JP3174518B2 - Simple diagnostic method for biological treatment of sewage - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for diagnosing biological treatment, and more particularly, to a simple method for diagnosing biological treatment of sewage such as sewage and human waste.

[0002]

2. Description of the Related Art In recent years, sewage such as sewage and human waste has been subjected to biological treatment using microorganisms. The microorganisms involved in these biological treatments are diverse and include microorganisms having various characteristics and functions, and the dominant bacterial species differs depending on the target of the biological treatment. In an actual sewage treatment plant or the like, it is necessary to grasp the state of biological treatment in order to efficiently utilize these mixed microorganism groups and perform excellent treatment. To grasp the situation appropriately and quickly, for example, if the processing is not progressing well, take appropriate measures,
It is necessary to prevent deterioration of biological treatment.

In order to grasp the state of biological treatment, it is necessary to measure the quality (characteristics) and quantity (density) of a wide variety of microorganisms from bacteria living in treatment tanks to protozoa. However, it is cumbersome and impractical to culture in a medium suitable for each characteristic and perform various physiological tests after isolation and culture.

Conventionally, among the biologically treated microorganisms, a bacterium that plays a central role in decomposing and assimilating organic matter is a gram-positive bacillus, a useful bacterium having an endospore-forming ability, and a degrading ability of this organic matter. Useful bacteria in the reaction tank
0 ⁶ cells / ml desirable in conducting superior biological treatment that occurs more than is known.

The use of Bacillus bacteria for sewage treatment is also described in Japanese Patent Publication No. 426834.

[0006] Accordingly, the inventor of the present invention has determined the progress of the biological treatment by measuring the quality (characteristics) and quantity (density) of the Bacillus bacterium as an important indicator bacterium in the biological treatment. Thought that you can grasp.

However, it has been confirmed that there are more than 30 Bacillus bacteria involved in biological treatment. To confirm the decomposability of each Bacillus bacterium, first, a test sample is diluted and plated. The medium is usually cultured for 4 to 12 days,
After germination / proliferation, microscopic inspection is performed for each colony that has occurred, and degradation / degradation function tests for each nutrient are performed after isolation / culture. It took weeks, and it was not possible to grasp the current state of Bacillus spp., Which is changing daily, and it was not suitable for daily facility management. In addition, since this method requires specialized knowledge and inspection technology, it is necessary to allocate a specialized staff, and it is difficult to use it for daily facility management in terms of cost.

[0008]

SUMMARY OF THE INVENTION Therefore, the present invention can easily and quickly determine a bacterium belonging to the genus Bacillus, which is an indicator bacterium for biological treatment, and can easily determine its nutritional resolution and the like. It is an object of the present invention to provide a simple diagnosis method of biological treatment of sewage that can be used for daily facility management.

[0009]

A simple diagnostic method for biological treatment of sewage according to the present invention, which solves the above-mentioned problems, comprises receiving sewage in a reaction tank in which useful bacteria which are aerobic and form spores inhabit and receive the sewage. When biologically treating sewage by a biological treatment system having a reaction step of biologically treating the components and a solid-liquid separation step of solid-liquid separation of the sludge sent from the reaction step, the sludge in the biological treatment system And diluting the sludge at different magnifications to produce a plurality of test samples, and the plurality of test samples are used as nutrients required by various microorganism groups involved in biological treatment of sludge. The culture medium A suitable for the examination of the multi-species coexisting sample comprising the source and the culture medium A separately cultured in the culture medium B obtained by adding starch and yeast extract,
From one medium A, it is visually determined whether or not an early colony is formed. If an early colony is formed, whether the colony is a colony of a Bacillus bacterium which is a useful bacterium is determined. The presence of Bacillus bacteria, a useful bacterium in the sludge, is visually determined by judging whether
When the colony is a colony of the bacterium belonging to the genus Bacillus, it is visually determined whether or not spores are formed, and then the number of colonies of the bacterium belonging to the genus Bacillus on the medium A is counted. By measuring the density of the bacteria and measuring the appearance rate of bacteria having the starch-degrading ability from the other medium B, of the colonies of the genus Bacillus among the colonies formed on the medium B, the useful bacteria in the sludge were measured. It is characterized by judging the quality of biological treatment of wastewater without performing isolation culture.

