JP5705448B2 - Microbial activity evaluation method, and water-based microorganism control method using the evaluation method - Google Patents

Description

  The present invention relates to a microbial activity evaluation method for evaluating microbial activity in a sample, and an aqueous microbial control method using such a microbial activity evaluation method.

  In the fields of food hygiene and biological treatment of wastewater, microbial activity in samples is regarded as important for management, and microbial activity has been measured by various methods. Specific methods for measuring such microbial activity include a method for measuring respiration rate (oxygen consumption rate) (Japanese Patent Laid-Open No. 06-181742, etc.), a method for measuring dehydrogenase activity, and adenosine triphosphate (ATP). ) A method for measuring the amount has been adopted.

  Moreover, as a recent new technique, a method for detecting a biological font (weak light generated from an ecological tissue, a cell, or a biological substance) (JP 05-23169, JP 05-168461), dielectrophoresis and Examples include a method in combination with electrical impedance (Japanese Patent Laid-Open No. 2003-224).

  Among these methods, the method for measuring the amount of ATP is simple and capable of on-site measurement, so it has been widely applied to technical fields that require microbial control. Microbial control in water systems including open circulation cooling water systems The evaluation of microbial activity based on this ATP measurement has also been used for (slime control).

  All of the above methods for evaluating microbial activity measure the relative amount of microorganisms present in a sample. However, when only a small amount of highly active microorganisms are present, There is a disadvantage that it cannot be distinguished from the case where a large amount of is present. In particular, in an aqueous system such as an open circulation cooling water system that is treated with a microbial control agent, there are a large amount of low-activity microorganisms that are weakened by the microbial control agent, and there are highly active microorganisms that are resistant to the microbial control agent. There is a big difference in the direction of subsequent microorganism control when it is present in a small amount. In other words, in such an aqueous system, it is important to determine the proportion of highly active microorganisms with respect to the total amount of microorganisms in the sample, but there has been no evaluation method as an index for such information. It was.

Japanese Patent Laid-Open No. 06-181742 JP 05-23169 A JP 05-168461 A Japanese Patent Laid-Open No. 2003-224

  Here, when there is an evaluation method for evaluating the proportion of highly active microorganisms with respect to the total amount of microorganisms in the sample as described above, the evaluation method is used as a microorganism control means such as drug addition. By applying to an aqueous system that controls microorganisms, the applied microorganism control means (for example, the concentration and frequency of the added drug, and the type of drug, etc.) are functioning sufficiently effectively, or It is possible to determine whether there is an excess. However, if the evaluation method takes time and labor, the measures are naturally delayed, which is not preferable.

  For this reason, in order to apply the above evaluation method to the water-based microorganism control method, it is necessary to be able to be performed simply and in a short time.

  The present invention improves the above-mentioned conventional problems, that is, it can be carried out simply and in a short time, and the proportion of highly active microorganisms is present relative to the total amount of microorganisms in the sample. It is an object of the present invention to provide a method for evaluating microbial activity, which is a method for knowing whether or not to do so.

  The present inventors conducted various studies in order to obtain the above-described method for evaluating microbial activity.

  In order to solve the above problems, it is necessary to measure the total amount of microorganisms in a sample separately from the conventionally measured microorganism activity, and to compare and evaluate the total amount of microorganisms and the activity of microorganisms.

  Here, as a method of measuring the total amount of microorganisms in the sample, turbidity measurement of the sample water and measurement of the number of viable bacteria are performed. In the turbidity measurement, turbidity other than microorganisms and microorganisms are measured. Indistinguishable, measurement of the number of viable bacteria requires a culture operation of microorganisms, which requires time and labor. In the field of wastewater treatment, MLSS (activated sludge suspended matter) and MLVSS (activated sludge organic suspended matter) are used as an indicator of the total amount of microorganisms in activated sludge, but these methods are at least 1000 mg / L. It is used for the above high-concentration microbial suspensions. To apply to microbial disorders such as open circulation cooling water systems, a large amount of microbial mass (slime) is required, which is impractical and can be used for measurement operations. However, operations such as filtration, drying, and combustion are required, which requires labor.

