hemicellulase, aminopeptidase, chymotrypsin, urokinase, urease, amidase, glucose phosphatase Ribonuclease, various restriction enzymes (endodeoxy) Ribonuclease), phospholipases, saccharase and the like, amylase, cellulase, protease, lysozyme are preferred.

The function-enhancing gene suitably used in the embodiment is selected from the group consisting of a gene encoding a lipase enzyme, a gene encoding a cellulase enzyme, a gene encoding a protease enzyme, and a gene encoding an amylase enzyme It is mentioned that it is any 1 type or more.
The lipase enzyme is a group of enzymes having a function of decomposing lipid, for example, triacylglyceride lipase. The cellulase enzyme is an enzyme group having a function of hydrolyzing a glycosidic bond, and examples thereof include cellulase and hemicellulase. Protease enzymes are a group of enzymes having a function of hydrolyzing peptide bonds. An amylase enzyme is an enzyme group having a function of hydrolyzing a glycosidic bond, for example, α-amylase and glucoamylase.

Examples of the microorganisms to be genetically modified include Pseudomonas genus bacteria such as Pseudomonas alcaligenes, P. putida, P. dacunhae, etc .; Gluconobacter melanogenes, G. oxydans, etc .; Gluconobacter genus gram negative bacteria; Alcaligenes eutrophus Gram-negative bacteria; acetic acid bacteria such as Acetobacter suboxydans; coliform bacteria such as Escherichia coli, E. freundii, Enterobacter aerogenes; other gram-negative bacteria such as Erwinia carotovora, Serratia marcescens, Protaminobacter rubrum, Proteus mirabilis .
Also, lactic acid bacteria such as Streptococcus faecalis, Leuconostoc mensenteroides, and Lactobacillus delbruckii; Gram-positive bacteria of the genus Bacillus such as Bacillus subtilis and B. megaterium; Other Gram-positive bacteria such as Corynebacterium glutamicum, Brevibacterium ammoniagenes, B. flavum, Propionibacterium sp.
Furthermore, Nocardia rhodocrous, Streptomyces phaeochromogenes, S. rimosus, S. roseochromogenes, S. tendae, S. rimosus and other actinomycetes, Saccharomyces sp., Hansenula jadinii, Candida tropicalis, Rhodotorula minuta, etc. And filamentous fungi such as Curvularia lunata, Aspergillusochraceus, A. niger, and Penicillium chrysogenum.
Two or more different microorganisms may be used as the microorganism to be genetically modified.

The genetically modified microorganism is preferably one that secretes or presents the enzyme on the cell surface.
The enzyme produced by the genetically modified microorganism may be secreted from the genetically modified microorganism. In addition, as shown in FIG. 2, the enzyme produced by the genetically modified microorganism may be presented (armed) on the cell surface of the microorganism.

By adding a command sequence to be displayed on the cell surface of the microorganism to the function-enhanced gene, the synthesized enzyme can be presented on the cell surface.
As an example of the method for causing the microorganism to present the enzyme, a method in which a gene encoding a function-enhanced gene and a cell surface display protein is introduced into a specific microorganism by genetic manipulation can be mentioned. Enzymes are synthesized from the cells of microorganisms by gene expression and then presented on the cell surface. More specifically, the following sequence, secretion signal, target enzyme, agglutinin protein, and a sequence containing a gene encoding the anchor attachment signal may be introduced into a microorganism (yeast) to be genetically modified. Agglutinin protein serves as an anchor for fixing the enzyme. An anchor attachment signal includes glycosyl phosphatidylinositol (GPI). By introducing a sequence having the above characteristics into yeast, the target enzyme can be armed into the microorganism. The microorganism on which the degrading enzyme is presented serves as a biocatalyst and acts to lower the molecular weight of organic substances. Utilizing the above technology, using a microorganism such as commonly used yeast, Escherichia coli, etc., producing a biocatalyst having the ability to reduce the molecular weight of organic matter by the above method, and reducing the molecular weight of the organic matter in the wastewater Can be used.
Moreover, when using microorganisms which produce valuables, such as yeast, as a biocatalyst, these microorganisms themselves can also use low molecular organic substances as food. For example, by the action of cellulase presented on the surface of the yeast, the cellulose in the treated water is broken down into low molecular sugars, and the yeast uses this low molecular sugar to perform alcoholic fermentation and produce bioethanol. Can be produced. One or more kinds of enzymes may be expressed in the microorganism. From the viewpoint of expression efficiency, it is preferable to express one type of protein, and it is more preferable to express different types of proteins one by one in a plurality of microorganisms.
By presenting an enzyme suitable for the degradation of the hardly degradable target on the surface layer of the microorganism, the efficiency of reducing the molecular weight by the microorganism can be improved.

