Methods for evaluating biodegradability

Abstract

To provide a biodegradability evaluation method that is both highly accurate in assessing the biodegradability of organic matter and easy to measure, the proportion of organic carbon oxidized by microbial catabolism of organic matter (C) is measured. BOD ) and the proportion of organic carbon assimilated by microorganisms (C BIO The biodegradability is evaluated by calculating the total of ( ) and ( ) as the degree of biodegradation, and evaluating the biodegradability based on the said degree of biodegradation.

Description

[Technical Field]

[0001] This invention relates to a method for evaluating the biodegradability of organic matter, and more specifically, to a method for evaluating biodegradability more accurately than conventional methods. Furthermore, it relates to a method for evaluating the changes in the degree of biodegradation over time more accurately and simply. [Background technology]

[0002] Traditionally, methods for evaluating the biodegradability of organic matter have widely utilized methods that primarily measure indicators related to microbial catabolism. For example, the OECD 301B test method and JIS K6951 measure the amount of carbon dioxide produced, while the OECD 301F test method evaluates biodegradability by measuring oxygen consumption.

[0003] In evaluating the biodegradability of biodegradable resins, the biodegradability is also evaluated based on the evaluation method described above (for example, Patent Documents 1-3).

[0004] However, these conventional evaluation methods only assess the change in organic carbon content associated with microbial catabolism, and therefore were sometimes insufficient for understanding biodegradation as a whole. For example, even organic substances that are actually highly biodegradable, such as starch, may only be evaluated as being around 60% biodegradable using conventional methods, leading to a tendency to underestimate their biodegradability.

[0005] On the other hand, methods have been proposed to comprehensively evaluate biodegradability by considering not only the amount of organic carbon oxidized by catabolism, but also the amount of organic carbon in the biomass due to assimilation by microorganisms and the amount of dissolved organic carbon that remains undegraded (undegraded residual polymer). However, considering all of these factors would require conducting numerous tests, which would not only make the procedure cumbersome but also increase the time required for measurement. Therefore, it is not practical to apply this as a general indicator for evaluating the biodegradability of various organic materials.

[0006] Furthermore, such comprehensive methods make it difficult to track and evaluate the degree of biodegradation over time. For example, quantifying the amount of organic carbon in the biomass produced by assimilation, or the amount of organic carbon remaining in the test solution, requires sampling each time, making continuous or non-destructive measurement difficult. In other words, it is not easy to evaluate the biodegradation process over time using the same test system. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2015-174979 [Patent Document 2] Special Publication No. 2023-504142 [Patent Document 3] International Publication No. 2024 / 203672 [Overview of the project] [Problems that the invention aims to solve]

[0008] Therefore, against this background, the present invention provides a biodegradability evaluation method that allows for more accurate evaluation of the biodegradability of organic matter while also being easy to measure. [Means for solving the problem]

[0009] However, the inventors of this invention have focused on the fact that in order to accurately evaluate biodegradability, it is important to consider not only microbial catabolism but also the amount of organic carbon incorporated through assimilation by microorganisms. However, since measuring multiple items with different indicators is cumbersome, we conceived the idea that a method for accurately and simply evaluating biodegradability based on organic carbon balance, while limiting the number of measurement items, would be effective. In light of these circumstances, after diligent research, we found that the proportion of organic carbon oxidized during microbial catabolism (C) BOD ) and the proportion of organic carbon (C) incorporated by assimilation into microorganisms. BIO) By calculating the biodegradation degree using these, it has been found that the biodegradability of organic substances can be evaluated more accurately and simply.