  After such studies, the present inventors have arrived at the present invention.

  That is, in order to solve the above problems, the method for evaluating microbial activity of the present invention measures the amount of protein and adenosine triphosphate contained in a sample as described in claim 1, and the adenosine triphosphate is measured. It is a method for evaluating microbial activity, wherein the activity of microorganisms in the sample is evaluated based on the microbial activity obtained by dividing the amount by the amount of protein.

  In addition, the water-based microorganism control method of the present invention is a sample collected from an aqueous system as described in claim 2, and the microorganism activity obtained by the method for evaluating microorganism activity according to claim 1 In the case of a predetermined upper limit value or more, the suppression level of the water-based microorganisms is enhanced.

  Further, according to the aqueous microorganism control method of the present invention, as described in claim 5, the sample is a sample collected from the aqueous system, and the microorganism activity obtained by the microorganism activity evaluation method according to claim 1 is obtained. The water-based microorganism control method is characterized in that the level of inhibition of the water-based microorganism is reduced when it is equal to or lower than a predetermined lower limit value.

  According to the method for evaluating microbial activity of the present invention, it is possible to measure and evaluate microbial activity while being a very simple method.

  In addition, according to the water-based microorganism control method of the present invention, it is possible to prevent microbial damage in the water system and to optimize the use of microorganism control means such as a microorganism treatment agent, thereby performing excessive microorganism control. Waste can be prevented.

  In the method for evaluating microbial activity of the present invention, the total amount of microorganisms in a sample is measured by the amount of protein, the activity of microorganisms is measured by the amount of adenosine triphosphate (ATP), and the microbial activity (= ATP amount / protein amount) is calculated. This is a method of evaluating based on the value.

  The sample in the present invention is any sample that requires measurement / evaluation of microbial activity, such as activated sludge, aqueous water that requires microbial control, biofilm (slime), slime containing microorganisms, and sludge. I do not care.

  Here, when the sample is an aqueous solution or an aqueous suspension, the microbial activity is measured as it is or after dilution. In the case of solid (including slime or sludge), suspend in an appropriate amount of sterilized water and, if necessary, grind the particles using a homogenizer or the like to form an aqueous suspension. The sample is used as it is or after dilution. Furthermore, in the case of a sample that is difficult to collect directly, such as an aqueous biofilm, wipe the sample with a sterilized cotton swab, etc., suspend the collected sample in sterilized water, and measure the amount of protein and ATP. It can be.

  Here, since the ratio of the amount of adenosine triphosphate to the amount of protein is used in the present invention, the microbial activity does not depend on the sample concentration of the sample as described above. Evaluation is possible not only for slime-like samples that can be collected at the same time, but also for samples that have a small amount of collection, such as biofilms that are thinly attached to equipment and piping.

  For protein measurement, known protein quantification methods such as Bradford method, Lowry method, BCA method and the like can be used. Among these, the lower limit of quantification is low, the operation is simple, and there are few interfering substances. From the point of view, it is preferable to use the Bradford method.

  The Bradford method is a method in which Coomassie blue is mixed with a sample and the change in absorbance when Coomassie blue and protein are bound is measured. According to this method, if there is an absorptiometer, Quick and simple measurement at the sampling site is possible.

  On the other hand, ATP can be measured by extracting ATP contained in bacteria in a sample with an ATP extraction reagent, causing the extracted ATP to emit light with luciferase, and measuring the amount of light emitted at this time. Various measuring devices for measuring ATP are known, and simple measuring devices are also commercially available, so that quick measurement at the sampling site is possible.

  In the method for evaluating microbial activity of the present invention, the activity of microorganisms in a sample is evaluated based on the microbial activity obtained by dividing the amount of adenosine triphosphate in the sample by the amount of protein in the sample. The ratio of adenosine triphosphate concentration to protein concentration is substantially the same, and is included in the present invention.