[0010] Preferably, the medium A contains at least meat extract, peptone, glucose and salt.

The sewage is sewage or night soil, and when counting the number of colonies of Bacillus bacteria and measuring the density, the dilution ratio x and the count number y of the test sample in which the number of colonies were counted are as follows. It is preferable to simply determine the quality of the biological treatment status of the wastewater using the satisfaction of the expression as an index. [Formula] y × 10 ^x ≧ 10 ⁵

[0012] More preferably, the appearance rate of bacteria having starch degradability is 10% or more.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a biological treatment system is a system in which sewage such as sewage and human waste is received in a reaction tank in which useful bacteria that form aerobic spores inhabit, and components in the sewage are subjected to biological treatment. And a solid-liquid separation step of solid-liquid separation of the sludge sent from the reaction step.

An important indicator bacterium in these biological treatment systems is a bacillus belonging to the genus Bacillus. The bacterium belonging to the genus Bacillus has mainly four kinds of properties, and is capable of selectively decomposing only carbohydrates and proteins. There are species that selectively degrade, those that degrade both carbohydrates and proteins, and those that do not degrade both carbohydrates and proteins.

In the biological treatment of sludge, although it depends on the type of nutrient, the species that selectively decomposes only the above-mentioned proteins dominates, and the next most species are those that selectively degrade only carbohydrates. is there. Most of the species that degrade proteins also degrade (solubilize) lipids. Bacillus bacteria have endogenous spore-forming properties and can sporulate under adverse environments such as oligotrophic conditions and can withstand low pH, drying, low oxygen, high and low temperatures, pressure changes, and the like. On the contrary, in a favorable environment such as a eutrophic state, the germination and propagation again occur. In addition, the generation time is 14 to 45 minutes, and it becomes dominant in an appropriate environment.

Further, other characteristics of the bacterium belonging to the genus Bacillus include:
During its growth and sporulation, it secretes antibiotics, synthesizes special enzymes, suppresses the growth of fine dust, fly and mosquitoes, and directly attacks the cell wall (membrane) of spoilage bacteria, It also has the effect of preventing the growth of.

Bacteria belonging to the genus Bacillus used for the biological treatment described above have three characteristics depending on their properties, properties, functions and biological characteristics.
0 or more types have been classified and confirmed. Among these various types of Bacillus bacteria, there is a difference in their ability to degrade nutrients,
According to the present invention, it is possible to easily and quickly determine the difference in the ability to decompose nutrients.

Hereinafter, the method for simple diagnosis of biological treatment of the present invention will be described in detail.

First, sludge in a reaction step or a solid-liquid separation step in a biological treatment system is sampled. The sampled sludge is diluted at different magnifications. When diluting or culturing, use all equipment that has been sterilized,
All operations are performed aseptically.

As the water for diluting sludge, sterilized distilled water is used. The sludge is thoroughly stirred so as to be uniform, 1 ml is collected, diluted water is added to make 10 ml, and a 10-fold diluted sample is prepared. Then stirred well the 10-fold diluted samples were aliquoted 1 ml, to create the 10 ^two-fold dilution samples in the 10ml by the addition of dilution water. This is sequentially repeated to prepare a plurality of test samples.

Next, this test sample is mixed with a nutrient source required by various microorganisms involved in biological treatment of sludge, which will be described below. A and A are cultured using a medium A obtained by adding a starch and a yeast extract to the medium A.

