A method for efficiently and rapidly removing microcystis aeruginosa and ctab by bacterial polysaccharide
By using bacterial polysaccharide flocculants to form flocs with Microcystis aeruginosa and CTAB, the problem of difficult removal of Microcystis aeruginosa and CTAB in water was solved, achieving a highly efficient and environmentally friendly removal effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SOUTH CHINA UNIV OF TECH
- Filing Date
- 2023-10-13
- Publication Date
- 2026-06-02
AI Technical Summary
Existing technologies are insufficient for efficiently removing Microcystis aeruginosa and CTAB from water, and traditional methods may lead to environmental pollution and harm to organisms.
Bacterial polysaccharides were used as flocculants. They were mixed with Microcystis aeruginosa and CTAB to form flocs, which were then separated and removed by sieve. The electrostatic neutralization effect of bacterial polysaccharides was used to efficiently adsorb CTAB.
It achieved the inactivation of Microcystis aeruginosa and the removal of CTAB, reducing the risk of environmental pollution, simplifying the operation process, and reducing harm to organisms.
Smart Images
Figure CN117602748B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of CTAB wastewater treatment, specifically to a method for efficiently and rapidly removing Microcystis aeruginosa and CTAB using bacterial polysaccharides. Background Technology
[0002] The overgrowth of harmful cyanobacteria in aquaculture ecosystems threatens human and animal health. Microcystis aeruginosa is a representative source of this pollution. Its inactivation and removal have become a challenging task for the sustainable recycling of marine aquaculture water. When the amount of CTAB added is sufficient to form micelles, CTAB can act as an algaecide, directly inactivating microalgae by disrupting cell membranes. Zhong Chenghua et al. prepared an algaecide by modifying sediment with CTAB. However, the algae-removing effect was not significant (Patent No.: CN 102276032 A).
[0003] However, discarded CTAB can still be a pollutant because it can kill beneficial microorganisms and even small animals, potentially disrupting aquatic micro-ecosystems if not adequately remediated. Zhou Yu et al. used organic solvents such as DDT, acetone, methanol, toluene, and MHA to remove CTAB from the surface of gold nanorods. While this method can remove CTAB, it uses large amounts of toxic organic solvents, causing secondary pollution to the environment (Patent No.: CN112535886 A).
[0004] From a broader perspective, CTAB and many other quaternary ammonium surfactants can be considered biohazardous substances, emitted in large quantities from numerous chemical products, including bactericides / disinfectants, laundry detergents, and cleaning products. These products easily deposit in water systems and therefore require timely removal. Summary of the Invention
[0005] The purpose of this invention is to propose a method for efficiently and rapidly removing Microcystis aeruginosa and CTAB using bacterial polysaccharides, thereby inactivating Microcystis aeruginosa while simultaneously removing CTAB.
[0006] The objective of this invention is achieved through the following technical solution:
[0007] A method for efficiently and rapidly removing Microcystis aeruginosa and CTAB using bacterial polysaccharides includes the following steps:
[0008] (1) Mix CTAB with the water sample to be treated to obtain a mixed solution and co-culture it. The water sample to be treated contains Microcystis aeruginosa.
[0009] (2) Add the bacterial polysaccharide to the mixture described in step (1) and mix evenly so that the bacterial polysaccharide, Microcystis aeruginosa and CTAB flocculate to form flocs;
[0010] (3) The bacterial polysaccharide described in step (2) is flocculated with Microcystis aeruginosa and CTAB to form flocs for separation.
[0011] Furthermore, in step (1), the concentration of CTAB is 1.25–110 mg / L.
[0012] Preferably, in step (1), the concentration of CTAB is 50 mg / L.
[0013] Furthermore, in step (1), the co-culture temperature is 25-35℃, the co-culture light intensity is 900-2000 lux, the co-culture humidity is 40-65% RH, and the co-culture time is 0.5-6 days.
[0014] Preferably, in step (1), the co-culture temperature is 28°C, the co-culture light intensity is 1100 lux, the co-culture humidity is 50%RH, and the co-culture time is 3 days.
[0015] Preferably, the concentration of Microcystis aeruginosa in the water sample to be treated in step (1) is 34–89 mg / L.
[0016] Furthermore, in step (2), the bacterial polysaccharide is Agrobacterium sp. extracellular polysaccharide (AMP), xanthan gum (XG), and Alcaligenes sp. extracellular polysaccharide (ALP).