[0010] That is, the present invention has the following aspects. [1] A method for evaluating the biodegradability of an organic substance, comprising: calculating the total of the ratio (C BOD ) of the organic carbon oxidized along with the dissimilation by microorganisms and the ratio (C BIO ) of the organic carbon assimilated by microorganisms as the biodegradation degree, and evaluating the biodegradability based on the biodegradation degree. A method for evaluating biodegradability. [2] The method for evaluating biodegradability according to [1], including the following steps (a) to (d). (a) A step of calculating the ratio (C BOD ) of the organic carbon oxidized along with the dissimilation of microorganisms at a plurality of time points including the end point of the biodegradation test. (b) A step of calculating the ratio (C BIO ) of the organic carbon assimilated by microorganisms at the end point of the biodegradation test. (c) By equally dividing the ratio (C BIO ) of the organic carbon assimilated by the microorganisms among the plurality of time points at which the ratio (C BOD ) of the organic carbon oxidized along with the dissimilation of the microorganisms was obtained and cumulatively adding at each time point, calculating the estimated C BIO at each time point. (d) At each time point at which the ratio (C BOD ) of the organic carbon oxidized along with the dissimilation of the microorganisms was obtained, adding the estimated C BOD calculated in the step (c) to the ratio (C BIO ) of the organic carbon oxidized along with the dissimilation of the microorganisms to calculate the biodegradation degree. [3] Further, in addition to the end point of the biodegradation test, calculating the ratio (C BIO ) of the organic carbon assimilated at one or more time points during the biodegradation test period, and based on the calculated values at these multiple measurement time points, the ratio (C BIOA method for evaluating biodegradability as described in [1] or [2], which involves interpolating and estimating the values ​​of the following: [4] When the organic substance is water-soluble, the standard value of the total amount of organic carbon contained in the organic substance (C CAL ) From this, the proportion of organic carbon remaining in the test solution (C DOC By subtracting the aforementioned catabolized organic carbon (C), the proportion of C BOD ) and the proportion of organic carbon assimilated (C BIO A method for evaluating biodegradability described in any of [1] to [3], which calculates the total percentage of ). A method for evaluating biodegradability, comprising creating a biodegradation curve based on the degree of biodegradation at each measurement point calculated by the biodegradability evaluation method described in [5] [2] or [3], and evaluating the biodegradability of organic matter over time using the biodegradation curve. [Effects of the Invention]

[0011] This invention relates to the proportion of organic carbon assimilated by microorganisms (C BIO By taking these factors into consideration, it becomes possible to calculate the degree of biodegradation that reflects the overall picture of the biodegradation process. This allows for a more accurate assessment of biodegradability that reflects the actual situation, compared to conventional methods that only evaluate changes in organic carbon content associated with microbial catabolism.

[0012] Furthermore, when displaying a biodegradation curve using the calculated biodegradation rate at each measurement point, the time-dependent changes in the biodegradation rate of organic matter can be evaluated more accurately and simply.

[0013] In particular, for water-soluble resins, the proportion of organic carbon in insoluble organic matter in the test solution at the start of the biodegradation test (C POL In systems where the initial value of ) is extremely small, the proportion of organic carbon in the insoluble organic matter in the test solution (C POL By assuming that ) is 0 and measuring the amount of carbon dioxide generated by catabolism, it is also possible to easily perform an evaluation that is close to the true degree of biodegradation.

[0014] This invention is effective as a practical evaluation method due to its excellent operability and reproducibility. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 is a graph showing the biodegradation curve created based on the degree of biodegradation at each measurement point, calculated using the method for evaluating the biodegradability of the examples and reference examples. [Figure 2] Figure 2 is a graph showing the biodegradation curve created based on the degree of biodegradation at each measurement point, calculated using the method for evaluating the biodegradability of the examples and reference examples. [Modes for carrying out the invention]

[0016] The present invention will be described below based on examples of embodiments for carrying out the present invention. However, the present invention is not limited to the embodiments described below.

[0017] In this specification, when "X~Y" (where X and Y are any numbers) is used, unless otherwise specified, it means "greater than or equal to X and less than or equal to Y," and also includes the meanings of "preferably greater than X" or "preferably less than Y." In this specification, when we use the expressions "X or greater" (where X is any number) or "Y or less" (where Y is any number), we also mean "preferably greater than X" or "preferably less than Y." In this specification, the numerical ranges described in stages may be arbitrarily combined with the upper or lower limits of the numerical ranges in any stage. Furthermore, in the numerical ranges described herein, the upper or lower limits may be replaced with the values ​​shown in the examples.

[0018] In this specification, "organic matter" means a compound having a carbon skeleton, and includes naturally occurring or synthetically derived polymers, low molecular weight compounds, or mixtures thereof. Specifically, examples include natural polymers such as starch, cellulose, pullulan, casein, gelatin, chitin, chitosan, and lignin, as well as synthetic biodegradable polymers such as polylactic acid, polybutylene succinate, polybutylene adipate terephthalate, polycaprolactone, polyhydroxyalkanoic acid, and polyglycolic acid. Furthermore, the intention is to include organic low molecular weight substances such as glucose, organic acids, fatty acids, sugar alcohols, and surfactants, as well as additives such as plasticizers, dispersants, and stabilizers that have a carbon skeleton. Furthermore, the organic material can be in any form of substance, such as solid, liquid, gel, or film, that can be subjected to biodegradation testing.

