A method for detecting plant growth stimulant-like substances in sludge stabilization products
By combining plant roots with CaCl2-treated sludge extract and using bromocresol purple indicator, the complexity of detecting plant growth stimulants in sludge stabilization products was solved, enabling rapid and low-cost qualitative detection that reflects the expression activity of growth stimulants.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHINA UNIV OF GEOSCIENCES (WUHAN)
- Filing Date
- 2023-09-15
- Publication Date
- 2026-05-29
Smart Images
Figure CN117214161B_ABST
Abstract
Description
Technical Field
[0001] This application belongs to the field of rapid organic matter detection technology, and more specifically, relates to a method for detecting plant growth stimulants in sludge stabilization products. Background Technology
[0002] With the rapid pace of urbanization, wastewater treatment loads have increased dramatically, generating large amounts of excess activated sludge. This sludge is rich in natural organic matter, including amino acids, polypeptides, sugars, fulvic acid, and humic acid—all active organic matter that promotes plant growth and enhances plant resistance. Sludge stabilization treatment brings the excess sludge to the sanitary standards for land use, while simultaneously stabilizing and transforming the organic matter in the sludge, generating susceptible substances (such as amino acids and aromatic carboxylic acids), fulvic acid, and humic acid—plant growth stimulants.
[0003] Currently, the detection of (-)plant growth stimulants mainly focuses on liquid chromatography-mass spectrometry (LC-MS), which involves steps such as sludge sample drying, target substance extraction, solid-phase extraction purification, redissolution, rotary evaporation, purification, nitrogen blowing reconstitution, and LC-MS. For example, patent CN113125591B discloses a method for quantitative detection of bioactive plant growth hormones in sludge using LC-MS. This method is cumbersome and requires sophisticated equipment and operation. The extraction and solid-phase extraction methods need to be changed for different types of plant growth stimulants. Furthermore, while this method can quantitatively detect plant growth stimulants, it cannot predict their expression activity. Currently, there is no simple and rapid method for detecting plant growth stimulants in sludge stabilization products. Summary of the Invention
[0004] The purpose of this application is to provide a method for detecting plant growth stimulants in sludge stabilization products, so as to solve the technical problem of complex detection methods for plant growth stimulants in the prior art.
[0005] To achieve the above objectives, this application provides a method for detecting plant growth stimulants in sludge stabilization products, comprising the following steps:
[0006] The plant roots were brought into contact with a sludge extract containing CaCl2 to activate the messenger.
[0007] The plants treated with the activated messenger were transferred to an agar gel plate containing bromocresol purple indicator. During cultivation, the color development of the agar gel plate was observed, and the amount of plant growth stimulants in the sludge extract was determined based on the color development.
[0008] Furthermore, the agar gel plate is prepared according to the following method:
[0009] The blank agar gel layer was adjusted to pH 6.8 using tris(hydroxymethyl)aminomethane buffer, and the bromocresol purple indicator was added.
[0010] Furthermore, the amount of bromocresol purple indicator added is 0.04 g / L.
[0011] Furthermore, the concentration of CaCl2 in the sludge extract containing CaCl2 is 20~100 mmol / L.
[0012] Furthermore, the concentration of CaCl2 in the sludge extract containing CaCl2 is 50 mmol / L.
[0013] Furthermore, the processing time for activating the messenger is 10-30 minutes.
[0014] Furthermore, the sludge extract is prepared according to the following method:
[0015] Add the sludge sample to an aqueous solution, stir, centrifuge, and take the supernatant as the sludge extract.
[0016] Furthermore, the plant is bok choy, and the plant is cultivated according to the following method:
[0017] The seeds of Chinese cabbage were sterilized with sodium hypochlorite solution and then washed with distilled water.
[0018] Place the bok choy seeds on a plate lined with autoclaved filter paper and cultivate them in distilled water until they germinate, with a root length of 3-4 cm.
[0019] Furthermore, the mass concentration of the sodium hypochlorite solution is 1%.
[0020] Furthermore, the sterilization process takes 10 to 30 minutes.
[0021] Compared with the prior art, this application has the following technical effects:
[0022] This application proposes a method for detecting plant growth stimulants in sludge stabilization products based on the acidification degree of plant roots and using bromocresol purple as an indicator. It proposes a method that combines colorimetric analysis with hydroponic plant experiments to achieve simple, rapid, and low-cost qualitative detection of plant growth stimulants in sludge stabilization products.