  Here, such a microorganism activity evaluation method can be applied to an aqueous system to control an aqueous microorganism.

  That is, when the microbial activity is high, it is determined that there are many highly active microorganisms in the microorganisms present in the aqueous system, and when the microbial activity is low, it is determined that there are few active microorganisms among the microorganisms present in the aqueous system.

  Specifically, in the case of a sample obtained from an aqueous system, generally, the boundary value (predetermined upper limit value) of the microbial activity at which it is determined that there are many highly active microorganisms among the microorganisms present in the aqueous system is, for example, 1000 nmol. When the microbial activity is greater than or equal to this value, it is judged that there are relatively many highly active microorganisms, and the suppression level of the water-based microorganisms is strengthened to suppress the highly active microorganisms. On the other hand, the boundary value (predetermined lower limit value) of the microbial activity at which it is judged that there are few highly active microorganisms among the microorganisms present in the water system is, for example, 100 nmol / g, and the microbial activity is below this value. Can determine that there are relatively few highly active microorganisms, and can reduce the suppression level of the water-based microorganisms.

  Water-based microbial control (control) includes water-based physical sterilization (ultraviolet treatment, electrolytic sterilization, etc.), ozone treatment, etc. Any applicable microbial control means can be used.

  Examples of the microorganism control agent include isothiazolines such as 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, and 1,2-benzisothiazolin-3-one. Compounds, glutaraldehyde, aldehydes such as phthalaldehyde, hydrogen peroxide, hydrazine, chlorinated fungicides (sodium hypochlorite, etc.), bromine and iodine fungicides, pyrithione compounds, dithiol compounds, Examples include thiocyanate compounds such as methylene bis thiocyanate, ionene polymers, bis-type quaternary ammonium salts, quaternary ammonium salts other than bis-type quaternary ammonium salts, and cation-type compounds such as quaternary phosphonium salts.

  Examples of the chemical cleaning agent used when performing the aqueous chemical cleaning include hydrogen peroxide, a high concentration chlorine agent, and a high concentration glutaraldehyde.

  Here, at least one selected from an increase in the concentration of the microorganism control agent added to the aqueous system, an increase in the frequency of addition of the microorganism control agent to the aqueous system, and a change in the type of the microorganism control agent added to the aqueous system. Or by performing chemical cleaning of the aqueous system, the level of suppression of microorganisms in the aqueous system can be enhanced, and the concentration of the microorganism control agent added to the aqueous system can be reduced, and the microorganism control agent can be added to the aqueous system. By suppressing the addition of at least one selected from the decrease in the frequency of addition or the microorganism control agent, the level of suppression of water-based microorganisms can be reduced.

  Examples of the microbial activity evaluation method and the water-based microbial control method of the present invention will be specifically described below.

<Example 1>
Slime collected from a cooling water system refrigerator tube at a factory in Ibaraki Prefecture that had been treated continuously for 5 years with 5-chloro-2-methyl-4-isothiazolin-3-one as a microorganism control agent; and Samples wiped off with a sterilized cotton swab from a part of the adhering material (biofilm) adhering to the cooling tower filler of the same cooling water system were each suspended in sterilized water and then divided into three parts. Sample.

  To each of these samples, 5-chloro-2-methyl-4-isothiazolin-3-one (hereinafter referred to as “CMI”) added at a concentration of 20 mg / L (CMI added system), 2,2- After preparing dibromo-3-nitrilopropionamide (DBNPA) at a concentration of 20 mg / L (DBNPA added system) and no drug (added system), each was allowed to stand at 27 ° C. for 3 days. , ATP concentration (ATP analyzer (using Toa DKK AF-100)) and protein concentration (by Bradford method) were measured.

  The ATP concentration thus obtained was divided by the protein concentration to determine the microbial activity. In addition, the number of heterotrophic bacteria (based on JIS K0101 63.3) of each sample was measured. These results are shown in Table 1.