[0019] In this specification, "biodegradation" refers to the phenomenon in which organic matter is mineralized into carbon dioxide and water, etc., by the action of microorganisms. On the other hand, in this specification, "biodegradability" refers to the property of organic matter to be decomposed by microorganisms. Furthermore, in this specification, "biodegradability" refers to the percentage (%) of organic carbon that is decomposed by microorganisms within a certain period of time relative to the total amount of organic carbon contained in the organic matter. In other words, it refers to the percentage (%) at which organic matter is decomposed by microorganisms within a certain period of time.

[0020] In this specification, "catabolism" refers to the process by which organic matter is broken down by the metabolic activity of microorganisms and converted into inorganic compounds such as carbon dioxide and water. Catabolism is primarily caused by the respiratory activity of microorganisms, where organic carbon contained in organic matter is oxidized and released as carbon dioxide, leading to a decrease in organic matter.

[0021] Furthermore, in this specification, "assimilation" refers to the process by which organic carbon in organic matter is taken up as a component of microorganisms, i.e., as cells or biomass, and retained within the microbial body. This is a process in which microorganisms utilize organic matter as a nutrient source and use it for their own growth and proliferation. Unlike catabolism, it does not involve the release of organic carbon through decomposition, and organic carbon is accumulated within the microorganism.

[0022] In this specification, C CAL (Calculated Carbon) refers to a standard value (100%) based on the total amount of organic carbon contained in the organic substance in question. Also, C BOD (Carbon from Biochemical Oxygen Demand) refers to the proportion of organic carbon released as carbon dioxide by microorganisms, calculated based on the amount of oxygen consumed by the respiratory metabolic activity of microorganisms; in other words, it indicates the proportion of organic carbon that has been decomposed. BOD Although it is usually not possible to measure organic carbon directly, the amount of oxygen consumed, measured as biochemical oxygen demand (BOD), can be converted based on the correspondence between the theoretical oxygen demand (ThOD) of the organic substance in question and the amount of organic carbon. This conversion allows for the calculation of the percentage of organic carbon resulting from decomposition. And C BIO (Carbon from Biomass Incorporation) refers to carbon produced by microorganisms, specifically the proportion of organic carbon that is incorporated into the microbial body through assimilation of organic matter and retained within the microbial cells.

[0023] In this specification, C DOC "(Carbon from Dissolved Organic Carbon)" refers to the percentage of organic carbon dissolved in the test solution at the end of the biodegradation test. Also, C POL "Carbon from Polymer Residue" refers to the proportion of organic carbon in insoluble organic matter (polymers, etc.) that remains suspended or precipitated in the test solution.

[0024] <First Embodiment> A method for evaluating the biodegradability of organic matter according to one embodiment of the present invention involves converting the amount of oxygen consumed due to catabolism of the organic matter by microorganisms into the amount of organic carbon, and determining the proportion of catabolized organic carbon (C BOD ) is calculated, and the proportion of catabolized organic carbon (C BOD ) and the proportion of assimilated organic carbon (C BIOThe total of ( ) and ( ) is calculated as the degree of biodegradation, and the biodegradability is evaluated based on the said degree of biodegradation.

[0025] Organic carbon in organic matter can be broadly classified into "decomposed" and "undecomposed" organic carbon (C). Decomposed organic carbon is catabolic organic carbon (C). BOD ) and assimilated organic carbon (C BIO ) consists of organic carbon (C) dissolved in the test solution. DOC ) and insoluble organic matter (C POL ) consists of.

[0026] Of these, (C BOD ) and (C BIO As mentioned above, all of these indicate that the target organic matter has been decomposed by microorganisms, and (C BOD ) and (C BIO The sum of these can be considered to represent the proportion of biodegraded organic carbon, i.e., the degree of biodegradation.

[0027] Thus, (C BOD ) and (C BIO By using the sum of ( ), it is possible to calculate the degree of biodegradation (%) that takes both catabolism and assimilation into account. This allows for a higher proportion of catabolized organic carbon (C) compared to conventional methods. BOD Compared to methods that use only ) as an evaluation indicator, this method allows for a simpler assessment of biodegradability that reflects the actual situation.

