JP6199080B2 - Method for provisional determination of ballast water quality, method for treatment of ballast water, method for provisional determination of biological composition, method for evaluating biological activity, and method for estimating biological concentration - Google Patents

Description

  The present invention relates to a provisional determination method for ballast water quality that tentatively determines whether or not a biological composition in ballast water satisfies a standard, a ballast water treatment method for treating ballast water based on the determination method, and a test sample A provisional determination method of a biological composition for tentatively determining whether or not a biological composition in the medium satisfies a standard, a biological activity evaluation method for evaluating an organism present in a test medium as an activity, and a test medium The present invention relates to a biological concentration estimation method for estimating the concentration of a biological organism present in a living body.

  A cargo ship that transports crude oil or the like is provided with a ballast tank to maintain the stability of the hull during navigation. Usually, when crude oil or the like is not loaded, the inside of the ballast tank is filled with ballast water, and when the crude oil or the like is loaded, the ballast water is discharged to adjust the buoyancy of the hull and stabilize the hull. As described above, the ballast water is water necessary for the safe navigation of the ship, and the seawater of the port that performs cargo handling is usually used. The amount is said to exceed 10 billion tons per year worldwide.

  Ballast water is mixed with microbes and eggs of small and large organisms that inhabit the port from which the water was taken, and along with the movement of the ship, these microbes and eggs of small and large organisms are transported to foreign countries at the same time. Become. Therefore, the destruction of ecosystems, such as the replacement of existing species with species that did not originally live in the sea, has become serious.

  Against this background, if the duty to receive periodic inspections related to ballast water treatment equipment, etc. is adopted at a diplomatic meeting of the International Maritime Organization (IMO), it will be applied from 2009 and later construction ships.

  For example, the discharge standards (water quality standards) specified in the D-2 regulation ballast water discharge standards of the Convention for the Regulation and Management of Ship Ballast Water and Sediment are intended only for living things, such as bacteria. Since the unit is indicated by cfu (colony forming unit), the test for type approval of the ballast water treatment apparatus is evaluated by visual confirmation of colonies by a culture method. Therefore, it is usual that a ship is equipped with a ballast water treatment device having a capability of removing or killing microorganisms that can satisfy such a standard.

  On the other hand, Patent Document 1 describes a method for detecting microorganisms in a test material in the field of environmental health inspection or sanitary inspection such as food, hygiene, medical-related products, or a site handling them. That is, the test material is filtered through a filtration membrane, the microorganisms in the test material are captured on the filtration membrane, the captured microorganisms are reacted with a signal generating substance, and the signal obtained is optically read. In the method for inspecting microorganisms in a material, (a) the microorganisms in the test material react with the signal generating substance on the filtration membrane, and (b) the filtration membrane of (a) is on the device or the device A microbiological test method is described in which the intensity of a signal is read in place of an electrical signal using an apparatus suitable for the device of (b), thereby lysing the microbe in the microbiological test. The microorganism, its components and / or its activity (nucleic acid, respiratory activity, esterase activity, etc.) can be detected easily and rapidly.

JP 2002-281998 A

  As described above, the ballast water treatment apparatus usually has a performance that can satisfy the standard. However, from the viewpoint of satisfying the discharge standard more reliably, the inventor of the present application has in advance about the ballast water to be discharged. We considered measuring whether the emission standards could be met. This is to cope with an unexpected situation such as an abnormality in the ballast water treatment apparatus and a re-growth of organisms in the ballast tank.

  However, if it is attempted to visually check colonies by the culture method based on the provision of D2, it takes about 4 days from the start of the culture until the colonies can be visually confirmed. For example, it is conceivable that the sample is collected and cultured 4 days before the discharge of the ballast water, but there is a problem that it is difficult to cope with the case where the organism re-growth in the ballast tank after the sample is collected.

  Although the technique described in Patent Document 1 detects the activity of microorganisms, it only detects the amount of substances constituting the microorganisms themselves or the microorganisms. Since the activity of the microorganism itself is not dynamically detected, there is a limit in terms of directly evaluating the activity.

