Method of evaluating organic substance constituting aquatic environment preservation material together with iron-containing substance

Abstract

To provide a method of evaluating an organic substance constituting an aquatic environment preservation material together with an iron-containing substance.SOLUTION: A method of evaluating an organic substance constituting an aquatic environment preservation material together with an iron-containing substance is provided, the method involving evaluating capability of eluting iron from the iron-containing substance and stabilizing the eluted iron of the organic substance that constitutes the aquatic environment preservation material when mixed with the iron-containing substance, and comprises quantifying total amino acids contained in an eluate obtained by immersing the organic substance in water.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating the ability of an organic substance to stabilize iron eluted from the iron-containing substance by quantifying the amount of amino acids contained in the organic substance to be mixed with the iron-containing substance. [Background technology]

[0002] In recent years, biological production in waters has declined due to a lack of iron, which is necessary for the growth of living organisms. For example, in coastal waters, seaweed beds have disappeared and become covered with calcareous algae, a phenomenon known as rocky shore denudation. This has led to a decline in not only seaweed but also many of the marine resources that live there, such as sea urchins, abalone, and krill.

[0003] One of the causes of rocky shore bleaching is the decrease in iron in seawater. It is thought that iron in coastal areas was originally supplied to the sea through rivers, but it has been suggested that the felling and devastation of trees upstream of rivers flowing into the sea made it difficult for humic acid in the humus soil, which had previously been formed from the accumulation of fallen leaves, to combine with iron in the soil to form humic acid iron, reducing the supply of iron to the sea that is necessary for algae growth.

[0004] By the way, seawater is an environment in which it is difficult for ferrous iron to be eluted from iron-containing substances because its pH is maintained at 8.0 to 8.3. Even if ferrous iron is eluted from iron-containing substances, it is immediately converted into hydroxide ions (OH -) and precipitate as colloidal iron hydroxide. Thus, from a chemical perspective, simply supplying ferrous iron to seawater with the aim of supplying iron to the ocean is thought to be insufficient to provide algae with the iron they need for growth in seawater. However, in nature, ferrous iron is thought to exist in a state that is available to organisms by stabilizing it by forming a chelate with humic acid produced in forest humus soil. Focusing on this mechanism, a fertilization technique has been proposed in which a mixture of a weakly acidic humic acid-containing substance and an iron-containing substance is supplied to a water body, eluting ferrous iron from the iron-containing substance, and then supplying humic acid from the humic acid-containing substance as a ligand to form a complex, thereby supplying dissolved iron to algae.

[0005] For example, Patent Document 1 proposes an aquatic environment conservation material in which a bivalent iron-containing substance and a humus-containing substance are packed into a water-permeable bag material, and a method for conserving aquatic environments using the same.

[0006] Furthermore, Patent Document 2 proposes an iron ion supply material containing iron oxide and / or metallic iron-containing material (A) and one or more organic acids selected from gluconic acid and glutamic acid (B) in order to promote the elution of iron from an iron-containing material. Furthermore, Patent Document 3 proposes an iron supply material containing an iron-containing substance, a phenolic acid substance having two hydroxyl groups bonded adjacent to each other on a benzene ring, and an amino acid-containing substance.

[0007] As mentioned above, various organic substances are considered to have the function of dissolving and stabilizing iron in water, and it is considered preferable to select a more suitable substance. However, since there has been no conventional method for evaluating the function of promoting iron dissolution in water and stabilizing iron, the only way to select an organic substance with excellent functions has been through repeated experimentation and trial and error. Patent Documents 2 and 3 show that adding specific amino acids to iron-containing substances promotes the amount of iron dissolution, but no method for evaluating the function of promoting and stabilizing iron dissolution was known. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-212036 [Patent Document 2] Japanese Patent Application Laid-Open No. 2011-160764 [Patent Document 3] Japanese Patent Application Laid-Open No. 2016-104001 Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for evaluating organic matter which, together with iron-containing substances, constitutes aquatic environment conservation materials. [Means for solving the problem]