[0023] Furthermore, conventional chromatography-mass spectrometry (GC-MS) methods often detect only the absolute content of a particular class of plant growth stimulants, without considering the proportion of expression activity of these stimulants. In contrast, the detection method in this application uses hydroponic plant experiments to detect plant growth stimulants, yielding results that identify the plant growth stimulants with expression activity. Moreover, compared to conventional GC-MS methods, this method does not require expensive instruments or cumbersome pretreatment procedures, significantly reducing the difficulty of detecting plant growth stimulants in sludge stabilization products. Attached Figure Description
[0024] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0025] Figure 1 A flowchart illustrating a method for detecting plant growth stimulants in sludge stabilization products, provided in this application embodiment;
[0026] Figure 2 The color development results are shown in Examples 1-3 of this application;
[0027] Figure 3 The color development results are shown in Examples 4-6 of this application and the positive control group.
[0028] Figure 4 The color development is shown in Examples 7-11 of this application. Detailed Implementation
[0029] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.
[0030] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.
[0031] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.
[0032] Example 1
[0033] Embodiment 1 of this application provides a method for detecting plant growth stimulants in sludge stabilization products, the flowchart of which is shown below. Figure 1 As shown, it includes the following steps:
[0034] (1) Extraction of organic matter from sludge stabilization products: Weigh 1 g of sludge sample, stir continuously with 25 mL of aqueous solution for 6 h, and centrifuge to obtain supernatant.
[0035] (2) Selection and cleaning of Chinese cabbage seeds: Select plump Chinese cabbage seeds by visual inspection, sterilize the Chinese cabbage seeds with 1% sodium hypochlorite solution for 20 min, and wash them with distilled water 6 times for 2 min each time after sterilization.
[0036] (3) Seed germination experiment: Place the seeds on a plate covered with high-pressure sterilized filter paper and culture them in distilled water for 2 days (the roots are about 3-4 cm long) to germinate.
[0037] (4) Activation of related messengers: Seedlings were placed on autoclaved filter paper and four experimental groups were set up. Each group was vertically placed into a 2 mL sterilized tube containing 50 mmol / L CaCl2 aqueous solution to activate the second messenger and accelerate the subsequent proton pump response and expression. In the four experimental groups, 1.5 mL of distilled water, 1.5 mL of 200 μmol / L sodium vanadate (Na3VO4) solution, 1.5 mL of 1 μmol / L indoleacetic acid (IAA) and 1.5 mL of sludge extract from step (1) were added for posttreatment for 30 min. Distilled water was used as the normal control group; sodium vanadate (Na3VO4) solution was (PM) H + -A specific inhibitor of ATPase was used as a negative control. IAA, the most common plant growth stimulant, was used as a positive control.
[0038] (5) pH adjustment and indicator addition: Prepare a blank agar (1%) gel layer, adjust the pH of the blank agar gel layer to 6.8 using 100 mM Tris buffer, and add bromocresol purple indicator (0.04 g / L).
[0039] (6) Color development experiment: The seedlings treated in step (4) were transferred to a 5 mm agar (1%) gel plate. The seedlings were cultured on each plate for 24 h and the color development was observed. The expression activity of plant-like active substances (plant growth stimulants) in the sludge extract sample was judged based on the color development.
[0040] Example 2
[0041] The difference between this and Example 1 is that the 2 mL sterilization tube in step (4) contains a 20 mmol / L CaCl2 aqueous solution, while the rest is the same as in Example 1.
[0042] Example 3
[0043] The difference between this and Example 1 is that the 2 mL sterilization tube in step (4) contains 100 mmol / L CaCl2 aqueous solution, while the rest is the same as in Example 1.
[0044] Examples 4-6
[0045] The difference between this example and Example 1 is that the sludge samples in step (1) are traditional compost sludge, original sludge, and earthworm compost sludge, respectively, while the rest is the same as in Example 1.
[0046] Examples 7-11
[0047] The difference between this and Example 1 is that: in step (1), the sludge samples are aerobic composted sludge samples from different stages, namely, sludge samples from day 0 (i.e., the initial sludge sample before composting), day 5, day 9, day 21, and day 35 are tested, and the rest is the same as in Example 1.
[0048] The color development of Examples 1-3 of this application is shown in the attached figures. Figure 2 As shown, Figure 2 This indicates that adding an appropriate amount of CaCl2 can activate the second messenger in plant roots and promote the colorimetric response of plant growth stimulants to plant roots. Using IAA as a model plant growth hormone, the addition of different doses of CaCl2 revealed that both high and low concentrations of CaCl2 were detrimental to the occurrence of the colorimetric response, with the optimal colorimetric effect observed at a CaCl2 solution concentration of 50 mmol / L.
[0049] Proton pumps, present in the vacuolar membrane and plasma membrane of plants, are key factors in regulating electrochemical gradients, providing energy for the plant cell's nutrient absorption system and growth. During the germination stage, plant cells absorb exogenous plant growth stimulants to promote root development, leading to (PM)H... +-ATPase response is intense, promoting root acidification in plants. An embodiment of this application proposes a method for detecting plant growth stimulants in sludge stabilization products based on the degree of root acidification. Using bromocresol purple as an indicator, it proposes a method combining colorimetric analysis with a hydroponic experiment on Chinese cabbage to achieve simple, rapid, and low-cost qualitative detection of plant growth stimulants in sludge stabilization products.