  Accordingly, an object of the present invention is to provide a provisional determination method for ballast water quality that tentatively determines whether or not a biological composition in the ballast water satisfies a standard, and a ballast water treatment method for treating ballast water based on the determination method , Provisional determination method of biological composition that tentatively determines whether or not the biological composition in the test sample satisfies the standard, evaluation of biological activity that directly and quickly evaluates the organism present in the test medium as activity It is an object of the present invention to provide a method and an organism concentration estimation method that quickly estimates the concentration of an organism present in a test medium.

  Other problems of the present invention will become apparent from the following description.

  The above problems are solved by the following inventions.

1. The amount of decrease in the concentration of the substrate measured by culturing a test medium containing at least a predetermined substrate and a sample obtained by concentrating raw water collected from the ballast water treated by the ballast water treatment device for a predetermined time. The provisional determination method of the ballast water quality characterized by tentatively determining whether or not the biological composition of the ballast water satisfies a predetermined standard.

  2. At the time of the determination, the amount of decrease in the concentration of the substrate is compared with a predetermined value, and when it is equal to or less than the predetermined value, it is tentatively determined that the biological composition of the ballast water satisfies the standard, and when it exceeds the predetermined value The method for provisional determination of ballast water quality according to the item 1, wherein it is provisionally determined that the biological composition of the ballast water does not satisfy the standard.

  3. The predetermined value corresponds to a decrease in the concentration of the substrate measured in a reference medium having a known biological composition corresponding to the standard in which the culture conditions are substantially the same as the test medium. 3. The method for provisional determination of ballast water quality according to 1 or 2 above.

  4). The provisional ballast water quality according to any one of 1 to 3 above, wherein when measuring the amount of decrease in the concentration of the substrate, a region outside the body of the organism is selectively measured in the test medium. Judgment method.

  5. As a preliminary test for selecting the substrate to be measured, the amount of decrease in the concentration of any substrate that accompanies the culture in at least two media having substantially the same culture conditions and different biological concentrations at the start of the culture 1 to 4, wherein the substrate is used as the substrate when it is confirmed that the amount of decrease in the concentration of the substrate changes according to the biological concentration at the start of culture. The temporary determination method of the ballast water quality in any one of.

  6). 6. The provisional determination method for ballast water quality according to any one of 1 to 5, wherein glucose is used as the substrate.

  7). The provisional determination method for ballast water quality according to any one of the above items 1 to 6, which is performed in a ship.

  8). When it is tentatively determined that the ballast water does not meet the standard by the provisional determination method for ballast water quality described in 1 to 7 above, before the ballast water is discharged out of the ship, the biological removal treatment or the biological A method for treating ballast water, comprising performing killing treatment.

9. Based on the consumption of the substrate in the culture medium in the test medium containing the sample obtained by concentrating the raw water collected from the ballast water treated by the ballast water treatment device, the proliferation ability of the organism containing the ballast water as a whole is increased. A method for evaluating the biological activity of ballast water, characterized by estimating whether the activity of a plurality of types of organisms present in the test medium is high or low .

  According to the present invention, a provisional determination method for ballast water quality that tentatively determines whether or not a biological composition in the ballast water satisfies a standard, a ballast water treatment method for treating ballast water based on the determination method, A provisional determination method of a biological composition for tentatively determining whether or not a biological composition in a test sample satisfies a standard, a biological activity evaluation method for directly and rapidly evaluating an organism present in a test medium, In addition, it is possible to provide a biological concentration estimation method for quickly estimating the concentration of a biological organism present in a test medium.

Process drawing which shows an example of the provisional determination method of the ballast water quality which concerns on this invention The figure which shows the result of the example The figure which shows the result of the example The figure which shows the result of the example The figure which shows the result of the example The figure which shows the result of the example

When discharging ballast water to the outside of the ship (the ocean, etc.), if there are a large amount of aquatic organisms in the ballast water, there is a concern of causing environmental destruction. On the other hand, the drainage standard like D2 mentioned above is defined. The definition of D2 is as follows.
・ Living organisms with a minimum diameter of 50 μm (mainly zooplankton): fewer than 10 surviving individuals in 1 cubic meter ・ Living organisms with a minimum diameter of 10 to 50 μm (mainly phytoplankton): Less than 10 surviving individuals in 1 ml (O-1, O-139): 100 ml or less than 1 colony formation in 1 g of zooplankton (not detected)
・ Escherichia coli: Less than 250 colonies in 100 ml ・ Enterococci: Less than 100 colonies in 100 ml

  According to the present invention, it can be tentatively determined whether or not the biological composition in the ballast water satisfies such criteria.