The medium A used in the present invention is a medium capable of easily germinating and growing a wide variety of microorganisms in sludge, particularly more than 30 Bacillus bacteria which are useful bacteria for biological treatment of sewage. It is. That is, in the medium A, the nutrients required by the above-mentioned microorganism group, particularly the bacterium belonging to the genus Bacillus, are present in the medium in a well-balanced manner, so that the nutrient degradation is completed 24 to 48 hours after the inoculation, and spores are formed. It is a nutrient concentration that is suitable for germination and is capable of germinating and growing all Bacillus bacteria useful for biological treatment.

According to the present invention, the state of the microorganisms present in the sludge is grasped by culturing the diluted sludge using the above-mentioned medium A, and the presence of Bacillus bacterium which is an effective bacterium is further investigated. Can be confirmed.

The medium B used in the present invention is the same as the above-mentioned medium A
It is a medium to which starch and yeast extract are added, and similarly to the above-mentioned medium A, a variety of microorganisms in sludge, particularly more than 30 kinds of Bacillus bacteria that are useful bacteria for biological treatment of sewage are easily germinated, Furthermore, it is a medium that can easily measure the appearance rate of bacteria having starch resolution among the nutritional resolution of Bacillus bacteria. With this medium B, the resolution of useful bacteria in sludge by nutrient source can be easily estimated. That is,
Among the Bacillus bacteria, the predominant species are those that mainly degrade proteins or those that mainly degrade carbohydrates. If the appearance rate can be measured, it can be inferred that other species are species capable of decomposing proteins.

The above-mentioned medium A contains at least a meat extract,
It contains peptone, glucose and salt. Specifically, 0.5 to 1.0% of nutrient broth, glucose
It can be composed of 0.5-1.0% and salt 0.3-1.0%.
Preferably, the nutrient broth is composed of 0.8%, glucose 0.8%, and salt 0.6%. 1.5% of agar is added to this medium and heated (80 ° C.) to completely dissolve the agar. The agar is dispensed into petri dishes in 20 ml portions, spread on the petri dishes and cooled to prepare a plate medium A.

[0026] The above-mentioned medium B is prepared by adding starch and yeast extract to the above-mentioned medium A. Specifically,
In the above-mentioned medium A, starch 0.3-1.5% and yeast extract 0.03
~ 0.1% can be added. Preferably, 0.8% nutrient broth, 0.8% glucose, 0.6% salt,
It consists of 0.5% starch and 0.05% yeast extract. Add 1.5% agar to this medium and heat (80 ℃)
Then, the agar was completely dissolved, dispensed in a Petri dish in 20 ml portions, spread on the Petri dish, and cooled to prepare a plate medium B.

A test sample of the diluted sludge is taken in an amount of 0.1 ml, spread over the entire plate medium A and the plate medium B, and cultured at a temperature of 30 to 32 ° C. in a thermostat.

In the present invention, using the above-mentioned plate medium A,
Perform the stepwise certification described below. The first stage certification is
It is visually determined whether an early colony is formed on the plate medium A, and if an early formed colony is present, whether the colony is a colony of a bacterium belonging to the genus Bacillus, which is a useful bacterium, is determined. Visually approve or not. Early means up to about 48 hours from the start of culture.
It is first necessary that a Bacillus bacterium colony be formed on the medium at this early stage.

This can be easily identified with the naked eye and a shape microscope. Specifically, the colony formed on the medium is observed with the naked eye, and the specific color tone of the colonies of Bacillus bacteria as shown in Table 1 is obtained. And the shape and the like, it can be determined that the colony is a Bacillus bacterium.

[0030]

[Table 1]

In the present invention, as a result of the first stage certification,
When colonies are not formed early or when colonies are confirmed early but the colonies cannot be determined to be Bacillus bacteria colonies, the biological treatment of sludge is judged to be extremely poor. Can be. As a countermeasure, minerals such as silicates, aluminum salts, calcium salts, and magnesium salts are added to promote the cultivation and growth of Bacillus bacteria and to change the amount of aeration in the reaction process. It is also important to control the amount of sludge that has been solid-liquid separated and returned. In addition, it is necessary to control the amount of wastewater input.