[0010] In order to solve the above problems, the present invention employs the following configuration. [1] A method for evaluating the ability of organic matter to dissolve iron from iron-containing substances and stabilize the iron after dissolution, when mixed with iron-containing substances to form aquatic environment conservation materials, comprising: The method for evaluating organic matter that constitutes aquatic environment conservation materials together with iron-containing substances comprises quantifying the total amino acids contained in an eluate obtained by immersing the organic matter in water or seawater. [2] A method for evaluating organic matter that constitutes aquatic environment conservation materials together with iron-containing substances described in [1], in which the quantification of the total amino acids is carried out by quantifying the total amount of free amino acids contained in the eluate and amino acids derived from proteins contained in the eluate. [3] The quantification of the total amino acids is an eluate preparation step of mixing the organic matter and pure water in a weight ratio of organic matter:pure water=1:200 to 1:20, and shaking the mixture for 48 to 36 hours under conditions of a dissolved oxygen content of 0.2 mg / L or less, to prepare the eluate; a first measurement step of quantifying free amino acids contained in a first sample by liquid chromatography using a part of the eluate obtained by the eluate adjustment step as a first sample; a second measurement step in which a part of the eluate obtained in the eluate adjustment step is concentrated to dryness and then subjected to acid decomposition to obtain a second sample, and another part of the eluate is concentrated to dryness and then subjected to alkali decomposition to obtain a third sample, and then amino acids contained in the second sample and the third sample are quantified by liquid chromatography; A method for evaluating organic matter that constitutes aquatic environment conservation materials together with the iron-containing substance described in [2], by: [4] The quantification of the total amino acids is an eluate preparation step of mixing the organic matter with seawater in a weight ratio of organic matter:seawater=1:200 to 1:20, and shaking the mixture for 48 to 36 hours under conditions of a dissolved oxygen content of 0.2 mg / L or less, to prepare the eluate; a first measurement step of quantifying free amino acids contained in a first sample by liquid chromatography using a part of the eluate obtained by the eluate adjustment step as a first sample; a second measurement step in which a part of the eluate obtained in the eluate adjustment step is concentrated to dryness and then subjected to acid decomposition to obtain a second sample, and another part of the eluate is concentrated to dryness and then subjected to alkali decomposition to obtain a third sample, and then amino acids contained in the second sample and the third sample are quantified by liquid chromatography; A method for evaluating organic matter that constitutes aquatic environment conservation materials together with the iron-containing substance described in [2], by: [5] A method for evaluating organic matter that constitutes aquatic environment conservation materials together with iron-containing substances according to [1], characterized in that the organic matter contains raw materials of animal origin. [Effects of the Invention]

[0011] According to the present invention, a method for evaluating organic matter that, together with iron-containing substances, constitutes aquatic environment conservation materials can be provided, which makes it possible to select the organic matter that meets the purpose or demand. [Brief explanation of the drawings]

[0012] [Figure 1] Correlation diagram between the amount of humic acid in plant-derived organic matter and the amount of iron leaching from a mixture with iron-containing substances. [Figure 2] Correlation diagram between the amount of humic acid in organic matter, including animal-derived matter, and the amount of iron leaching from a mixture with iron-containing substances. [Figure 3] This is a correlation diagram between the total amount of amino acids in organic matter obtained by using pure water as the eluent and the amount of iron eluted from a mixture with an iron-containing substance. [Figure 4] This is a correlation diagram between the total amount of amino acids in organic matter obtained by using natural seawater as the eluent and the amount of iron eluted from a mixture with iron-containing substances. DETAILED DESCRIPTION OF THE INVENTION

[0013] The inventors focused on the correlation between the amount of humic acid in organic matter containing humic acid-containing substances and the amount of iron eluted from the iron-containing substances in aquatic environment conservation materials composed of a mixture of iron-containing substances and humic acid-containing substances. Based on this correlation, they attempted to evaluate the ability of organic matter containing humic acid-containing substances to promote iron elution and stabilize the eluted iron. However, while organic matter derived from plants had a relatively high linear correlation between the amount of humic acid and the amount of iron elution, the correlation significantly decreased when the organic matter contained organic matter derived from animal sources (see Figures 1 and 2).

[0014] Therefore, the inventors investigated plant- and animal-derived organic matter as materials that have the function of promoting and stabilizing the supply of iron from iron-containing substances, and after extensive research into methods for evaluating the iron elution-promoting and stabilizing functions of organic matter, they discovered that it is effective to measure the amount of amino acids that migrate from organic matter into water or seawater when the organic matter is immersed in water or seawater.

[0015] The amino acids in the present invention are classified into two types: free amino acids and protein-derived amino acids. Free amino acids are amino acids that are eluted into an eluate obtained by immersing organic matter in water or seawater and that are not bound to other amino acids by peptide bonds. Protein-derived amino acids are amino acids that are obtained by treating proteins that have been migrated into an eluate by immersing organic matter in water or seawater with an acid or alkali. In the present invention, the term "total amino acids" refers to a mixture of the above-mentioned free amino acids and amino acids derived from proteins.