[0028] The proportion of catabolized organic carbon (C BOD (%) can be measured by a publicly known method, and is the value obtained by dividing the amount of oxygen consumed by microbial catabolism of organic matter by the theoretical oxygen demand (ThOD) of the organic matter. BOD Let it be (%).

[0029] The proportion of assimilated organic carbon (C BIO )(%) can be measured by publicly known methods, but it is preferable to calculate it by the following method, for example. The amount of dissolved organic carbon (DOC0) at the start of the experiment and the amount of dissolved organic carbon (DOCt) at the end of the experiment were measured, and C DOC First, calculate DOCt / DOC0. Next, add 0.5 N hydrochloric acid to the test solution after the specified time to lower the pH to 3 or below, centrifuge at 3500 rpm for 15 minutes, then suction filter using 1 μm glass filter paper that has been previously filtered with purified water and dried, and dry at 105°C for 2 hours. From the mass of the residue on the filter paper after drying and the mass of the organic matter used in the test, calculate the percentage of organic carbon (C) in the remaining insoluble organic matter (polymer, etc.) suspended and precipitated in the test solution. POL ) was sought. And the proportion of assimilated organic carbon (C BIO ) is the standard value for total organic carbon (C CAL ) From this, the proportion of organic carbon oxidized by microbial catabolism (C BOD ), the proportion of organic carbon that remains dissolved (C DOC ), and the proportion of organic carbon in the insoluble organic matter remaining as suspension or precipitate (C POL By subtracting ), the proportion of organic carbon assimilated by microorganisms can be calculated.

[0030] When the aforementioned organic matter exhibits high water solubility, a more accurate assessment of biodegradability can be made more easily. In other words, in the present invention, if the water solubility of organic matter is high, it can be assumed that all organic matter is uniformly dissolved in the test solution. This allows for the determination of the proportion of insoluble organic matter remaining in the test solution (C POL ) can be considered as 0.

[0031] Under these conditions, the standard value for the total organic carbon content of organic matter at the start of the test (C CAL ) the proportion of organic carbon catabolized by microorganisms (C BOD ) and the percentage of organic carbon remaining in the test solution at the end of the test (C DOC Subtracting the total of (C) gives the percentage of organic carbon assimilated by microorganisms. BIO ) is calculated, and the percentage of organic carbon catabolized by microorganisms (C BOD The degree of biodegradation can be calculated by adding () and ().

[0032] Furthermore, under these conditions, the standard value for the total organic carbon content of organic matter at the start of the test (C CAL ) to the percentage of remaining organic carbon in the test solution at the end of the test (C DOC By subtracting ), the proportion of organic carbon catabolized by microorganisms (C BOD ) and the proportion of organic carbon assimilated (C BIO The sum of these, i.e., the degree of biodegradation, can be calculated. Thus, for highly water-soluble organic substances, the proportion of organic carbon dissolved in the test solution at the end of the test (C) DOC By measuring only ), the degree of biodegradation can be easily estimated.

[0033] In this specification, "an organic substance is water-soluble" means an organic substance that has a solubility of 100 mg / L or more at 20°C, as determined by a water solubility test in accordance with OECD Guideline 120. The water solubility can be determined, for example, by the following procedure. Specifically, 2 g (Wg) of the test substance is accurately weighed using an electronic balance, placed in a 300 mL stoppered Erlenmeyer flask, and 200 mL (V mL) of pH 7.0 buffer solution is added. The mixture is then shaken for 24 hours in a constant temperature shaking incubator set to 20°C, and the same procedure is repeated three times to prepare three test solutions. The test solutions are filtered through a 5 μm pore size membrane filter, the residue and filter are placed in a weighing bottle, dried under reduced pressure in a vacuum dryer (40°C), and then cooled to room temperature in a desiccator. The total mass Z (g) is then measured using an electronic balance. Let X (g) be the mass of the weighing bottle containing the pre-dried and weighed membrane filter. The water solubility concentration C (g / L) in the test solution is calculated from the relationship with the sample mass W (g) using the following formula (1). C = [W - (ZX)] / V × 1000 ... (1) Here, C represents the water solubility concentration in the test solution (g / L), W represents the sample mass (g), Z represents the mass of the weighing bottle including the residue and filter (g), X represents the mass of the weighing bottle including the filter (g), and V represents the volume of the test solution (mL). The average of the three obtained measurements is taken as the water solubility concentration, and if the average value is 100 mg / L or higher, the organic substance is considered to be water soluble.