  FIG. 1 is a process diagram showing an example of a provisional determination method for ballast water quality according to the present invention.

  In FIG. 1, A is a step of preparing a test medium, B is a step of measuring a decrease amount of the substrate concentration, and C is a provisional test whether or not the biological composition in the ballast water satisfies a predetermined (arbitrary) standard. It is the process of determining to.

  In the step A of preparing the test medium, a test medium including at least a sample collected from the ship's ballast water and a predetermined substrate is prepared.

  Since the amount of organisms in the ballast water may be relatively small, in the present invention, it is also preferable to concentrate the raw water appropriately to obtain a sample. The concentration method is not particularly limited. For example, the raw water is passed through a filtration membrane to concentrate organisms that cannot permeate the membrane onto the membrane surface, or gravity is loaded on the raw water using a gravity settling means such as a cyclone. A method of concentrating organisms by the method can be preferably used. When concentration is performed using a filtration membrane, it is also preferable to add the filtration membrane to which the organism is attached to the test medium.

  In the present invention, by such concentration, in addition to the effect of increasing the reduction amount of the substrate in the test medium and improving the measurement accuracy, the effect of shortening the analysis time can be obtained. In addition, even if falling bacteria or the like are mixed, an effect of relatively reducing the influence can be obtained.

  In this example, glucose is added to the test medium so as to have a predetermined concentration as a substrate to be measured (predetermined substrate). The substrate will be described in detail later.

  The test medium may contain any medium component from the viewpoint of promoting the growth of an organism. As a result, the substrate can be significantly reduced in a shorter culture time, and the ballast water quality can be quickly determined. For example, as a medium component, those that have been confirmed to have an effect of promoting the growth of organisms by a preliminary culture test can be suitably used.

  In the present invention, the test medium can be, for example, a liquid medium or a solid medium, and is particularly preferably a liquid medium.

  As the components constituting the test medium (excluding the above sample to be tested) and the container (culture vessel) for containing it, those that are sterile (sterilized) are preferably used. .

  Next, in the step B of measuring the decrease amount of the substrate concentration, the test medium is cultured for a predetermined time, and the decrease amount of the substrate concentration accompanying the culture is measured.

  The amount of decrease in substrate concentration corresponds to the difference in substrate concentration at two different times after the start of culture, and is preferably, for example, the difference between the substrate concentration at the start of culture and the substrate concentration after culturing for a predetermined time.

  In the present invention, the amount of decrease in the substrate concentration is preferably determined for a predetermined time within 10 hours from the start of culture, more preferably within 4 hours from the start of culture, further within 2 hours, within 1 hour. It is preferable to measure. In the present invention, a significant decrease in the substrate concentration can be observed within such a relatively short time.

  The substrate concentration at the start of the culture may be confirmed by measurement, or may be calculated from the volume of the medium and the content of the substrate. The substrate concentration after culturing for a predetermined time is preferably confirmed by measurement. When measuring the substrate concentration after culturing for a predetermined time, the test medium in the culture container may be measured directly, or a part of the test medium taken out from the culture container may be measured. . The method for measuring the substrate concentration will be described in detail later.

  The culture temperature is not particularly limited, but is preferably set according to the optimal temperature of the organism, and for example, it is preferably cultured at a predetermined temperature in the range of 30 ° C to 40 ° C. Moreover, if it is a liquid culture medium, it is preferable to use shaking culture. Further, the culture is preferably batch culture.

  It is preferable that the process from collection of the sample described above to measurement of the amount of decrease in the substrate concentration is performed in a state in which contamination with bacteria (such as falling bacteria) is prevented.