[0017] Preferably, in step (2), the bacterial polysaccharide is ALP.
[0018] Furthermore, in step (2), the concentration of the bacterial polysaccharide is 5–60 mg / L.
[0019] Preferably, in step (2), the concentration of the bacterial polysaccharide is 40 mg / L.
[0020] The innovation of this invention compared with the prior art lies in:
[0021] 1. A novel method for removing CTAB from water is provided. The operation is simple and efficient, requiring only the uniform mixing of CTAB with Microcystis aeruginosa algal solution, the addition of bacterial polysaccharides to form flocs, and separation through a sieve or screen. This simple and easy-to-implement preparation method potentially reduces preparation costs and production cycles.
[0022] 2. Traditional flocculants mostly use chemical substances, while this method utilizes natural bacterial polysaccharides (Agrobacterium extracellular polysaccharides, Alcaligenes polysaccharides, and xanthan gum) as flocculants. Bacterial polysaccharides are negatively charged macromolecules that can efficiently adsorb positively charged CTAB small molecules through electrostatic neutralization. This not only reduces environmental pollution but also minimizes harm to organisms. Attached Figure Description
[0023] Figure 1 1-1 in the figure represents the CTAB quantitative standard curves of Examples 1-6; Figure 1 Figure 1-2 shows the inhibition rate of CTAB on chlorophyll a (Chl a) of Microcystis aeruginosa under different conditions in Examples 1-6.
[0024] Figure 2 This is a graph showing the changes in the content of CTAB before and after treatment with different concentrations in Example 1, as well as the changes in the inhibition rate of Vibrio fischeri activity.
[0025] Figure 3 This is a graph showing the changes in CTAB content and inhibition rate against Vibrio fischeri activity before and after treatment with different concentrations in Example 2.
[0026] Figure 4 This is a graph showing the changes in the content of CTAB before and after treatment with different concentrations in Example 3, as well as the changes in the inhibition rate of Vibrio fischeri activity.
[0027] Figure 5 This is a graph showing the changes in the content of CTAB before and after treatment with different concentrations in Example 4, as well as the changes in the inhibition rate of Vibrio fischeri activity.
[0028] Figure 6 This is a graph showing the changes in the content of CTAB before and after treatment with different concentrations in Example 5, as well as the changes in the inhibition rate of Vibrio fischeri activity.
[0029] Figure 7 This is a graph showing the changes in the content of CTAB before and after treatment with different concentrations in Example 6, as well as the changes in the inhibition rate of Vibrio fischeri activity.
[0030] Figure 8 This is a graph showing the changes in zebrafish embryo mortality rate after treatment with 5.5 mg / L CTAB using different methods in Example 7.
[0031] Figure 9 The different methods used in Example 7 to treat zebrafish embryos with 80 mg / L CTAB followed by LC 50 Graph showing the change in concentration. Detailed Implementation
[0032] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.
[0033] Preparation of Agrobacterium polysaccharide (AMP)
[0034] Fermentation medium formula: mannitol 5g, MgSO4 0.2g, KH2PO4 0.2g, NaCl 0.2g, CaCO3 3g, distilled water 1L, pH 7.0~7.2. The bacterial strain is Agrobacterium sp. The culture conditions are 37℃, 125rpm, for 5 days.
[0035] Bacteria were removed by centrifugation (1.2 × 10⁴ rpm, 10 min) and filtration through a 0.22 μm membrane. The cell-free fermentation supernatant was concentrated and dialyzed. Proteins in the dialysate were removed by treatment with 10% trichloroacetic acid. Precipitation was then performed using different volumes of 95% ethanol, and the precipitate was dissolved in water to obtain a crude extracellular mucopolysaccharide solution. The crude extracellular mucopolysaccharide solution was stored at 4°C overnight and then dialyzed using a 10 kDa membrane. Finally, the dialysate was freeze-dried under vacuum to obtain the extracellular mucopolysaccharide sample, labeled AMP (Patent No.: CN114891673A).
[0036] Example 1
[0037] (1) In a 34 mg / L Microcystis aeruginosa algal solution (OD 680 =0.1) CTAB was added to make the final CTAB concentrations 1.25, 5.5 and 10 mg / L, respectively. The mixture was cultured for 10 min at 30℃, 1500 lux light intensity and 45% RH humidity. The chlorophyll a (Chl a) content of Microcystis aeruginosa was tested.