[0016] The investigations conducted by the present inventors will be described below.

[0017] After the divalent iron in iron-containing materials is dissolved into the aquatic environment, especially in marine areas with a pH of 8.0 to 8.3, the divalent iron becomes trivalent iron, which then combines with OH groups to form colloidal iron and precipitate (Fe 3+ +3OH - →Fe(OH)3). Therefore, for iron to exist in a dissolved state in the aquatic environment, divalent iron must bind to a ligand to form a complex.

[0018] The inventors investigated the correlation between the amount of humic acid in organic matter composed of various raw materials and the amount of dissolved iron eluted when the organic matter was mixed with an iron-containing substance. As a result, as shown in Figure 1, a linear correlation was confirmed between the amount of humic acid and the amount of dissolved iron in organic matter derived from plants. On the other hand, when organic matter derived from animals was included, the correlation between the amount of humic acid and the amount of dissolved iron was not clear. This indicates that a new indicator is needed to evaluate diverse organic matter.

[0019] Next, based on the knowledge that specific amino acids derived from the reagent promote iron elution, we attempted to quantify the free amino acids eluted from the organic matter. As a result, as shown in Table 1, we found that free amino acids eluted from the organic matter when it contained organic matter of animal origin, but almost no elution occurred from organic matter of plant origin.

[0020] Therefore, the inventors focused on proteins eluted from organic matter. Free amino acids are components of proteins in living organisms, and are presumed to be produced by the decomposition of proteins as the decay of organic matter progresses. As the decomposition of organic matter progresses, the amount of eluted free amino acids increases, which is thought to contribute to the promotion and stabilization of iron elution. Furthermore, proteins eluted from organic matter are thought to be rapidly decomposed into amino acids. In fact, as shown in Table 1, proteins were also detected in the eluates of organic matter examined in this embodiment.

[0021] The amount of free amino acids in Table 1 is a value measured by the method described in the Examples.

[0022] [Table 1]

[0023] Based on the above results, the inventors considered that proteins could also potentially be evaluated as having the ability to stabilize eluted iron, and came up with the idea of ​​quantifying the total amino acids as the sum of the amino acids produced by decomposition of proteins and free amino acids by immersing the organic matter to be mixed with the iron-containing substance in water or seawater and subjecting the eluate of the organic matter to acid or alkali hydrolysis treatment.

[0024] The present invention aims to evaluate organic substances that improve the amount of iron eluted into water bodies, and therefore evaluates the eluate obtained by immersing organic substances in water or seawater. Therefore, the total amino acids effective for iron stabilization are those that elute into the eluate.

[0025] Various organic materials were soaked in water, and the resulting eluates were subjected to acid or alkali hydrolysis. The quantified amino acid content was used as the total amino acid content. Furthermore, the amount of iron eluted from aquatic environment conservation materials obtained by mixing each organic material with an iron-containing substance was measured, and the correlations between these values ​​are shown in Figures 2 and 3. As shown in Figure 2, no clear correlation was found between the amount of humic acid in the organic material and the amount of iron eluted from mixtures of organic material and iron-containing substances, regardless of whether the organic material was plant- or animal-derived. On the other hand, as shown in Figure 3, a linear correlation was found between the total amount of amino acids eluted from organic material and the amount of iron eluted from mixtures with iron-containing substances, regardless of whether the organic material was plant- or animal-derived.

[0026] Furthermore, this correlation was found to be higher than that of humic acid contained in the same organic matter as above, and to be superior in evaluating the ability to stabilize iron.

[0027] We also found that it is preferable to use water or seawater as the liquid in which the organic matter is soaked to obtain the eluate. In particular, seawater has pH buffering properties, which reduces pH fluctuations during soaking of the organic matter, thereby stabilizing the amount of eluted amino acids. As a result, we found that the total amount of amino acids obtained using seawater has a relatively high correlation with the amount of eluted iron compared to the total amount of amino acids obtained using pure water.

[0028] Hereinafter, a method for evaluating the iron elution-promoting and stabilizing functions of organic substances according to an embodiment of the present invention will be described.

[0029] The organic matter to be evaluated in this embodiment includes raw materials of plant origin or animal origin. In particular, it is preferable that the organic matter includes raw materials of animal origin. Furthermore, the organic matter may be a mixture of raw materials of plant origin and raw materials of animal origin.

[0030] Plant-derived organic matter is organic matter that uses plants as raw materials and has been processed or decomposed or altered by organisms such as microorganisms. Examples include rice bran, oil cake, wood ash, thinnings, fallen trees, tree bark, fallen leaves, rice and wheat, lignite, brown coal, and sewage sludge.