[0034] In this specification, "highly water-soluble" means that in the initial stages of the biodegradation test (for example, within 24 hours of the start of the test), most of the total organic carbon content of the organic substance (for example, 80% by mass or more) is dissolved in water. In such a state, the proportion of insoluble organic matter in the test solution (C POL ) is very small, therefore the proportion of insoluble organic matter (C POL It is reasonable to treat ) as 0 (zero).

[0035] If the organic matter is insoluble in water or has low solubility in water, the percentage of insoluble organic matter remaining in the solution at the end of the test (C POL ) and the percentage of dissolved organic carbon in the solution at the end of the test (C DOC ) Measure. Furthermore, the oxygen consumption is converted based on the theoretical oxygen demand (ThOD) to determine the proportion of catabolized organic carbon (C). BOD Calculate (%) and these C DOC (%), C BOD (%) and C POL (%) is the standard value for total organic carbon content (C CAL By subtracting from ), the proportion of assimilated organic carbon (C BIO It is possible to calculate ). The proportion of insoluble organic carbon obtained (C POL ) and the proportion of dissolved organic carbon (C DOC The total of ) is the standard value for total organic carbon (C CAL By subtracting from ), the proportion of organic carbon catabolized by microorganisms (C BOD ) and the proportion of organic carbon assimilated (C BIO It is also possible to calculate the total of ) and, i.e., the degree of biodegradation.

[0036] <Second Embodiment> Furthermore, in addition to the final biodegradability, evaluating the time change in the degradation behavior, i.e., the transition of biodegradability over time, is useful for more accurately understanding the biodegradability of organic substances. In recent years, devices capable of continuously measuring the transition of biodegradability have been developed. However, even when using these devices, due to the measurement principle, processes such as microorganism separation and dry mass measurement cannot be completely omitted, and there is a problem that it takes time and effort. On the other hand, for the dissimilatory fraction (C BOD ), in an experiment in a closed system, since the amount of generated carbon dioxide can be continuously measured, data over time can be obtained relatively easily.

[0037] Therefore, in the present invention, the time point for calculating C BIO is limited, for example, it is calculated only at the end point or at a plurality of time points including the end point. Then, by evenly distributing the calculated C BIO along the time axis, the estimated C BIO value at each time point is calculated. By adding this estimated C BIO to the time-course data of C BOD respectively, the total of C BOD and C BIO (C BOD + C BIO ) can be simply calculated as the biodegradability over time, and for example, the change over time can be visually evaluated by a graph or the like.

[0038] That is, the method for evaluating the change over time of the biodegradability of the present invention preferably includes the following steps (a) to (d). (a) A step of calculating the ratio (C BOD ) of the organic carbon released as carbon dioxide due to the dissimilation of microorganisms at a plurality of time points including the end point of the biodegradation test. (b) A step of calculating the ratio (C BIO ) of the organic carbon assimilated by microorganisms at the end point of the biodegradation test. (c) The ratio (C BIO ) of the organic carbon assimilated by the microorganisms is measured by measuring the oxygen consumption to obtain C BODDividing at a plurality of time points where the obtained value is determined and cumulatively adding at each time point, the estimated C at each time point BIO is calculated. (d) At each time point where the above C BOD is determined, the ratio of the oxidized organic carbon (C BOD ) and the estimated C BIO calculated in the above step (c) are added to calculate the biodegradability.

[0039] The above method will be described in more detail. Figure 2 is a graph showing the change over time by comparing the biodegradability (%) (Reference Example 2) based only on the conventional C BOD with the biodegradability (%) (Example 2) used in the present invention, using water-soluble gelatinized starch as the target organic substance.

[0040] That is, in the present embodiment, in order to evaluate the biodegradability of the organic substance, the test start date was set as day 0, and the oxygen consumption amount associated with the oxidation by microorganisms was measured at each time point of 8 days, 16 days, 24 days, and 32 days (the final day). The oxygen consumption amounts at these time points were converted into carbon amounts, and the ratio of the oxidized organic carbon (C BOD ) at each time point was calculated. On the other hand, the ratio of the assimilated organic carbon (C BIO ) was calculated only at the end of the test, on the 32nd day. The value of this C BIO was divided into four equal parts, and by adding them in a form of sequentially accumulating at each measurement time point, the estimated C BIO at each time point was calculated.