[0038] The activity changes of *Microcystis aeruginosa* were assessed by measuring the chlorophyll a (Chl a) content of *Microcystis aeruginosa* using a UV-1900 UV-Vis spectrophotometer. In each experiment, 3 mL of algal solution was filtered through 0.22 μm glass fiber filter paper to obtain algal biomass, which was then transferred to a 2 mL cryopreservation tube along with 0.1 mm and 0.5 mm zirconium beads and 1.5 mL of 90% (v / v) acetone. The samples were then stored in the dark at 4 °C for 12 h and centrifuged at 10,000 rpm. The absorbance values of the samples at 630, 647, 664, and 750 nm were measured to assess the Chl a content and reflect changes in *Microcystis aeruginosa* activity. The Chl a content (CC) was calculated using Equation 1. Results are as follows: Figure 1 As shown in 1-2.
[0039]
[0040] (2) Add bacterial polysaccharide AMP to the mixture of Microcystis aeruginosa and CTAB in step (1) so that the concentration of AMP in the mixture reaches 40 mg / L, mix evenly to flocculate bacterial polysaccharide, Microcystis aeruginosa and CTAB.
[0041] (3) Separate the flocs formed by the bacterial polysaccharide, Microcystis aeruginosa and CTAB in step (2) through a 30-mesh sieve and obtain the supernatant.
[0042] (4) The CTAB content in the supernatant was tested using the cyclophosphamide reagent kit.
[0043] Preparation of CTAB quantitative standard curve. CTAB standard solutions were prepared at concentrations of 0 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, and 6 mg / L. CTAB was quantified using a quaternary ammonium salt reagent kit from Guangdong Huankai Biotechnology Co., Ltd. The reaction solution was prepared according to the instructions. After thorough mixing, the concentration at 535 nm (A) was measured using a UV-1900 spectrophotometer within 10 seconds. 535 Absorbance. All data are the average of three parallel measurements. Bacterial polysaccharides and Microcystis aeruginosa culture media did not interfere with CTAB quantification. Standard curves were obtained in CTAB solutions of known concentrations (0-6 mg / L). The CTAB quantification standard curve is shown below. Figure 1 As shown in 1-1. The calibration equation is as follows.
[0044] A 535 =0.044×C CTAB R 2 =0.998, n=7 (2)
[0045] The concentration of CTAB in the subclass can be obtained by measuring the absorbance value of the subclass and substituting it into the calibration equation.
[0046] (5) The inhibitory effect of the subsoil on Vibrio luminescence was tested using the Boluteng water quality test kit.
[0047] Inhibitory effect of CTAB on Vibrio fischeri. 1 mL of test sample, 0.1 mL of NaCl solution (21 wt%), and 0.5 mL of V. fischeri preparation were transferred to test tubes. The test sample for the experimental group was the subnavigation liquid, and the test sample for the control group was a mixture of Microcystis aeruginosa and CTAB prepared in step (1). After incubation at 24℃ for 15 min, the incubation time (S0) and time (S2) were measured. tThe luminescence intensity of the sample was measured using a BLT Lux-T010 detector. Simultaneously, the luminescence values of ultrapure water were measured as a control (C0 and C2). t Based on the activity of V. fischeri, its inhibition percentage (IP) was calculated using the following formula. vf ).
[0048]
[0049]
[0050] from Figure 1 From 1-2, it was found that 10 mg / L CTAB could inactivate 20% of Microcystis aeruginosa after 10 min. Figure 2 This example illustrates the changes in CTAB content before and after treatment with different concentrations in Example 1, as well as the inhibition rate of CTAB against Vibrio fischeri. For CTAB concentrations of 1.25, 5.5, and 10 mg / L, 40 mg / L AMP removed 98%, 95%, and 75%, respectively. Furthermore, it reduced CTAB toxicity against Vibrio fischeri, with the toxicity reduction rates in the supernatant reaching 99%, 95%, and 84%, respectively.
[0051] Example 2
[0052] (1) CTAB was added to a 68 mg / L Microcystis aeruginosa algal solution to make the final CTAB concentrations 15, 25 and 45 mg / L, respectively. The algae were cultured for 0.5 days at a temperature of 32℃, a light intensity of 1800 lux and a humidity of 45% RH. The chlorophyll a (Chl a) content of Microcystis aeruginosa was then tested.