A gene that promotes the ability to synthesize polyphosphate in a genetically modified microorganism can be preferably exemplified as a function-enhancing gene that is preferably used in the embodiment.
Recombinant genes that promote synthesis ability of polyphosphoric acid in microorganisms has been incorporated recombinant Escherichia coli (polyphosphoric acid synthesis E. coli), the phosphorus phosphorus Santai in waste water ^- polyphosphoric captures in the cells (PO4) Synthesize acid. In this reaction process, phosphorus is taken into the cells of polyphosphate-synthesizing Escherichia coli and phosphorus in the waste water is removed. Since the genetically modified microorganism is Escherichia coli, it is propagated by the organic matter contained in the waste water. For this reason, phosphorus removal reaction proceeds with high efficiency inside the biological reaction tank.
In this way, since phosphorus in the waste water can be stably removed, the problem of eutrophication of the treated water discharge destination can be reduced. Since excess sludge contains phosphorus in a high concentration, it can be used as a fertilizer. In addition, phosphorus can be removed more efficiently by incorporating it into E. coli having a high growth rate.

As a function-enhanced gene suitably used in the embodiment, a gene encoding a protein having metal adsorption ability can be suitably exemplified. The genetically modified microorganism is preferably one that presents the protein having the metal adsorption ability on the cell surface. A metallothionein is mentioned as a protein which can adsorb | suck a metal.
In a genetically modified yeast in which a gene encoding metallothionein is incorporated, metallothionein is applied to the cell surface layer, and heavy metals in the waste water are adsorbed by metallothionein, thereby removing heavy metals from the water. Metallothionein can adsorb heavy metals such as zinc (Zn ²⁺ ), copper (Cu ⁺ ), cadmium (Cd ²⁺ ), lead (Pb ²⁺ ), silver (Ag ⁺ ), mercury (Hg ²⁺ ) it can. Since the yeast with enhanced functions is grown using the organic matter in the wastewater, heavy metals can be adsorbed and removed with high efficiency.
In this way, heavy metals in the waste water can be stably removed, so that the treatment of heavy metals usually performed by chemical treatment can be carried out by biological treatment which is an economical treatment.
In addition, the metal adsorption protein imparted to the cell surface by gene recombination is not limited to metallothionein, but any protein known to have a function of adsorbing heavy metals such as cysteine, cystine, histidine, etc. It may be.

  FIG. 5 shows a configuration example of a conventional processing apparatus that performs a membrane separation activated sludge process (MBR). In MBR, the amount of microorganisms can be maintained at a higher concentration than in the conventional activated sludge method, so that space saving can be realized, and furthermore, since solid-liquid separation of activated sludge is performed with a membrane, extremely high processing performance (BOD, SS) , Nitrogen). However, in this MBR method, in order to recycle treated water, in many cases, in order to remove persistent oxygen-degraded COD (Chemical Oxygen Demand), which is an organic component that is hardly biodegradable, oxidation such as ozone is subsequently performed. There is room for improvement in terms of operating costs because it requires an apparatus, or a chemical treatment such as agglomeration treatment or another membrane treatment such as a reverse osmosis membrane. Oil components and cellulosic organic components that are difficult to decompose by microorganisms present in activated sludge must be used in combination with other treatments. There is a problem that it is necessary to add a flocculant such as PAC (polyaluminum chloride) for stable phosphorus removal.

  On the other hand, according to the processing apparatus and the processing method of the embodiment, since a genetically modified microorganism in which a function-enhanced gene that expresses a processing function for a processing target substance is incorporated by genetic recombination is used, conventional biological processing has been difficult. Even substances can be processed in a biological reactor. Thereby, the processing equipment required for processing a substance that has conventionally been difficult to biologically process can be simplified.

  According to at least one embodiment described above, by having a fluorescence measuring means, it is possible to detect a genetically modified microorganism in which a fluorescent protein gene is incorporated, and to prevent the genetically modified microorganism from being released into the environment. Leads to.