[0041] The estimated C BIO at each time point Ti can be calculated by the following formula (2). Estimated C BIO (T i ) = i / N × (C BIO )(final day)…(2) i: Order of the measurement time point (an integer from 1 to N) N: Total number of all measurement time points (including the final day) C BIO (final day): Ratio of the assimilated carbon measured on the final day

[0042] In other words, on the 8th day C BIO (On the final day) 1 / 4 is added, 1 / 4 x 2 is added on the 16th day, 1 / 4 x 3 is added on the 24th day, and 1 / 4 x 4 (i.e., the total amount) is added on the 32nd day, and this is the estimate at each measurement point. CBIO That's what I decided.

[0043] Subsequently, C at each point in time BOD Estimated C corresponding to BIO The biodegradation rate (%) at each point in time was calculated by adding these values ​​together.

[0044] As a result, Reference Example 2 (C BOD The biodegradability (based on individual samples) increases over time, but has plateaued at a low level. On the other hand, the present invention is "C BOD and C BIO The biodegradability based on the "total of" (Example 2) showed generally high values, approaching 100% on day 32 (the final day), indicating that the biodegradability of starch was appropriately evaluated.

[0045] Thus, according to this embodiment, C BIO Without directly calculating it at multiple points in time, C can be obtained from the final value. BOD Estimated C at each time point measured BIO Because this calculation reduces the burden of testing, it is possible to appropriately grasp the overall biodegradability.

[0046] <Third Embodiment> Furthermore, in order to more accurately understand the changes in biodegradability over time, C BIO Measurements may be taken not only on the 32nd day, which marks the end of the period, but also at multiple points in time. For example, if measurements are taken at an intermediate point in the test period (e.g., the 16th day), the change over time can be interpolated between the two points, further improving the accuracy of correction and reducing evaluation errors. In this invention, the proportion of assimilated organic carbon C BIO To more accurately estimate the change over time, C was measured at multiple (two or more) points in time during the test period. BIOIt is preferable to determine C at points 2-4. BIO It is preferable to determine the C between each time point, and it is even more desirable that these measurement points be evenly distributed in time. By appropriately setting the measurement points, the C between each time point can be determined. BIO This allows for highly accurate estimation of changes using linear interpolation, thereby improving the reliability of the time-dependent changes in biodegradation rates.

[0047] Specifically, the C obtained throughout the test period BIO The measurement points are listed in ascending order as t1, t2, ..., t n (However, t n (≧2) and the (C) measured at each point in time BIO The values ​​of C1, C2, ..., C n When this is the case, at any point in time t(t i ≦t≦t i+1 ) in C BIO This can be estimated based on the following equation (3). Estimated C BIO (t)=C i +((C i+1 -C i ) / (t i+1 -t i ))×(tt i )...(3) However, i is an integer of any value between 1 and n-1.

[0048] The above method will be explained in more detail. C BIO Since it increases over time due to microbial assimilation, we can assume that assimilation has not occurred at the start of the experiment (day 0), C BIO The initial value can be set to 0. For example, C BIO If we determine the values ​​on day 16 and day 32, and denote the respective measured values ​​as A(%) and B(%), then C on day 8 and day 24 BIO This can be estimated by linear interpolation as follows. Day 8 :C BIO =A × (8 / 16) = 0.5A Day 16:C BIO =A Day 24:CBIO =A + (BA) × (8 / 16) = A + 0.5 (BA) Day 32:C BIO =B In this way, C between measurement time points BIO By linearly interpolating the time change of C at any given time, BIO This allows us to estimate the time-dependent biodegradation rate and calculate a more accurate representation of the actual situation.

[0049] C BIO Measuring at multiple points in time allows for highly accurate temporal evaluation without relying on interpolation, but on the other hand, C BIO The measurement process involves time-consuming steps such as sample collection, biomass separation, and drying. As the number of measurements increases, the experimental procedure becomes more complicated, and it also presents the problem of difficulty in performing measurements while maintaining the same test system. In this invention, C BIO By measuring the biodegradability only at limited points during the test period and estimating it through interpolation at other points, it is possible to calculate a highly reliable biodegradability while maintaining a balance between ease of evaluation and accuracy.