[0053] The activity changes of *Microcystis aeruginosa* were assessed by measuring the chlorophyll a (Chl a) content using a UV-1900 UV-Vis spectrophotometer. In each experiment, 3 mL of algal solution was filtered through 0.22 μm glass fiber filter paper to obtain algal biomass, which was then transferred to a 2 mL cryopreservation tube along with 0.1 mm and 0.5 mm zirconium beads and 1.5 mL of 90% (v / v) acetone. The samples were then stored in the dark at 4 °C for 12 h and centrifuged at 10,000 rpm. The absorbance values of the samples at 630, 647, 664, and 750 nm were measured to assess the Chl a content and reflect changes in *Microcystis aeruginosa* activity. The Chl a content (CC) was calculated using Equation 1.
[0054]
[0055] (2) Add bacterial polysaccharide XG to the mixture of Microcystis aeruginosa and CTAB in step (1) so that the concentration of XG in the mixture reaches 40 mg / L, mix evenly to flocculate the bacterial polysaccharide, Microcystis aeruginosa and CTAB.
[0056] (3) Separate the flocs formed by the bacterial polysaccharide, Microcystis aeruginosa and CTAB in step (2) through a 30-mesh sieve and obtain the supernatant.
[0057] (4) The CTAB content in the subnatus was tested using the Cyclocarya quaternary ammonium salt kit.
[0058] Preparation of CTAB quantitative standard curve. CTAB standard solutions were prepared at concentrations of 0 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, and 6 mg / L. CTAB was quantified using a quaternary ammonium salt reagent kit from Guangdong Huankai Biotechnology Co., Ltd. The reaction solution was prepared according to the instructions. After thorough mixing, the concentration at 535 nm (A) was measured using a UV-1900 spectrophotometer within 10 seconds. 535 Absorbance. All data are the average of three parallel measurements. Bacterial polysaccharides and Microcystis aeruginosa culture media did not interfere with CTAB quantification. Standard curves were obtained in CTAB solutions of known concentrations (0-6 mg / L). The CTAB quantification standard curve is shown below. Figure 1 As shown in 1-1. The calibration equation is as follows.
[0059] A 535 =0.044×C CTAB R 2 =0.998, n=7 (2)
[0060] Since this kit can only test CTAB concentrations from 0 to 6 mg / L, the subnaphthalene is diluted, and the absorbance value of the diluted subnaphthalene is measured. Substituting this value into the calibration equation yields the CTAB concentration of the diluted subnaphthalene. Multiplying this by the dilution factor gives the CTAB concentration of the undiluted subnaphthalene.
[0061] (5) The inhibitory effect of the subsoil on Vibrio luminescence was tested using the Boluteng water quality test kit.
[0062] Inhibitory effect of CTAB on Vibrio fischeri. 1 mL of test sample, 0.1 mL of NaCl solution (21 wt%), and 0.5 mL of V. fischeri preparation were transferred to test tubes. The test sample for the experimental group was the subnavigation liquid, and the test sample for the control group was a mixture of Microcystis aeruginosa and CTAB prepared in step (1). After incubation at 24℃ for 15 min, the incubation time (S0) and time (S2) were measured. tThe luminescence intensity of the samples was measured using a BLT Lux-T010 detector. Simultaneously, the luminescence values of bacteria in ultrapure water were measured as controls (C0 and C10). t Based on the activity of V. fischeri, its inhibition percentage (IP) was calculated using the following formula. vf ).
[0063]
[0064]
[0065] from Figure 1 From 1-2, it was found that 15 mg / L CTAB could inactivate more than 62% of Microcystis aeruginosa after 12 hours. Figure 3 This example illustrates the changes in CTAB content before and after treatment with different concentrations in Example 2, as well as the inhibition rate of CTAB against Vibrio fischeri. For CTAB concentrations of 15 mg / L, 25 mg / L, and 45 mg / L, 40 mg / L XG removed 43%, 40%, and 37%, respectively. At CTAB concentrations of 15 and 25 mg / L, XG reduced the toxicity of CTAB against Vibrio fischeri, with reduction rates of 35% and 11%, respectively.