  Next, in the step C for tentatively determining whether or not the biological composition in the ballast water satisfies the standard, it is tentatively determined whether or not the biological composition of the ballast water satisfies the standard based on the amount of decrease in the substrate concentration. To do.

  Specifically, the amount of decrease in the measured substrate concentration is compared with a predetermined value, and if it is less than the predetermined value, it is tentatively determined that the biological composition of the ballast water satisfies the standard, and if it exceeds the predetermined value It is preferable to tentatively determine that the biological composition of the ballast water does not satisfy the standard.

  The predetermined value is the substrate concentration measured in a reference medium whose culture conditions are substantially the same as the test medium and has a known biological composition corresponding to the above-mentioned standard related to the biological composition of ballast water. It is preferable to correspond to the amount of decrease.

  Note that the fact that the culture condition of the reference medium is substantially the same as that of the test medium means that the setting of the predetermined culture time for measurement is also the same as that of the test medium. In the test medium, when the raw water is in a concentrated state (X times), it is preferable that the reference medium contains an organism having a concentration X times the standard related to the biological composition of ballast water.

  The present inventor has found that when the culture conditions are substantially the same, the amount of decrease in the substrate concentration varies depending on the biological composition at the start of the culture, particularly the biological concentration. In the present invention, using this property, it is tentatively determined whether or not the test medium satisfies the criteria related to the biological composition of ballast water based on the decrease amount (predetermined value) of the substrate concentration in the reference medium. Can be determined.

  According to the present invention, since a significant decrease in the substrate concentration is observed in a short time from the start of culture, the determination is quicker than in the case of visual confirmation of colonies by the culture method (which takes about 4 days). The effect that can be performed is also obtained. Thereby, it can implement within the limited time and there also exists an effect which is hard to affect the navigation schedule etc. of a ship. Moreover, since a complicated apparatus and process are not required, the effect which can be implemented easily also in a ship is acquired.

  In the ballast water treatment method according to the present invention, when it is temporarily determined that the ballast water does not satisfy the standard by the above-described provisional determination method for ballast water quality, the ballast water is discharged outside the ship. Prior to this, a biological removal process or a biological killing process is performed. This contributes to the ship meeting the drainage standard more reliably.

  Ballast water treatment devices are usually designed so that they can essentially meet drainage standards. Therefore, as long as there are no unforeseen problems such as abnormalities in the ballast water treatment device or regrowth in the ballast tank, the drainage standards can usually be met. According to the present invention, whether or not such a problem has occurred can be quickly detected by making a provisional determination of the water quality of the ballast water before drainage. It is also possible to tentatively determine whether or not the biological composition of the ballast water satisfies a predetermined standard based on the presence or absence of such a problem.

  In the present specification, the "predetermined standard" in the present invention has been described mainly with respect to the discharge standard (water quality standard) defined in the D-2 rule ballast water discharge standard, but the standard itself has a property that can be changed. Even if the standard becomes stricter than the standard related to the D-2 rule, or the standard becomes looser, those standards are included in the “predetermined standard” in the present invention. In addition, the predetermined standards do not necessarily have to be established by the treaty, etc. For example, by appropriately setting standards that are stricter than the standards prescribed in the treaty, the standards prescribed in the treaty can be more reliably established. It is also preferable in the present invention to satisfy the requirements.

  In the above description, glucose is exemplified as the substrate to be measured, but the present invention is not limited to this.

  The “substrate” that can be measured by the present invention is a substance that can be consumed with the activity of a living organism in a broad sense, and a substance (reactant) that can catalyze a chemical reaction by an enzyme derived from a living organism in a narrow sense. It can be.

  The concentration of the substrate in the medium causes a significant decrease relatively early after the start of the culture. This is considered to be due to the fact that the consumption of the substrate by the organism can occur significantly even in the induction period (period before cell division begins to proceed actively) in a general growth curve, for example. According to the present invention using such properties, provisional determination of ballast water quality can be performed more rapidly than, for example, observing the amount of living organisms.