  Although several embodiments of the present invention have been described, these embodiments are presented by way of example and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, replacements, and changes can be made without departing from the spirit of the invention. These embodiments and their modifications are included in the scope and gist of the invention, and are also included in the invention described in the claims and the equivalents thereof.

DESCRIPTION OF SYMBOLS 1 ... Processing apparatus, 1a ... Processing apparatus, 1b ... Processing apparatus, 1c ... Processing apparatus, 2 ... Piping, 2a ... Screen, 3 ... Piping, 4 ... Piping, 4a ... Pump, 5 ... Piping, 5a ... Pump, 6 ... Return pipe, 7 ... pipe, 8 ... pipe, 9 ... pipe, 10 ... biological reaction tank, 10a ... washing blower, 10b ... auxiliary diffuser, 11 ... biological reaction tank, 11a ... washing blower, 11c ... discharge port 11d ... Inlet, 11e ... Exhaust port, 20 ... Filtration membrane, 30 ... Reservoir, 40 ... Fluorescence measuring device, 50 ... Ultraviolet irradiation device, 60 ... Dehydrator, 70 ... Dryer, 80 ... Control unit, 90 ... Microorganism culture tank, 100 ... shut-off valve

Claims (11)

A biological reaction tank in which a fluorescent protein gene and a function-enhanced gene that expresses a treatment function for a substance to be treated hold a genetically modified microorganism incorporated by genetic recombination, and biologically treat the treated water by the genetically modified microorganism; ,
A filtration membrane for filtering the treated water biologically treated in the biological reaction tank;
A fluorescence measuring means for measuring fluorescence of the treated water discharged from the biological reaction tank through the filtration membrane;
A sterilization means for sterilizing the treated water, provided in a subsequent stage of the fluorescence measuring means, or a blocking means for temporarily blocking discharge of the treated water;
A processing apparatus comprising: a control unit that controls the operation of the sterilization unit or the blocking unit based on a fluorescence measurement result obtained by the fluorescence measurement unit. With the sterilizing means, the sterilizing means is a UV irradiation apparatus, the control unit, on the basis of the fluorescence measurement means fluorescence measurement results obtained, the processing apparatus according to claim 1 for controlling the UV irradiation. Furthermore, the processing apparatus of Claim 1 or 2 provided with the sludge sterilization means which sterilizes the sludge containing the said genetically modified microorganisms discharged | emitted from the said bioreaction tank. The processing apparatus according to claim 3 , wherein the sludge sterilization means is a dry sterilization apparatus. The enhancements gene is a gene encoding an enzyme having a processing function for processing target material, the genetically modified microorganism, claim 1-4 is intended to be presented to the secretion or cell surface the enzyme The processing apparatus according to one item. The function-enhancing gene is at least one selected from the group consisting of a gene encoding a lipase enzyme, a gene encoding a cellulase enzyme, a gene encoding a protease enzyme, and a gene encoding an amylase enzyme. The processing apparatus according to claim 5 . The processing apparatus according to any one of claims 1 to 4 , wherein the function-enhanced gene is a gene that promotes the ability to synthesize polyphosphate in the genetically modified microorganism. The enhancements gene is a gene encoding a protein having a metal-adsorbing ability, the genetically modified microorganism, claim 1-4 proteins having the metal adsorption capacity is intended to be presented on the cell surface The processing apparatus according to one item. Furthermore, the processing apparatus as described in any one of Claims 1-8 provided with the culture tank which culture | cultivates the said gene recombinant microorganism. Furthermore, a dehydrating means for dewatering the sludge containing the genetically modified microorganisms discharged from the biological reaction tank,
Processing apparatus according to any one of claims 1 to 9, and a returning means for returning the eluate obtained by the dehydration unit to the bioreactor. A treatment step of biologically treating the treated water with a genetically modified microorganism in which a fluorescent protein gene and a function-enhanced gene that expresses a treatment function for a substance to be treated are incorporated by genetic recombination
A separation step of passing at least a part of the biologically treated water through a filter membrane for filtering the treated water;
A fluorescence measurement step of measuring the fluorescence of the treated water that has passed through the filtration membrane;
Based on the fluorescence measurement result obtained in the fluorescence measurement step, a control step for controlling the operation of the sterilization means or the blocking means,
Processing method.

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