[0066] Example 3
[0067] (1) CTAB was added to a 34 mg / L Microcystis aeruginosa algal solution to make the final CTAB concentrations 80, 100 and 115 mg / L, respectively. The algae were cultured for 1 day at a temperature of 30℃, a light intensity of 2000 lux and a humidity of 55%RH. The chlorophyll a (Chl a) content of Microcystis aeruginosa was determined.
[0068] The activity changes of *Microcystis aeruginosa* were assessed by measuring the chlorophyll a (Chl a) content using a UV-1900 UV-Vis spectrophotometer. In each experiment, 3 mL of algal solution was filtered through 0.22 μm glass fiber filter paper to obtain algal biomass, which was then transferred to a 2 mL cryopreservation tube along with 0.1 mm and 0.5 mm zirconium beads and 1.5 mL of 90% (v / v) acetone. The samples were then stored in the dark at 4 °C for 12 h and centrifuged at 10,000 rpm. The absorbance values of the samples at 630, 647, 664, and 750 nm were measured to assess the Chl a content and reflect changes in *Microcystis aeruginosa* activity. The Chl a content (CC) was calculated using Equation 1.
[0069]
[0070] (2) Add bacterial polysaccharide ALP to the mixture of Microcystis aeruginosa and CTAB in step (1) so that the ALP concentration in the mixture reaches 40 mg / L, and mix evenly to flocculate the bacterial polysaccharide, Microcystis aeruginosa and CTAB.
[0071] (3) Separate the flocs formed by the bacterial polysaccharide, Microcystis aeruginosa and CTAB in step (2) through a 30-mesh sieve and obtain the supernatant.
[0072] (4) The CTAB content in the subnatus was tested using the Cyclocarya quaternary ammonium salt kit.
[0073] Preparation of CTAB quantitative standard curve. CTAB standard solutions were prepared at concentrations of 0 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, and 6 mg / L. CTAB was quantified using a quaternary ammonium salt reagent kit from Guangdong Huankai Biotechnology Co., Ltd. The reaction solution was prepared according to the instructions. After thorough mixing, the concentration at 535 nm (A) was measured using a UV-1900 spectrophotometer within 10 seconds. 535 Absorbance. All data are the average of three parallel measurements. Bacterial polysaccharides and Microcystis aeruginosa culture media did not interfere with CTAB quantification. Standard curves were obtained in CTAB solutions of known concentrations (0-6 mg / L). The CTAB quantification standard curve is shown below. Figure 1 As shown in 1-1. The calibration equation is shown in Equation 1.
[0074] A 535 =0.044×C CTAB R 2 =0.998, n=7 (2)
[0075] Since this kit can only test CTAB concentrations from 0 to 6 mg / L, the subnaphthalene is diluted, and the absorbance value of the diluted subnaphthalene is measured. Substituting this value into the calibration equation yields the CTAB concentration of the diluted subnaphthalene. Multiplying this by the dilution factor gives the CTAB concentration of the undiluted subnaphthalene.
[0076] (5) The inhibitory effect of the subsoil on Vibrio luminescence was tested using the Boluteng water quality test kit.
[0077] Inhibitory effect of CTAB on Vibrio fischeri. 1 mL of test sample, 0.1 mL of NaCl solution (21 wt%), and 0.5 mL of V. fischeri preparation were transferred to test tubes. The test sample for the experimental group was the subnavigation liquid, and the test sample for the control group was a mixture of Microcystis aeruginosa and CTAB prepared in step (1). After incubation at 24℃ for 15 min, the incubation time (S0) and time (S2) were measured. tThe luminescence intensity of the samples was measured using a BLT Lux-T010 detector. Simultaneously, the luminescence values of bacteria in ultrapure water were measured as controls (C0 and C10). t Based on the activity of V. fischeri, its inhibition percentage (IP) was calculated as shown in Equation 2. vf ).
[0078]
[0079]
[0080] from Figure 1 From 1-2, it was found that 80 mg / L CTAB could inactivate more than 90% of Microcystis aeruginosa after 72 hours. Figure 4 This describes the changes in CTAB content and inhibition rate against *Vibrio fischeri* activity before and after treatment with different concentrations in Example 3. For CTAB concentrations of 80 mg / L, 100 mg / L, and 115 mg / L, 40 mg / L ALP removed 49%, 52%, and 35%, respectively. Due to the high toxicity of CTAB at concentrations of 80–115 mg / L, even after removal treatment, the inhibition rate against *Vibrio fischeri* could not be significantly reduced.