  The substrate used in the present invention is preferably one that is difficult to be released from the culture system (medium) outside the system or unintentionally mixed into the culture system from outside the system during culture. For example, oxygen can be used as a substrate because it is a substance that can decrease with the activity of living organisms. However, when the culture system is a liquid (liquid medium) or solid (solid medium), Due to the equilibrium with the adjacent gas phase, it may be released from the culture system or unintentionally mixed into the culture system, resulting in an error in the concentration measurement value.

  From the above viewpoint, as a substrate preferably used in the present invention, sugars such as glucose and fructose, coenzymes such as acetyl CoA, organic substances such as pyruvic acid, and inorganic substances (including inorganic ions) are also included. Preferable examples are glucose, and glucose is particularly preferable.

  Glucose is a highly versatile substrate because it is a glycolytic starting material, and is highly available in various organisms. Moreover, since the consumption of glucose is comparatively difficult to depend on the size of the living thing, there is also an effect that the determination of the ballast water quality can be made more accurate. Furthermore, since glucose is difficult to ionize (it is difficult to influence pH), even if it is added to the medium, there is an effect that the influence on the culture due to pH change does not easily occur.

  Further, in the present invention, as a preliminary test for selecting a substrate to be measured, in any at least two media having substantially the same culture conditions and different biological concentrations at the start of the culture, any arbitrary accompanying the culture is used. When the amount of decrease in the concentration of the substrate is measured and it is confirmed that the amount of decrease in the concentration of the substrate changes according to the biological concentration at the start of culture, the substrate is preferably used as the substrate to be measured. Can be used.

  In the present invention, the consumption of the substrate can be grasped mainly in two stages. First, there is a first stage in which the substrate is taken into the living body, and then there is a second stage in which the substrate taken into the living body is chemically changed to another substance by a chemical reaction. In the present invention, it may be considered that consumption of the substrate occurs (concentration decreases) at any of these stages.

  In the present invention, from the viewpoint of more rapidly observing a significant decrease in the substrate concentration, it is preferable that the substrate is consumed in the first stage in which the substrate is taken into the body of the organism. Thus, a significant decrease in the substrate can be observed without waiting for the substrate to chemically change to another substance in the organism.

  From the viewpoint of suitably measuring substrate consumption in the first stage, a substrate concentration measuring method that can be preferably used is a method in which the substrate concentration is selectively measured for a region outside the organism in the medium.

  As such a measurement method, for example, a method requiring a step of directly contacting a substrate and an element (substance or object) for detecting the substrate can be preferably exemplified. Here, it is preferable that the element is disposed outside the living body in the medium and is difficult to be taken into the living body or is not taken up substantially. Thereby, the measured value preferentially reflects the substrate concentration in the region outside the organism in the culture medium.

  Preferred examples of the element for detecting a substrate include a catalyst (enzyme) having substrate specificity for the substrate.

  As an example of enzyme utilization, there can be mentioned a method of measuring a substrate concentration using a biosensor that detects the substrate by contact with the substrate in a medium (or a measurement sample collected from the medium). Such a biosensor is configured so that, for example, an enzyme immobilized on an electrode directly detects the substrate by directly contacting the substrate, so that the substrate taken into the body of the organism Is hard to detect. The measured value preferentially reflects the substrate concentration in the area outside the organism in the medium. For example, if the substrate is glucose, a biosensor (glucose sensor) that specifically detects this concentration electrochemically and measures the concentration is commercially available.

  Alternatively, as another example of enzyme utilization, there is a method in which an enzyme is added to a medium (or a measurement sample collected from the medium), the substrate is treated with an enzyme, and a change in optical properties accompanying the enzyme treatment is optically measured. be able to. Since the enzyme is not easily taken into the living body, a substrate existing in a region outside the living body of the medium selectively contributes to a change in optical properties. Therefore, the measured value preferentially reflects the substrate concentration in the region outside the organism in the culture medium. For example, if the substrate is glucose, a method for measuring the absorbance of NADPH produced by enzymatic treatment with hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) in the presence of ATP and NADP is mentioned. Can do. Kits for performing such enzyme treatment are commercially available, for example, as “F-kit” manufactured by Roche.