Next, as a result of the first stage recognition, if the colony is a colony of a bacterium belonging to the genus Bacillus, a second stage determination is made as to whether or not spores have been formed. this is,
Although it can be confirmed with the naked eye that spores have been formed from the center of the colony, the details are confirmed by microscopic observation.

The study of the present inventors has revealed that there is a proportional relationship between the rate of spore formation in the plate medium A and the rate of decomposition of nutrients, which is a characteristic of Bacillus bacteria. Therefore, according to the present invention, since the Bacillus bacterium forms spores early (from about 48 hours after culture), it can be determined that the Bacillus bacterium is a species having a high ability to decompose nutrients. . This eliminates the trouble of isolating the bacteria from the colonies cultured on the plate medium and performing a nutritional degradation test on each of them, as in the past, and is extremely simple and quick, and can be used to quickly and rapidly degrade the bacterium belonging to the genus Bacillus. Can be determined. In a Bacillus bacterium having a relatively high nutritional resolution, the colony itself can be identified in only 24 hours.
Spore formation is confirmed after approximately 48 hours of culture.

Therefore, if the bacterium belonging to the genus Bacillus has a high nutritional resolution, the diagnosis result of the biological treatment can be obtained in about 48 hours after the sampling, so that the bacterium can be sufficiently used for daily facility management such as a treatment plant.

The ability of Bacillus bacteria to degrade nutrients is as follows:
It can also be confirmed by observing the dissolution state of the culture medium around the cultured medium A colony. Species with high vegetative resolution have the characteristic that the medium around the colony is dissolved and the center of the colony sinks down,
In addition to the above-mentioned confirmation of spore formation, it is possible to know the nutritional resolution of Bacillus bacteria in more detail.

As a result of the second stage of certification, early (4
If spore formation is not confirmed by about 8 hours), Bacillus bacterium is present, but biological treatment of sludge may not proceed well because it is a species with low vegetative resolution. . Therefore, it is necessary to take appropriate measures so that species having excellent nutritional resolution can be dominant. Appropriate processing is the same as the countermeasures described above. Further, it is preferable to seed (seed) Bacillus bacterium sludge with high resolution. If spore formation was not confirmed early (about 48 hours after culture), the culture was continued and the time point when spore formation was confirmed was recorded. Proceed to certification.

Next, the number of colonies of Bacillus bacteria is counted, and the cell density is measured. The density is measured by counting the number of colonies according to the method for measuring the number of viable bacteria by the plate culture method. By this measurement, the amount (density) of the bacterium belonging to the genus Bacillus determined to have a high nutritional degradation ability in the first and second stages can be easily measured.

Specifically, the number of colonies can be measured based on the dilution ratio x of the test sample in which the number of colonies has been counted and the count number y. In the case of sewage or human waste treatment, it can be determined whether or not the useful bacterial cell density of the genus Bacillus is appropriate or not depending on whether or not x and y satisfy (expression) y × 10 ^x ≧ 10 ⁵ . This is the third stage of certification. When counting the number of colonies, 5-10
It is preferable to select and count 0, preferably 10 to 30 culture media. If more than 100 colonies are present on the plate medium, counting errors are likely to occur, and if the number of colonies is less than 5, errors due to dilution will occur.

If the dilution ratio x and the count number y of the test sample obtained by counting the number of colonies do not satisfy the above expression as a result of the certification in the third step, the density of Bacillus bacteria in the biological treatment of sewage is not appropriate. You can determine that there is no.
Therefore, it is necessary to take appropriate measures to increase the density. Appropriate processing is the same as the countermeasures described above.