[0081] Example 4
[0082] (1) CTAB was added to a 34 mg / L Microcystis aeruginosa algal solution to make the final CTAB concentrations 1.25, 5.5 and 10 mg / L, respectively. The algae were cultured for 3 days at a temperature of 25℃, a light intensity of 900 lux and a humidity of 40% RH. The chlorophyll a (Chl a) content of Microcystis aeruginosa was determined.
[0083] The activity changes of *Microcystis aeruginosa* were assessed by measuring the chlorophyll a (Chl a) content using a UV-1900 UV-Vis spectrophotometer. In each experiment, 3 mL of algal solution was filtered through 0.22 μm glass fiber filter paper to obtain algal biomass, which was then transferred to a 2 mL cryopreservation tube along with 0.1 mm and 0.5 mm zirconium beads and 1.5 mL of 90% (v / v) acetone. The samples were then stored in the dark at 4 °C for 12 h and centrifuged at 10,000 rpm. The absorbance values of the samples at 630, 647, 664, and 750 nm were measured to assess the Chl a content and reflect changes in *Microcystis aeruginosa* activity. The Chl a content (CC) was calculated using Equation 1.
[0084]
[0085] (2) Add bacterial polysaccharide ALP to the mixture of Microcystis aeruginosa and CTAB in step (1) so that the ALP concentration in the mixture reaches 20 mg / L, mix evenly to flocculate bacterial polysaccharide, Microcystis aeruginosa and CTAB.
[0086] (3) Separate the flocs formed by the bacterial polysaccharide, Microcystis aeruginosa and CTAB in step (2) through a 30-mesh sieve and obtain the supernatant at the same time.
[0087] (4) The CTAB content in the supernatant was tested using the cyclophosphamide reagent kit.
[0088] Preparation of CTAB quantitative standard curve. CTAB standard solutions were prepared at concentrations of 0 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, and 6 mg / L. CTAB was quantified using a quaternary ammonium salt reagent kit from Guangdong Huankai Biotechnology Co., Ltd. The reaction solution was prepared according to the instructions. After thorough mixing, the concentration at 535 nm (A) was measured using a UV-1900 spectrophotometer within 10 seconds. 535 Absorbance. All data are the average of three parallel measurements. Bacterial polysaccharides and Microcystis aeruginosa culture media did not interfere with CTAB quantification. Standard curves were obtained in CTAB solutions of known concentrations (0-6 mg / L). The CTAB quantification standard curve is shown below. Figure 1 As shown in 1-1. The calibration equation is as follows.
[0089] A 535 =0.044×C CTAB R 2 =0.998, n=7 (2)
[0090] (5) The inhibitory effect of the subsoil on Vibrio luminescence was tested using the Boluteng water quality test kit.
[0091] Inhibitory effect of CTAB on Vibrio fischeri. 1 mL of test sample, 0.1 mL of NaCl solution (21 wt%), and 0.5 mL of V. fischeri preparation were transferred to test tubes. The test sample for the experimental group was the subnavigation liquid, and the test sample for the control group was a mixture of Microcystis aeruginosa and CTAB prepared in step (1). After incubation at 24℃ for 15 min, the incubation time (S0) and time (S2) were measured. t The luminescence intensity of the samples was measured using a BLT Lux-T010 detector. Simultaneously, the luminescence values of bacteria in ultrapure water were measured as controls (C0 and C10). t Based on the activity of V. fischeri, its inhibition percentage (IP) was calculated using the following formula. vf ).
[0092]
[0093]
[0094] from Figure 1 From 1-2, it was found that 10 mg / L CTAB could inactivate 35% of Microcystis aeruginosa after 3 days. Figure 5 This is Example 4, showing the changes in CTAB content before and after treatment with different concentrations, and the inhibition rate of CTAB against Vibrio fischeri activity. For CTAB concentrations of 1.25, 5.5, and 10 mg / L, 20 mg / L ALP removed 99%, 97%, and 78%, respectively. Furthermore, it significantly reduced the toxicity of CTAB against Vibrio fischeri, with reduction rates of 100%, 98%, and 98%, respectively. This indicates that after 3 days of co-culturing with Microcystis aeruginosa, the activity of Microcystis aeruginosa became even lower.