  In the above examples of enzyme utilization, the case where the substrate is glucose has been mainly described, but the present invention is not limited to this. Since the substrate can be inherently metabolized by the organism, an enzyme specific to this is usually present. Therefore, a substrate other than glucose can be appropriately carried out by the same method as described above.

  Alternatively, as another measurement method for measuring substrate consumption in the first stage, organisms contained in the medium (or a measurement sample collected from the medium) are separated and removed by separation means such as membrane separation or gravity sedimentation. Thereafter, a method of directly measuring the concentration of the substrate in the medium from which the organism has been removed by an optical measurement method such as an absorbance measurement method can be mentioned. Thereby, the substrate concentration in the region outside the organism in the medium can be measured. Here, as the separating means, those capable of holding the substrate in a medium (or a measurement sample collected from the medium) are used. For example, the separation operation of removing the organism and holding the substrate can be easily performed by setting the opening of the membrane in the case of the membrane separation method or by setting the gravity of the gravity sedimentation method.

  As mentioned above, the method for measuring the consumption of the substrate in the first stage has been exemplified. However, in the present invention, it is particularly preferable that the concentration is measured by electrochemical detection using an enzyme specifically corresponding to the substrate. It is a sensor.

  On the other hand, in the present invention, it may be considered that the consumption of the substrate occurs at a stage where the substrate taken into the body of the organism chemically changes to another substance by the chemical reaction (the above-described second stage). In order to measure the consumption of such a substrate, for example, the concentration of the substrate in a medium (or a measurement sample collected from the medium) is directly measured in the presence of cultured organisms by an optical method such as absorbance measurement. What is necessary is just to measure by a measuring method. Since the biological membrane is usually light-transmitting, it is possible to detect both the substrate existing outside the living body and the substrate after being taken into the living body. Therefore, the substrate consumption in the second stage can be measured by measuring the substrate concentration in the whole medium (including the region in the body of the organism).

<Method for evaluating biological activity>
The biological activity evaluation method according to the present invention evaluates an organism present in the test medium as an activity based on the consumption of the substrate accompanying the culture in the test medium.

  The consumption of the substrate corresponds to, for example, the amount of decrease in the substrate concentration associated with the culture in the medium, and can be measured in the same manner as described in the above-described provisional determination method for ballast water quality.

  When the consumption amount of the substrate is large or the consumption rate of the substrate is high, the activity of the organism can be evaluated as high, and when the consumption amount of the substrate is small or the consumption rate of the substrate is slow, the activity of the organism can be evaluated as low.

  For example, in the case where the test medium includes a sample collected from ballast water, if the amount of decrease in the substrate concentration is small, the ballast water has a low growth ability as a whole of the contained organism (in the present invention, this is active). It can be estimated that the risk of environmental destruction is small. On the other hand, if the amount of decrease in the substrate concentration is large, the ballast water can be estimated to have a high growth ability as a whole of the contained organism (in the present invention, this is evaluated as having high activity), and the risk of causing environmental destruction. Can be determined to be large.

  In this way, for example, the safety of a test sample such as ballast water can be evaluated more quickly and more directly by a new viewpoint (proliferation ability of the contained organism as a whole) that is not present in the past. can get.

<Method of estimating biological concentration>
The biological concentration estimation method according to the present invention estimates the concentration of organisms present in the test medium based on the consumption of the substrate accompanying the culture in the test medium.

  When the culture conditions are substantially the same, the consumption of the substrate (the amount of decrease in the substrate concentration) associated with the culture for a predetermined time varies depending on the biological concentration at the start of the culture. Using this correlation, the biological concentration at the start of culture (ie, the biological concentration in the test sample) can be estimated based on the amount of decrease in the substrate concentration. Specifically, for the test medium whose biological concentration is unknown at the start of culture and the reference medium whose biological concentration is known, the amount of decrease in the substrate concentration was observed, and by comparing these, The biological concentration at the start of culture in the medium can be estimated.