  From Table 1, the microbial activity in the slime adhering to the refrigerator tube is the same level as the microbial activity in the biofilm adhering to the filler, and the same type of microorganism as the biofilm adhering to the filler is frozen. It was suggested that it was breeding in the machine tube.

  It was also found that CMI has no inhibitory effect against this microorganism and DBNPA is effective.

  Therefore, the treatment of this cooling water system was changed from CMI to DBNPA, and the treatment was continued for 6 months while periodically measuring the microbial activity of the filler deposit. During this period, the microbial activity was maintained at less than 100 nmol / g, and almost no deposits on the packing material and no deposits on the cooling tank lower water tank were observed.

<Example 2>
The deposit deposited in the cooling tank lower water tank of the cooling water system of a building in Tokyo without microbial control was taken as a sample, and its microbial activity was measured and found to be as high as 3200 nmol / g. For this purpose, chemical cleaning with hydrogen peroxide was carried out, after which the aqueous operation was resumed without microbial control.

  One week after the restart of operation, deposits were observed again in the cooling tower lower water tank. The deposits were collected and the microbial activity was measured, and it was 75 nmol / g.

  When these deposits were observed with a microscope, the deposits before chemical cleaning were mainly bacteria, and the deposits after restarting operation were mainly inorganic substances such as earth and sand. The LTD (dirt index) of the refrigerator was 6.0 ° C. before chemical cleaning and 0.6 ° C. after chemical cleaning.

  Since the microbial activity of the sediment one week after the resumption of the operation was low as described above, the operation was continued for another two months after the lower aquarium was manually washed and the microbial control was not performed (no treatment). During this continuous operation, almost no deposits were found in the lower water tank, but the adhering matter adhering to the cooling tower packing was sampled with a sterile cotton swab every other week, and its microbial activity was measured. The microbial activity gradually increased and reached 1200 nmol / g two months after the start of continuous operation. Therefore, in the subsequent operation, 1,4-bis (3,3 ′-(1-decylpyridinium) methyloxy) butanedibromide as a microorganism control agent, so that the concentration in cooling water is maintained at 10 mg / L, Added continuously.

  Thereafter, the microbial activity of the deposits adhering to the cooling tower packing gradually decreased, and reached 60 nmol / g one month after starting the maintenance of the drug concentration.

Claims (7)

  Measuring the amount of protein and adenosine triphosphate contained in a sample, and evaluating the activity of microorganisms in the sample by the microbial activity obtained by dividing the amount of adenosine triphosphate by the amount of protein. A method for evaluating a characteristic microbial activity.   When the sample is a sample collected from an aqueous system and the microbial activity obtained by the microbial activity evaluation method according to claim 1 is a predetermined upper limit value or more, the suppression level of the microbial activity in the aqueous system is enhanced. A method for controlling an aqueous microorganism.   The enhancement of the suppression level of the microorganisms in the water system can be achieved by increasing the concentration of the microorganism control agent added to the water system, increasing the frequency of addition of the microorganism control agent to the water system, and the type of microorganism control agent added to the water system. The method of controlling an aqueous microorganism according to claim 2, wherein the method is at least one selected from modification, or chemical cleaning of the aqueous system.   The water-based microorganism control method according to claim 2 or 3, wherein the predetermined upper limit value is 1000 nmol / g.   When the sample is a sample collected from an aqueous system and the microbial activity obtained by the microbial activity evaluation method according to claim 1 is not more than a predetermined lower limit value, the suppression level of the microbial activity in the aqueous system is reduced. A method for controlling an aqueous microorganism.   The suppression level of the microorganisms in the water system is at least one selected from a decrease in the concentration of the microorganism control agent added to the water system and a frequency of addition of the microorganism control agent to the water system, or the microorganism control The method for controlling an aqueous microorganism according to claim 5, wherein the addition of the agent is stopped.   The water-based microorganism control method according to claim 5 or 6, wherein the predetermined lower limit value is 100 nmol / g.