[0050] Furthermore, conventional chromatography-mass spectrometry (GC-MS) methods often detect only the absolute content of a certain class of plant growth stimulants, without considering the expression activity ratio of these stimulants. In contrast, the detection method in this application uses hydroponic plant experiments to detect plant growth stimulants, yielding results that identify the plant growth stimulants with expression activity. Moreover, compared to conventional GC-MS methods, the detection method in this application does not require expensive instruments or cumbersome pretreatment procedures, significantly reducing the difficulty of detecting plant growth stimulants in sludge stabilization products.
[0051] In this application, Chinese cabbage was selected as the model plant. Chinese cabbage seeds are inexpensive and have a short germination period, making hydroponics a suitable method for accelerating the detection process. Prior to the colorimetric experiment, a 20-100 mmol / L CaCl2 solution was used to promote the expression of relevant messengers in the Chinese cabbage roots. Results showed that a 50 mmol / L CaCl2 solution achieved the best results, significantly shortening the colorimetric time in the presence of plant growth stimulants.
[0052] This application also includes negative and positive control groups. Sodium vanadate (Na3VO4) solution can inhibit the expression of the proton pump, thereby inhibiting the colorimetric reaction. A 200 μmol / L solution was used as the negative control, and a typical plant growth stimulant (IAA) was used as the positive control for the colorimetric experiment. By comparing the colorimetric effect with that of the experimental group, the equivalent IAA concentration of the plant growth stimulant in the experimental group can be obtained.
[0053] Examples 4-6 of this application are used to compare the content of plant growth stimulants in different sludge samples. The color development of Examples 4-6 and the positive control group are shown in the attached figures. Figure 3 As shown, Figure 3 The results showed that the content of plant growth stimulants was lowest in traditional compost sludge and raw sludge, while the expression activity of plant growth stimulants was highest in vermicompost sludge products, comparable to the positive control group. Quantitative analysis using colorimetric absorbance allowed for the further calculation of the IAA equivalent concentration of plant growth stimulants in vermicompost sludge.
[0054] Examples 7-11 of this application are used to compare the content of plant growth stimulants at different stages of aerobic sludge composting. The color development of Examples 7-11 is shown in the attached figure. Figure 4 As shown, Figure 4 This indicates that a noticeable color reaction occurred between days 5 and 9 of composting. However, the color disappeared as composting continued. This suggests that a large amount of plant growth stimulants are produced during days 5 to 9 of the composting process, but as the composting time increases, these organic matter is degraded and mineralized to form inorganic salts or less active organic matter, resulting in the disappearance of the color reaction.
[0055] The above embodiments merely illustrate several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of this patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.
Claims
1. A method for detecting plant growth stimulants in sludge stabilization products, characterized in that, Includes the following steps: The plant roots were brought into contact with a sludge extract containing CaCl2 to activate the messenger. The plants treated with the activated messenger were transferred to an agar gel plate containing bromocresol purple indicator. During the cultivation process, the color development of the agar gel plate was observed, and the amount of plant growth stimulants in the sludge extract was determined based on the color development. The concentration of CaCl2 in the sludge extract with added CaCl2 is 50 mmol / L; The processing time for the activation messenger is 10-30 minutes; The sludge extract is prepared according to the following method: The sludge sample was added to an aqueous solution, stirred, and then centrifuged. The supernatant was taken as the sludge extract. The agar gel plate was prepared as follows: The blank agar gel layer was adjusted to pH 6.8 using tris(hydroxymethyl)aminomethane buffer, and the bromocresol purple indicator was added.
2. The method for detecting plant growth stimulants in sludge stabilization products as described in claim 1, characterized in that, The amount of bromocresol purple indicator added is 0.04 g / L.
3. The method for detecting plant growth stimulants in sludge stabilization products as described in claim 1, characterized in that, The plant is bok choy, and the plant is cultivated according to the following method: The seeds of Chinese cabbage were sterilized with sodium hypochlorite solution and then washed with distilled water. Place the bok choy seeds on a plate lined with autoclaved filter paper and cultivate them in distilled water until they germinate, with a root length of 3-4 cm.
4. The method for detecting plant growth stimulants in sludge stabilization products as described in claim 3, characterized in that, The sodium hypochlorite solution has a mass concentration of 1%.
5. The method for detecting plant growth stimulants in sludge stabilization products as described in claim 3, characterized in that, The sterilization process takes 10 to 30 minutes.