  Using a plurality of media as reference media, create a correlation diagram (calibration curve) between the amount of decrease in substrate concentration and the biological concentration at the start of culture, and take this into consideration to start culture in the test media. It is also preferable to estimate the biological concentration in

  In the biological concentration estimation method according to the present invention, the above-described test medium and reference medium contain a single species of organism, or when multiple species of organisms are included, at the start of culture, Each of the plurality of kinds of organisms is included at substantially the same ratio (for example, the concentration of the bacteria A: the concentration of the bacteria B: the concentration of the bacteria C ... = a: b: c ... It is preferable from the viewpoint of further improving the density estimation accuracy.

  In the above description, the case where the test medium mainly includes a sample collected from the ballast water of the ship has been described as an example, but the present invention is not limited to this. By using an arbitrary test sample as a sample, provisional determination as to whether or not the biological composition in the sample satisfies a predetermined standard, evaluation of biological activity, and estimation of biological concentration can be appropriately performed.

  In the present invention, as the sample (test sample) contained in the test medium, it is preferable to use a sample containing one or two or more organisms, or a sample for which it is unknown whether or not organisms are contained. it can.

  The organism contained in (or can be contained in) the test sample is not particularly limited. For example, aquatic organisms defined by D2 described above can be preferably exemplified.

  In addition, from the viewpoint of prominently achieving the effects of the invention, microorganisms that can hardly be seen with the naked eye (specifically, organisms having a diameter of 0.1 mm or less) are included as organisms contained in the test sample. Preferable examples can be given.

  In addition, since this invention is invention which utilizes the phenomenon in which a substrate is consumed, a living organism is selectively made into object.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

Example 1
30 ml of seawater was sampled in a culture container, and 0.03 g of glucose (substrate) and 0.75 g of LB broth mirror (powdered medium component) manufactured by BD were added thereto to prepare a medium.

  The culture vessel was covered and shake-cultured at 37 ° C.

  The glucose concentration in the medium was measured by the following measurement method at the start of the culture and at 4 hours and 10 hours after the start of the culture. The measurement results are shown in FIG.

<Measurement method>
The glucose concentration was measured using an enzyme specific for glucose. Specifically, NADPH produced by enzymatic treatment of glucose with hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) in the presence of ATP and NADP using “F-kit” manufactured by Roche The glucose concentration was calculated from the measured value.

(Example 2)
30 ml of seawater was passed through a filtration membrane (MF membrane having an opening of 0.2 μm) for filtration, and then the filtration membrane was recovered.

  In a culture vessel, a medium formed by mixing 0.03 g of glucose (substrate) and 0.75 g of LB broth mirror (powder medium component) manufactured by BD with 30 ml of water was placed.

  The collected filtration membrane was put into this medium (here, since the amount of seawater flowed and the volume of the medium were the same, the bioconcentration magnification was equal (one time)).

  The culture vessel was covered and shake-cultured at 37 ° C.

  The glucose concentration in the medium was measured by the following measurement method at the start of the culture and at 4 hours and 10 hours after the start of the culture. The measurement results are shown in FIG.

(Example 3)
The glucose concentration was measured in the same manner as in Example 2, except that the amount of seawater passed during filtration was 300 ml (biological concentration ratio: 10 times) that was 10 times that of Example 2. The measurement results are shown in FIG.

<Evaluation>
As can be seen from FIGS. 2 to 4 showing the measurement results of Examples 1 to 3, it was observed that the concentration of glucose (substrate) in the medium decreased with the passage of the culture time.

  5 and 6 compare the glucose consumption after 4 hours and 10 hours from the start of cultivation in Examples 1 to 3. FIG. As is clear from these figures, it can be seen that a significant decrease in the substrate concentration can be measured in a relatively short time from the start of the culture, and the biological activity can be evaluated quickly.

  Further, by comparing the concentration of the ballast water (raw water) with the comparison of Example 2 (bioconcentration ratio: equal (1 ×) times) and Example 3 (bioconcentration ratio: 10 times), glucose consumption is increased. It can be seen that can be increased. This contributes to enabling early measurement and improving measurement accuracy.

  Furthermore, it can be seen from the results of Example 2 and Example 3 that the amount of decrease in the substrate concentration tends to increase according to the number of organisms in the sample (initial organism concentration). From this, for example, the following matters were confirmed.