Next, in the plate medium B, which is carried out in parallel with the culture in the plate medium A, the appearance rate of bacteria having a starch-decomposing ability among the bacteria belonging to the genus Bacillus can be seen. The method is
First, a diluted test sample is inoculated, and colonies appear after culture. On this medium, colonies of Bacillus bacteria and colonies of other germs are also present. Whether or not the colony is a Bacillus bacterium can be confirmed by the same method as the above-described visual observation. The number of colonies of the bacterium belonging to the genus Bacillus is counted and defined as the total number of colonies. Next, a Lugol's solution (2 g of potassium iodine, 1 g of iodine, 80 ml of distilled water) is sprayed onto the medium and the colonies, and the number of colonies that have turned blue-violet is counted, and the number of discolored colonies is determined. The number obtained by subtracting the number of discolored colonies from the total number of colonies is the number of colonies of starch-degrading bacteria. Therefore, the appearance rate (%) of bacteria having starch resolution can be obtained from (−) ÷ × 100.

In the present invention, in addition to the above-mentioned qualification on the plate medium A, the cultivation on the plate medium B is carried out in parallel, so that the bacterium has a carbohydrate (starch) degrading ability among the vegetative degradation ability. The appearance rate of bacteria can be known, and the status of biological treatment can be understood in more detail and easily.

The rate of appearance of bacteria having starch resolution is 10
%, It can be determined that the state of biological treatment is very good.

As a result of culturing on the plate medium B, if no bacteria having starch-degrading ability are found, dehydrated sludge, dried sludge, fermented sludge and the like containing Bacillus bacteria having starch-degrading ability as seed sludge are placed in a treatment tank. It is preferable to construct a means for seeding and growing them.

The simple diagnosis method of biological treatment according to the present invention makes it possible to absolutely judge the state of biological treatment by one test. A comparison can be made. It is also important that this diagnosis is not a one-time diagnosis but a continuous test. By performing the inspection continuously, the current situation (data) can be compared with the previous situation (data). By comparing with the previous data, it is possible to predict not only the temporary situation but also the state of biological treatment to some extent, and there is an advantage that appropriate measures can be taken if improvement is necessary .

According to the present invention, the state of the Bacillus bacterium can be easily confirmed by visual observation and microscopic observation, whereby the state of biological treatment can be easily grasped, and appropriate measures are taken in daily facility management. be able to. Also,
Even those who have no knowledge, expertise, or inspection technology can easily operate the present invention by short-term training. The diagnostic method of the present invention makes it possible to easily determine the superiority of the nutritional resolution of Bacillus bacteria using sludge that has only been diluted without isolating and culturing the sludge. Reduced, quickly and easily judge the quality of the biological treatment of wastewater, but in more detail, isolate the bacteria from the culture medium of the present invention, and perform the nutritional resolution and biological tests of each bacteria Is also good.

[0046]

An embodiment of the present invention will be described. The present invention is not limited by such embodiments.

Example 1 20 ml of a test sample collected from the outlet of the aeration tank outlet of a sewage treatment facility was mixed and crushed at a level 3 for 30 seconds using a homogenizer to prepare raw test water. 1 ml is taken from the test raw water, and diluted water is added to make 10 ml to prepare 10-fold diluted water. Further, 10-fold diluted water is mixed well, 1 ml is taken from the 10-fold diluted water, and diluted water is added to make 10 ml.
Prepare ^two- fold dilution water. This is sequentially repeated, 10 ¹²
Prepare up to double dilution water.

[0048] 0.1 ml of each diluted sample is added to the plate medium A and the plate medium B prepared as described below, in order of increasing dilution ratio, from lower to higher.
Spread using a cone large rod or the like so that the diluted sample spreads over the entire surface of the medium. Incubate at a temperature of 30 to 32 ° C. in a thermostat.

Composition of plate medium A Nutrient broth (CM-1 manufactured by Difco) 8 g Glucose 8 g Salt 6 g Distilled water is added to make 1 liter. Add 15 g of agar and heat (80 ° C) to completely dissolve the agar and add 20 ml
Each of them is separated into petri dishes, spread on a petri dish, and cooled to prepare a plate medium A.

Plate medium B composition Nutrient broth (CM-1 manufactured by Difco) 8 g Glucose 8 g Salt 6 g Starch 5 g Yeast extract 0.5 g Distilled water is added to make 1 liter. Add 15 g of agar and heat (80 ° C) to completely dissolve the agar and add 20 ml
Each of them is separated into petri dishes, spread on petri dishes, and cooled to prepare a plate medium B.