[0031] Organic matter of animal origin includes the waste that animals excrete during their lives, animal body parts, processed products of these, and organic matter that has undergone decomposition and transformation by organisms such as microorganisms. Specific examples include animal excreta such as cow manure, pig manure, and chicken manure, as well as the flesh, bones, shells, and excrement of fish and shellfish, including their internal organs.

[0032] Examples of iron-containing materials to be mixed with the organic matter include steel slag, dust, scale, iron powder, iron oxide powder, iron sand, iron ore, and coal fusion ash.

[0033] Steel slag includes steel slag produced by steelworks and electric furnace slag (electric furnace steelmaking slag) produced by electric furnace manufacturers. Steel slag produced by steelworks is a by-product of the steel manufacturing process.

[0034] Iron and steel slag can be broadly divided into blast furnace slag and steelmaking slag. Of these, steelmaking slag is the slag that is preferably used in this embodiment. Since the iron content of steelmaking slag (approximately 20% by mass) is higher than the iron content of blast furnace slag (approximately 0.4% by mass), it is preferable to use steelmaking slag when efficiently supplying iron. However, the iron-containing material is not limited to steelmaking slag.

[0035] Carbonated steelmaking slag is particularly desirable as the steelmaking slag. Steelmaking slag contains approximately 1 to 2 mass% of f-CaO (free lime), which has the property of easily temporarily raising the pH in water. For this reason, it is more desirable to use carbonated steelmaking slag that has been carbonated to convert the f-CaO into CaCO3. By using carbonated steelmaking slag, the degree of increase in pH in the leaching water can be suppressed, which in turn can further promote the leaching of iron from the steelmaking slag.

[0036] Carbonation of steelmaking slag can be carried out by contacting the steelmaking slag with carbon dioxide or carbonate-containing water. This process converts CaO into CaCO3, which is formed on the surface of the steelmaking slag. 2+ By subjecting steelmaking slag to such carbonation treatment, it is possible to prevent a temporary increase in pH in the water body, which is believed to enable the evaluation method of the present invention to be used with high accuracy.

[0037] A typical example of scale is scale generated in the steel manufacturing process (for example, mill scale).

[0038] Coal fusion ash includes pulverized boiler ash (fly ash) and molten ash (slag) from entrained-flow coal gasifiers. Fly ash contains approximately 0.6 to 23 mass% of iron as iron oxide (Fe2O3), but in molten ash generated in a reducing atmosphere in integrated coal gasification combined cycle (IGCC), approximately 80 mass% of the iron oxide exists as ferrous iron.

[0039] In the evaluation method of this embodiment, an organic substance is immersed in water or seawater to obtain an eluate. Then, the total amount of amino acids contained in the eluate is quantified. The total amount of amino acids is quantified by quantifying the total amount of free amino acids contained in the eluate and amino acids obtained by acid or alkali hydrolysis of proteins contained in the eluate (amino acids derived from proteins). Specifically, the total amino acids are quantified through an eluate preparation step, a first measurement step, and a second measurement step. Each step will be explained below.

[0040] In the eluate preparation stage, the organic matter to be evaluated and pure water are mixed in a weight ratio of organic matter:pure water = 1:200 to 1:20, and the eluate is prepared by shaking the mixture for 48 to 36 hours under conditions where the dissolved oxygen level is 0.2 mg / L or less.

[0041] By setting the blending ratio of organic matter to pure water within the above range, suppressing the amount of dissolved oxygen, and shaking for a predetermined period of time, it is possible to elute free amino acids and proteins from the organic matter being evaluated, thereby enabling appropriate evaluation of the ability to elute iron from iron-containing substances and the ability to stabilize the iron after elution.

[0042] To reduce the amount of dissolved oxygen to 0.2 mg / L or less, for example, a non-oxidizing gas such as nitrogen or argon may be blown into a mixture of organic matter and pure water.

[0043] The shaking conditions are, for example, a shaking speed of 100 rpm (rotations per minute) and an amplitude of 45 mm.

[0044] Furthermore, in the eluate preparation step, seawater may be used instead of pure water. Specifically, the eluate may be prepared by mixing the organic matter to be evaluated with seawater in a weight ratio of organic matter:seawater = 1:200 to 1:20, and shaking the mixture for 48 to 36 hours under conditions of a dissolved oxygen content of 0.2 mg / L or less. Seawater has a pH buffering effect compared to pure water, and can reduce pH changes during the preparation of the eluate. This allows for more stable elution of free amino acids and proteins from the organic matter to be evaluated. It also reduces variations in the amount of elution due to pH fluctuations.