[0050] According to the present invention, the degree of biodegradation that accurately reflects the actual situation can be easily calculated, and this degree of biodegradation can be used to accurately and precisely evaluate the biodegradability of the target material. Generally, it is assumed that the rate and degree of biodegradation differ depending on the material. However, based on the biodegradation rate according to the present invention, it is possible to set standard values ​​(thresholds) and evaluation criteria according to each material. For example, it becomes possible to evaluate a material as "highly biodegradable if its biodegradation rate is ○% or higher" (where ○ is a specific number). This allows for quantitative and objective evaluation tailored to the characteristics of each material, and can serve as an indicator in material development, selection, and application development.

[0051] Thus, the present invention provides a method for evaluating the biodegradability of biodegradable materials in a manner consistent with actual conditions, and is useful for reducing environmental impact, addressing microplastics, and supporting the performance evaluation and certification of biodegradable materials. Therefore, it can be widely used in the development, selection, and examination of compliance with standards for biodegradable films, packaging materials, and the like. [Examples]

[0052] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist of the invention. In the examples, "parts" and "%" refer to mass unless otherwise specified.

[0053] First, we prepared various organic substances to be evaluated, as described below. • Cellulose: Crystalline cellulose (20μ, manufactured by Sigma-Aldrich) • Starch: Pregelatinized starch (Waxy Alpha Y, manufactured by Sanwa Starch Industry Co., Ltd.) • Pullulan: Pullulan (manufactured by Nagase Vita Co., Ltd.) • Polyvinyl alcohol: Partially saponified polyvinyl alcohol (average degree of saponification 88 mol%, viscosity of 4% aqueous solution at 20°C 43 mPa·s)

[0054] [Example 1] (Experimental conditions) In this example, crystalline cellulose (hereinafter sometimes referred to as "cellulose") was used as the organic material to verify the biodegradability evaluation method according to the present invention. Activated sludge from a sewage treatment plant that treats household wastewater was used as the microbial source, and inoculated into the test solution to achieve an MLSS concentration of 30 mg / L. The test solution was prepared using a test culture solution specified in OECD 301F, with a crystalline cellulose addition concentration of 100 mg / L. The prepared test culture solution was dispensed into a 510 mL sealed test container, OxiTop-C® (manufactured by WTW), and incubated at 22°C and pH 7.4 ± 0.2 for 32 days under dark conditions with continuous stirring.

[0055] (Calculation of biodegradability) Oxygen consumption was measured according to OECD 301F, and the C value at each time point was calculated from the oxygen consumption at four time points on days 8, 16, 24, and 32 during the test period. BOD The result was calculated. Furthermore, using a TOC meter multi N / C 3100 (manufactured by Analytic Jena), the amount of dissolved organic carbon in the test culture medium was measured on the start day and the final day (day 32), and the C at the time of the final day (day 32) was measured. DOC The result was calculated. Next, on the final day (day 32), the residual insoluble organic matter in the test culture medium was measured, and C POL The result was calculated. And then, C on the last day of the exam. BIO (Day 32) The standard value for total organic carbon content at the start of the test (C CAL ) From this, the proportion of dissolved organic carbon (C DOC (Day 32), the proportion of catabolized organic carbon (C BOD (Day 32), percentage of remaining insoluble organic matter (C POL It was calculated by subtracting ) from the result. The C calculated above BIO (Day 32) and C BOD The sum of this and the data from day 32 was used as the degree of biodegradation. Also, the obtained C BIO (Day 32) C BOD The value is divided equally at each measurement point (4 points on days 8, 16, 24, and 32), and the cumulative sum is calculated at each point to estimate the value of C at each point. BIO The result was calculated. Note C CAL This is a reference value based on the total amount of organic carbon contained in the target organic material, that is, a value calculated based on the total amount of organic carbon contained in the cellulose supplied at the start of the test.

[0056] [Example 2] In Example 1, the test conditions and measurement methods were the same as in Example 1, except that pregelatinized starch (hereinafter sometimes referred to as "starch") was used instead of crystalline cellulose as the test organic material, and the degree of biodegradation was calculated in the same manner. Note that pregelatinized starch is a water-soluble organic substance, C POL No measurements were taken.