The colony formed on the plate medium A after 48 hours of culture has a white color when observed with the naked eye,
The shape was circular, the periphery was clear, the appearance of the bulge was raised at the center, the gloss was dull, and the surface was wrinkled,
It was found that colonies of Bacillus bacteria existed.

As for various bacteria other than Bacillus bacteria, some Nocardia-type bacteria and yeast were observed.

The colonies of Bacillus bacterium were diluted at a dilution factor of 1.
In a petri dish of 0 ^8, it was 15. When a colony of the Bacillus bacterium was collected with a platinum loop and observed with a microscope, two sporulated species, five endospore-formed species, and two rod-shaped species were observed.

Further, among the colonies generated after 24 hours of culture on the plate medium B, simple recognition as a Bacillus bacterium was performed with the naked eye. Thereafter, Lugol's solution (2 g of potassium iodide, 1 g of iodine, 1 g of distilled water, 80 ml).

As a result, out of 15 colonies which were identified as Bacillus bacteria, 12 discolored to blue-violet and 3 did not discolor. Therefore, the appearance rate of the bacteria having starch resolution was 20%. Judging from the results of Example 1, it can be diagnosed that the state of biological treatment in this sewage treatment facility is good and stable.

[0056]

As described above, according to the present invention, bacteria of the genus Bacillus, which is an indicator bacterium for biological treatment, can be determined easily and quickly, and their nutritional resolution can be easily determined. It is possible to provide a simple diagnosis method of biological treatment of sewage which can be used for daily facility management in a place or the like.

Claims (4)

(57) [Claims] 1. A reaction step of receiving sewage in a reaction tank in which useful bacteria that form aerobic and spores inhabit, and biologically treating components in the sewage, and converting the sludge sent from the reaction step into solid-liquid When biologically treating sewage by a biological treatment system having a solid-liquid separation step to separate, the sludge in the biological treatment system is sampled, and the sludge is diluted by varying the magnification,
A plurality of test samples are prepared, and the plurality of test samples are suitable for testing a multi-species coexisting sample containing a nutrient source required by various microorganism groups involved in biological treatment in sludge. The medium A and the medium A were separately cultured in a medium B obtained by adding starch and yeast extract. From one medium A, it was visually recognized whether or not a colony was formed at an early stage. If formed, the presence of Bacillus bacterium, a useful bacterium in the sludge, is determined by visually recognizing whether the colony is a colony of Bacillus bacterium, a useful bacterium, When the colony is a colony of the bacterium belonging to the genus Bacillus, it is visually determined whether or not spores are formed, and then the number of colonies of the bacterium belonging to the genus Bacillus on the medium A is counted. Measure the density of From the other medium B, of the colonies of the genus Bacillus among the colonies formed on the medium B, by measuring the appearance rate of bacteria having starch-degrading ability, without performing the isolation and culture of useful bacteria in the sludge A simple diagnostic method for biological treatment of sewage, comprising determining whether or not the biological treatment status of sewage is good. 2. The simple diagnostic method for biological treatment of sewage according to claim 1, wherein said medium A contains at least meat extract, peptone, glucose and salt. 3. The wastewater is sewage or night soil, and when counting the number of colonies of Bacillus bacterium and measuring the density, the dilution ratio x and the count number y of the test sample in which the number of colonies is counted are as follows. 3. The diagnostic method for biological treatment of sewage according to claim 1 or 2, wherein the quality of the biological treatment of sewage is easily determined using the satisfaction of the following equation as an index. [Formula] y × 10 ^x ≧ 10 ⁵ 4. The appearance rate of bacteria having a starch degrading ability is 1
4. The method for diagnosing biological treatment of sewage according to claim 1, wherein the quality of the biological treatment of sewage is easily determined by using 0% or more as an index.