[0045] The seawater may be either natural seawater or artificial seawater.

[0046] As a pretreatment, natural seawater is preferably filtered through a 0.22 μm filter after collection from the ocean. The filtered natural seawater is preferably used in the eluate preparation stage. Filtration can remove organisms such as microalgae and suspended matter such as colloids contained in the natural seawater.

[0047] In the case of artificial seawater, filtration through a 0.22 μm filter may be performed as a pretreatment, or filtration may not be performed.

[0048] (First measurement stage) In the first measurement step, a part of the eluate obtained in the eluate adjustment step is used as a first sample, and the amount of free amino acids contained in the first sample is quantified by liquid chromatography.

[0049] When taking a first sample from the eluate, the eluate after shaking is left to stand for, for example, about 30 minutes to allow suspended matter in the liquid to settle, and then the supernatant is sampled. The supernatant is then further filtered through a filter with a pore size of, for example, 0.45 μm to obtain the first sample.

[0050] The free amino acids contained in the first sample are then quantified by liquid chromatography. The measurement by liquid chromatography is as follows. The free amino acids to be measured include aspartic acid, serine, glutamic acid, glycine, histidine, arginine, threonine, alanine, proline, cystine, tyrosine, valine, methionine, lysine, isoleucine, leucine, phenylalanine, and tryptophan.

[0051] (Second measurement stage) In the second measurement step, a portion of the eluate obtained in the eluate adjustment step is concentrated to dryness and then subjected to acid hydrolysis to obtain a second sample, and another portion of the eluate is concentrated to dryness and then subjected to alkaline hydrolysis to obtain a third sample, and then the total amino acids, including amino acids obtained by hydrolyzing proteins contained in the second sample and the third sample, are quantified by liquid chromatography.

[0052] The procedure of concentrating the eluate to dryness and then subjecting it to acid hydrolysis to obtain a second sample, and the procedure of concentrating the eluate to dryness and then subjecting it to alkaline hydrolysis to obtain a third sample, can be carried out in accordance with the general acid or alkaline treatment methods used when analyzing all amino acids, including those constituting proteins. For example, either the liquid-phase method, in which the eluate is concentrated to dryness and then mixed with hydrochloric acid and then sealed under reduced pressure, or the gas-phase method, in which hydrochloric acid is distilled from a sealed vessel under reduced pressure, can be used. Cystine and methionine are easily decomposed by hydrochloric acid hydrolysis, so it is recommended to oxidize them with performic acid before hydrolysis. Tryptophan is easily decomposed by oxidative degradation in hydrochloric acid hydrolysis, so it is recommended to decompose them by alkaline hydrolysis or acid hydrolysis in the presence of a reducing agent.

[0053] Then, the total amino acids contained in the second and third samples are quantified using liquid chromatography. The measurement method using liquid chromatography is as follows. The protein-constituting amino acids measured include aspartic acid, serine, glutamic acid, glycine, histidine, arginine, threonine, alanine, proline, cystine, tyrosine, valine, methionine, lysine, isoleucine, leucine, phenylalanine, and tryptophan.

[0054] When measuring amino acids by liquid chromatography, the amino acids may be derivatized as necessary.

[0055] In addition, amino acids can be quantified using a post-column labeling method, in which amino acids are separated by ion exchange chromatography and then derivatized and detected, or a pre-column labeling method, in which amino acids are derivatized in advance and then separated by reverse phase chromatography.

[0056] The organic substance with a larger amount of total amino acids can be evaluated as having a superior function of eluting iron from iron-containing substances and stabilizing the eluted iron.

[0057] Next, we will describe the analytical method for ferrous iron eluted from aquatic environment conservation materials consisting of iron-containing substances and organic matter.

[0058] First, an eluate of a mixture of the organic matter to be evaluated and the iron-containing substance is prepared. The organic matter and iron-containing material are mixed in a 1:1 weight ratio, immersed in seawater at a solid-liquid ratio of 1:10 (weight ratio), and shaken at 100 rpm with an amplitude of 45 mm for 48 to 36 hours under conditions of dissolved oxygen of 0.2 mg / L or less. After leaving the mixture to stand for approximately 30 minutes to allow the suspended matter to settle, the supernatant is removed, new seawater is added, and the mixture is shaken again under the same conditions for 48 to 36 hours. The above procedure is repeated 10 times. The removed supernatant is used to evaluate the amount of iron elution.