[0057] [Example 3] In Example 1, the test conditions and measurement methods were the same as in Example 1, except that pullulan was used instead of crystalline cellulose as the test organic material, and the degree of biodegradation was calculated in the same manner. Note that since pullulan is a water-soluble organic material, C POL No measurements were taken.

[0058] [Example 4] In Example 1, the test conditions and measurement methods were the same as in Example 1, except that partially saponified polyvinyl alcohol (hereinafter sometimes referred to as "PVA") was used instead of crystalline cellulose as the test organic material, and the degree of biodegradation was calculated in the same manner. Note that since partially saponified polyvinyl alcohol is a water-soluble organic material, C POL No measurements were taken.

[0059] [Reference example 1] C measured in Example 1 (cellulose) BOD Using only C BIO The degree of biodegradation was calculated without considering this factor.

[0060] The biodegradation results for Examples 1-4 and Reference Example 1 (final day of the test: day 32) are shown in Table 1 below. Furthermore, biodegradation curves were created based on the degree of biodegradation at each measurement point calculated in Examples 1-4 and Reference Example 1, and are shown in Figures 1 and 2. Note that Figures 1 and 2 show the conventional method C BOD The biodegradation curves for Reference Example 2 (starch), Reference Example 3 (pullulan), and Reference Example 4 (PVA), which were created based solely on biodegradability, are also shown.

[0061] [Table 1]

[0062] The results in Table 1 and Figures 1 and 2 show that the present invention is useful as a means of evaluating biodegradability with higher accuracy compared to conventional methods. In other words, in Examples 1 to 4, the C at the end of the test was BIO By calculating this value and distributing it evenly across each point in time, it was possible to evaluate biodegradability based on a more accurate degree of biodegradation, and it was also possible to easily estimate the degree of biodegradation over time. On the other hand, as in Reference Example 1, C BOD If evaluated using only C, BIO Because this factor was not taken into consideration, the degree of biodegradation could not be accurately assessed.

[0063] While the above embodiments illustrate specific forms of the present invention, these embodiments are merely illustrative and should not be interpreted restrictively. Various modifications that are obvious to those skilled in the art are intended to fall within the scope of the present invention. [Industrial applicability]

[0064] The present invention's method for evaluating the biodegradability of organic materials can be used for evaluating materials such as biodegradable films, biodegradable resins, and biodegradable additives. It can also be widely applied to environmental fields, including supporting the development of environmentally friendly products, addressing microplastics, and establishing evaluation indicators in biodegradability certification systems.

Claims

1. A method for evaluating the biodegradability of organic matter, The proportion of organic carbon (C) that is oxidized as a result of catabolism by microorganisms in the aforementioned organic matter. ＢＯＤ ) and the proportion of organic carbon assimilated by microorganisms (C ＢＩＯ The sum of ) and is calculated as the degree of biodegradation. A method for evaluating biodegradability, comprising the following steps (a) to (d), wherein biodegradability is evaluated based on the degree of biodegradation. (a) A step of calculating the percentage of organic carbon oxidized by microbial catabolism (C BOD) at multiple time points, including the end of the biodegradation test. (b) A step of calculating the percentage of organic carbon assimilated by microorganisms (C BIO) at the end of the biodegradation test. (c) A step of calculating the estimated C BIO at each time point by dividing the proportion of organic carbon assimilated by the microorganism (C BIO) equally among multiple time points in which the proportion of organic carbon oxidized due to the catabolism of the microorganism (C BOD) is determined, and adding them cumulatively at each time point. (d) A step of calculating the degree of biodegradation by adding the estimated C BIO calculated in step (c) to the proportion of organic carbon oxidized due to microbial catabolism (C BOD) at each point in time in which the proportion of organic carbon oxidized due to microbial catabolism (C BOD).

2. Furthermore, in addition to the end of the biodegradation test, the percentage of assimilated organic carbon (C) at one or more points during the biodegradation test period. ＢＩＯ ) is calculated, and based on the calculated values ​​at these multiple measurement points, the proportion of assimilated organic carbon (C) at the unmeasured time point between the multiple measurement points is calculated. ＢＩＯ A method for evaluating biodegradability according to claim 1, wherein the biodegradability is estimated by interpolating the following.

3. A method for evaluating biodegradability, comprising creating a biodegradation curve based on the degree of biodegradation at each measurement point calculated by the biodegradability evaluation method described in either claim 1 or 2, and evaluating the biodegradability of an organic substance over time using the biodegradation curve.

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