  First, it was confirmed that it is possible to tentatively determine whether or not the biological composition of the ballast water satisfies a predetermined standard based on the amount of decrease in the substrate concentration in the test medium.

  That is, assuming that the biological composition contained in the sample of Example 2 corresponds to a predetermined standard, the decrease amount of the substrate concentration observed in Example 2 can be set as a “predetermined value”. If an amount of decrease in the substrate concentration exceeding the predetermined value is observed for an unknown sample, it is tentatively determined that the sample does not satisfy the predetermined standard. This is because the sample of Example 3 having a larger number of organisms than the sample of Example 2 (that is, assuming that the biological composition contained in the sample of Example 2 corresponds to a predetermined standard) This is supported by the fact that a greater decrease in substrate concentration was observed in samples that were clearly unfilled.

  Conversely, assuming that the biological composition contained in the sample of Example 3 corresponds to a predetermined standard, the amount of decrease in the substrate concentration observed in Example 3 can be set as a “predetermined value”. If an amount of decrease in substrate concentration below this predetermined value is observed for an unknown sample, it is tentatively determined that the sample does not meet a predetermined standard. This is because the sample of Example 2 having a smaller number of organisms than the sample of Example 3 (that is, assuming that the biological composition contained in the sample of Example 3 corresponds to a predetermined standard) This is supported by the observation of a smaller decrease in substrate concentration in samples that are clearly not filled.

  Furthermore, from the results of Example 2 and Example 3, it is confirmed that the activity (high growth ability) can be evaluated and the biological concentration can be estimated based on the decreased amount of the substrate concentration in the test medium. It was done.

  In the above examples, an example using the absorbance method was shown in measuring the substrate concentration. However, it has been confirmed that measurement can be performed with higher accuracy and ease by using a biosensor.

Claims (9)

The amount of decrease in the concentration of the substrate measured by culturing a test medium containing at least a predetermined substrate and a sample obtained by concentrating raw water collected from the ballast water treated by the ballast water treatment device for a predetermined time. The provisional determination method of the ballast water quality characterized by tentatively determining whether or not the biological composition of the ballast water satisfies a predetermined standard.   At the time of the determination, the amount of decrease in the concentration of the substrate is compared with a predetermined value. The provisional determination method for ballast water quality according to claim 1, wherein it is provisionally determined that the biological composition of the ballast water does not satisfy the standard.   The predetermined value corresponds to a decrease in the concentration of the substrate measured in a reference medium having a known biological composition corresponding to the standard in which the culture conditions are substantially the same as the test medium. The provisional determination method of the ballast water quality according to claim 1 or 2, characterized by the above.   The ballast water quality according to any one of claims 1 to 3, wherein when measuring the amount of decrease in the concentration of the substrate, a region outside the body of the organism is selectively measured in the test medium. Provisional judgment method.   As a preliminary test for selecting the substrate to be measured, the amount of decrease in the concentration of any substrate that accompanies the culture in at least two media having substantially the same culture conditions and different biological concentrations at the start of the culture When the amount of decrease in the concentration of the substrate is confirmed to change according to the biological concentration at the start of culture, the substrate is used as the substrate. 4. The provisional determination method for ballast water quality according to any one of 4 above.   The method for provisional determination of ballast water quality according to any one of claims 1 to 5, wherein glucose is used as the substrate.   The method for provisional determination of ballast water quality according to any one of claims 1 to 6, wherein the method is performed in a ship.   When it is temporarily determined that the ballast water does not satisfy the standard by the provisional determination method for ballast water quality according to any one of claims 1 to 7, before the ballast water is discharged outside the ship, the biological removal treatment or A method for treating ballast water, comprising performing biocidal treatment. Based on the consumption of the substrate in the culture medium in the test medium containing the sample obtained by concentrating the raw water collected from the ballast water treated by the ballast water treatment device, the proliferation ability of the organism containing the ballast water as a whole is increased. A method for evaluating the biological activity of ballast water, characterized by estimating whether the activity of a plurality of types of organisms present in the test medium is high or low .

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