[0059] The supernatant of the eluate obtained is filtered through a filter with a pore size of 0.45 μm, and the iron concentration of the filtered supernatant is measured using ICP atomic emission spectrometry.

[0060] When evaluating organic matter that forms aquatic environment conservation materials by being mixed with iron-containing substances, the amount of iron eluted can be predicted from the correlation shown in Figure 3 by quantifying the amino acids eluted from the organic matter, and the organic matter can be evaluated. Compared to the conventional method of evaluating the amount of humic acid, this method can more appropriately evaluate the amount of iron eluted when the organic matter is mixed with an iron-containing substance. In particular, the difference with conventional evaluation methods becomes more pronounced when the organic matter to be mixed with the iron-containing substance contains organic matter of animal origin. Using this evaluation method makes it possible to select the organic matter that matches the purpose or demand. [Example]

[0061] Converter steelmaking slag (particle size 25 mm or less) was used as the iron-containing material, and seven organic substances of different origin were used as the organic matter to be mixed with this iron-containing material. Leaching tests were conducted in seawater or pure water. The amounts of leached iron, free amino acids, and total amino acids were then quantified using the methods described below. Humic acid in the organic matter was also quantified. Details are described below.

[0062] (1) Amount of eluted iron Converter steelmaking slag (particle size 25 mm or less) was prepared as the iron-containing material. The iron-containing material and the organic matter listed in Table 1 were mixed at a weight ratio of 1:1 to prepare a mixed raw material. 100 g of the prepared mixed raw material was placed in a 2 L polypropylene container. Natural seawater collected from Tokyo Bay was filtered through a glass fiber filter to prepare filtered seawater (initial seawater). 1 L of the filtered seawater was placed in the polypropylene container containing 100 g of the mixed raw material. The dissolved oxygen content was adjusted to 0.2 mg / L or less by nitrogen aeration, and the container was shaken at 100 rpm with an amplitude of 45 mm for 48 to 36 hours. After allowing the suspended matter to settle for approximately 30 minutes, the supernatant was removed, and new initial seawater was added. The container was shaken under the same conditions for 48 to 36 hours. The above procedure was repeated 10 times. The initial seawater and the removed supernatant were each filtered through a 0.45 μm filter, and the iron concentration (mg / L) of the filtered seawater was measured. The iron concentration in the seawater was analyzed using ICP atomic emission spectrometry (Shimadzu Corporation, ICPE-9000). The final amount of iron elution was calculated by subtracting the iron concentration of the initial seawater from the iron concentration of each supernatant, and integrating this over 10 runs.

[0063] (2) Determination of humic acid The organic matter to be evaluated was shredded using a blender to prepare the analytical sample, and humic acid content was quantified according to the Fertilizer and Other Materials Testing Methods (2019). Specifically, 1 g of analytical sample (A1) was weighed to the nearest 1 mg and placed in a 300 mL baffled Erlenmeyer flask. 50 mL of hydrochloric acid (1+9) was added and shaken at 120 rpm for 1 hour. This suspension was transferred to a 50 mL polypropylene centrifuge tube and centrifuged at 1700 G for 5 minutes, after which the supernatant was removed. For washing, water was added again, the tube was shaken, and the tube was centrifuged at 1700 G for 5 minutes. The supernatant was removed and the insoluble matter was washed. This washing process was repeated three times. After washing, the insoluble matter was transferred to a crucible-type glass filter with water, filtered under reduced pressure, and dried at 105 °C for 3 hours. After cooling, the mass was measured to the nearest 1 g, and the weight of the insoluble matter (A2) was calculated.

[0064] The organic matter to be evaluated was then shredded in a blender to prepare the analytical sample. One gram of this analytical sample (B1) was weighed to the nearest 1 mg and placed in a 300 mL baffled Erlenmeyer flask. 50 mL of hydrochloric acid (1+9) was added and the mixture was shaken at 120 rpm for 1 hour. The resulting suspension was transferred to a 50 mL polypropylene centrifuge tube and centrifuged at 1700 G for 5 minutes. The supernatant was then removed. For the wash step, water was added, the mixture was shaken, and the mixture was centrifuged at 1700 G for 5 minutes. The supernatant was then removed to wash the insoluble material. This wash step was repeated three times. The insoluble material was then transferred to a 300 mL baffled Erlenmeyer flask, 50 mL of sodium hydroxide solution (10 g / L) was added, and the mixture was shaken at 120 rpm for 1 hour. The resulting suspension was then transferred to a 50 mL polypropylene centrifuge tube and centrifuged at 1700 G for 5 minutes. The supernatant was then removed. For the washing process, water was added, the mixture was shaken, and the mixture was centrifuged at a centrifugal force of 1700 G for 5 minutes, after which the supernatant was removed and the mixture was washed. The washing process was repeated three times. The insoluble matter was transferred to an aluminum dish and dried overnight at 105°C. After cooling, the mass was measured to the nearest 1 g, and the weight of the insoluble matter (B2) was calculated. Humic acid was calculated using the following formula:

[0065] Humic acid (mass%)=(A2) / (A1)×100-(B2) / (B1)×100

[0066] (3) Quantitative determination of free amino acids (using purified water as the eluent solvent) 50 g of the target organic matter was placed in a 2 L polypropylene container, 1 L of purified water was added, and the dissolved oxygen content was adjusted to 0.2 mg / L or less by nitrogen aeration. The container was then shaken at 45 mm amplitude and 100 rpm for 3 days. The supernatant was allowed to stand for 30 minutes to allow suspended matter to settle, and then sampled with a syringe. The supernatant was filtered through a 0.45 μm filter, and the amino acids in the solution were extracted using reflux extraction with 75% ethanol. The resulting sample was used for analysis. The dissolved amino acid content (mg / L) in the solution was measured using a Waters AcquityArc system.

[0067] (4) Quantitation of total amino acids 50 g of organic matter to be mixed with the iron-containing material was placed in a 2 L polypropylene container, 1 L of pure water was added, and the dissolved oxygen content was adjusted to 0.2 mg / L or less by nitrogen aeration. The container was then shaken at 45 mm amplitude and 100 rpm for 3 days. The supernatant was allowed to stand for 30 minutes to allow suspended matter to settle, and then sampled using a syringe. This supernatant was filtered through a 0.45 μm filter, and the total amino acids in the solution were measured. Specifically, the supernatant was concentrated to dryness, and all amino acids except tryptophan were hydrolyzed with acid, while tryptophan was hydrolyzed with alkali and then fluorescently derivatized. The analytical sample was then used. The dissolved amino acid content was measured using a Waters AcquityArc system.

[0068] [Table 2]

[0069] The results are shown in Table 2, Figures 2 and 3. Figure 2 shows the relationship between the amount of humic acid in plant-derived and animal-derived organic matter and the amount of iron eluted from a mixture of organic matter and an iron-containing substance. Figure 3 shows the relationship between the amount of total amino acids eluted from plant-derived and animal-derived organic matter using pure water and the amount of iron eluted from a mixture of organic matter and an iron-containing substance.

[0070] As shown in Figure 2, no clear correlation was found between the amount of humic acid in organic matter and the amount of iron eluted from mixtures of organic matter and iron-containing substances, for both plant- and animal-derived organic matter. On the other hand, as shown in Figure 3, a linear correlation was found between the amount of total amino acids eluted from organic matter and the amount of iron eluted from mixtures of organic matter and iron-containing substances, regardless of whether they were plant- or animal-derived. The R 2 The value was also high at 0.9746.

[0071] Based on this, we determined that the total amount of amino acids eluted from organic matter is a suitable indicator highly correlated with the amount of iron elution. In other words, it was predicted that the higher the total amount of amino acids eluted from organic matter, the higher the amount of iron eluted. Therefore, it was considered preferable to measure the total amount of amino acids eluted from organic matter as a method for evaluating organic matter that, together with iron-containing substances, constitutes aquatic environment conservation materials.

[0072] (5) Quantitative determination of total amino acids (using natural seawater as the eluent solvent) Natural seawater was collected and filtered through a 0.22 μm filter to remove organisms such as microalgae and suspended matter such as colloids. Next, 50 g of the organic matter to be mixed with the iron-containing material was placed in a 2 L polypropylene container, 1 L of filtered natural seawater was added, and the dissolved oxygen content was adjusted to 0.2 mg / L or less by nitrogen aeration. The container was then shaken at 45 mm amplitude and 100 rpm for 3 days. The supernatant was allowed to stand for 30 minutes to allow suspended matter to settle, and then sampled using a syringe. This supernatant was filtered through a 0.45 μm filter, and the total amino acids in the solution were measured. Specifically, the supernatant was concentrated to dryness, and all amino acids except tryptophan were hydrolyzed with acid, and tryptophan was hydrolyzed with alkali, followed by fluorescent derivatization, and the resulting sample was used for analysis. The dissolved amino acid content was measured using a Waters AcquityArc system.

[0073] In addition, the amount of corrosive acid in the organic matter to be evaluated was measured in the same manner as above.

[0074] [Table 3]

[0075] The results are shown in Table 3 and Figure 4. Figure 4 shows the relationship between the total amount of amino acids eluted from plant-derived and animal-derived organic matter using natural seawater and the amount of iron eluted from a mixture of organic matter and an iron-containing substance.

[0076] Of the fertilizer classifications shown in Table 3 and Figure 4, measurements were taken on products from a different production lot for humic acid magnesium fertilizer and compost 5 than those for humic acid magnesium fertilizer and compost 5 shown in Table 2 and Figure 3. Therefore, the amount of eluted Fe and the amount of humic acid differ from those in Table 2. For all fertilizers other than humic acid magnesium fertilizer and compost 5, measurements were taken on products from the same production lot. Therefore, the values ​​for the amount of eluted Fe and the amount of humic acid were taken from Table 2.

[0077] As shown in Figure 4, a linear correlation was found between the total amount of amino acids eluted from organic matter, regardless of whether it was plant- or animal-derived, and the amount of iron eluted from mixtures with iron-containing substances.

[0078] Furthermore, as shown in Figure 4, when amino acids were eluted using natural seawater, a high correlation was observed between the total amount of amino acids and the amount of eluted iron, compared to Figure 3, where amino acids were eluted using pure water. 2 The value was 0.9938, which was higher than that in Figure 3. Since natural seawater is less susceptible to pH changes than pure water, the elution of all amino acids was stable, resulting in a R 2 The value is likely to have increased.

[0079] As described above, in the eluate preparation step of the present invention, measurements were possible using either pure water or seawater as the solvent. Furthermore, it was found that for the quantification of total amino acid content, seawater is preferable as the solvent when accuracy of the evaluation is required, while pure water is preferable when simplification of the procedure is required.

Claims

1. A method for evaluating the ability of organic matter to elute iron from iron-containing substances and to stabilize the iron after elution, for organic matter that constitutes an aquatic environment conservation material by being mixed with iron-containing substances, comprising: The method for evaluating organic matter that constitutes aquatic environment conservation materials together with iron-containing substances comprises quantifying the total amino acids contained in an eluate obtained by immersing the organic matter in water or seawater.

2. The method for evaluating organic matter that constitutes aquatic environment conservation materials together with iron-containing substances as described in claim 1, wherein the quantification of the total amino acids is carried out by quantifying the total amount of free amino acids contained in the eluate and amino acids derived from proteins contained in the eluate.

3. The quantification of all amino acids is an eluate preparation step of mixing the organic matter and pure water in a weight ratio of organic matter:pure water=1:200 to 1:20, and shaking the mixture for 48 to 36 hours under the condition that the dissolved oxygen content is 0.2 mg / L or less; a first measurement step of quantifying free amino acids contained in a first sample by liquid chromatography using a part of the eluate obtained by the eluate adjustment step as a first sample; a second measurement step in which a part of the eluate obtained in the eluate adjustment step is concentrated to dryness and then subjected to acid decomposition to obtain a second sample, and another part of the eluate is concentrated to dryness and then subjected to alkali decomposition to obtain a third sample, and then amino acids contained in the second sample and the third sample are quantified by liquid chromatography; 3. A method for evaluating organic matter constituting an aquatic environment conservation material together with the iron-containing substance according to claim 2, wherein the method comprises:

4. The quantification of all amino acids is an eluate preparation step of mixing the organic matter and seawater in a weight ratio of organic matter:seawater=1:200 to 1:20, and shaking the mixture for 48 to 36 hours under conditions of a dissolved oxygen content of 0.2 mg / L or less, to prepare the eluate; a first measurement step of quantifying free amino acids contained in a first sample by liquid chromatography using a part of the eluate obtained by the eluate adjustment step as a first sample; a second measurement step in which a part of the eluate obtained in the eluate adjustment step is concentrated to dryness and then subjected to acid decomposition to obtain a second sample, and another part of the eluate is concentrated to dryness and then subjected to alkali decomposition to obtain a third sample, and then amino acids contained in the second sample and the third sample are quantified by liquid chromatography; 3. A method for evaluating organic matter constituting an aquatic environment conservation material together with the iron-containing substance according to claim 2, wherein the method comprises:

5. 2. The method for evaluating organic matter constituting aquatic environment conservation materials together with iron-containing substances according to claim 1, wherein the organic matter includes raw materials of animal origin.

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