[0095] Example 5
[0096] (1) CTAB was added to a 34 mg / L Microcystis aeruginosa algal solution to make the final CTAB concentrations 15, 25 and 45 mg / L, respectively. The algae were cultured for 3 days at a temperature of 28℃, a light intensity of 1100 lux and a humidity of 50% RH. The chlorophyll a (Chla) content of Microcystis aeruginosa was determined.
[0097] The activity changes of *Microcystis aeruginosa* were assessed by measuring the chlorophyll a (Chl a) content using a UV-1900 UV-Vis spectrophotometer. In each experiment, 3 mL of algal solution was filtered through 0.22 μm glass fiber filter paper to obtain algal biomass, which was then transferred to a 2 mL cryopreservation tube along with 0.1 mm and 0.5 mm zirconium beads and 1.5 mL of 90% (v / v) acetone. The samples were then stored in the dark at 4 °C for 12 h and centrifuged at 10,000 rpm. The absorbance values of the samples at 630, 647, 664, and 750 nm were measured to assess the Chl a content and reflect changes in *Microcystis aeruginosa* activity. The Chl a content (CC) was calculated using Equation 1.
[0098]
[0099] (2) Add bacterial polysaccharide AMP to the mixture of Microcystis aeruginosa and CTAB in step (1) so that the concentration of AMP in the mixture reaches 60 mg / L, mix evenly to flocculate bacterial polysaccharide, Microcystis aeruginosa and CTAB.
[0100] (3) Separate the flocs formed by the bacterial polysaccharide, Microcystis aeruginosa and CTAB in step (2) through a 30-mesh sieve and obtain the supernatant.
[0101] (4) The CTAB content in the subnatus was tested using the Cyclocarya quaternary ammonium salt kit.
[0102] Preparation of CTAB quantitative standard curve. CTAB standard solutions were prepared at concentrations of 0 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, and 6 mg / L. CTAB was quantified using a quaternary ammonium salt reagent kit from Guangdong Huankai Biotechnology Co., Ltd. The reaction solution was prepared according to the instructions. After thorough mixing, the concentration at 535 nm (A) was measured using a UV-1900 spectrophotometer within 10 seconds. 535 Absorbance. All data are the average of three parallel measurements. Bacterial polysaccharides and Microcystis aeruginosa culture media did not interfere with CTAB quantification. Standard curves were obtained in CTAB solutions of known concentrations (0-6 mg / L). The CTAB quantification standard curve is shown below. Figure 1 As shown in 1-1. The calibration equation is as follows.
[0103] A 535 =0.044×C CTAB R 2 =0.998, n=7 (2)
[0104] Since this kit can only test CTAB concentrations from 0 to 6 mg / L, the subnaphthalene is diluted, and the absorbance value of the diluted subnaphthalene is measured. Substituting this value into the calibration equation yields the CTAB concentration of the diluted subnaphthalene. Multiplying this by the dilution factor gives the CTAB concentration of the undiluted subnaphthalene.
[0105] (5) The inhibitory effect of the subsoil on Vibrio luminescence was tested using the Boluteng water quality test kit.
[0106] Inhibitory effect of CTAB on Vibrio fischeri. 1 mL of test sample, 0.1 mL of NaCl solution (21 wt%), and 0.5 mL of V. fischeri preparation were transferred to test tubes. The test sample for the experimental group was the subnavigation liquid, and the test sample for the control group was a mixture of Microcystis aeruginosa and CTAB prepared in step (1). After incubation at 24℃ for 15 min, the incubation time (S0) and time (S2) were measured. t The luminescence intensity of the samples was measured using a BLT Lux-T010 detector. Simultaneously, the luminescence values of bacteria in ultrapure water were measured as controls (C0 and C10). t Based on the activity of V. fischeri, its inhibition percentage (IP) was calculated using the following formula. vf ).
[0107]
[0108]
[0109] from Figure 1 From 1-2, it was found that 15 mg / L CTAB could inactivate more than 72% of Microcystis aeruginosa after 12 hours. Figure 6 This is Example 5, showing the changes in CTAB content and inhibition rate against *Vibrio fischeri* activity before and after treatment with different concentrations. For CTAB concentrations of 15, 25, and 45 mg / L, 60 mg / L AMP removed 67%, 40%, and 51%, respectively. At CTAB concentrations of 15 and 25 mg / L, AMP reduced the toxicity of CTAB against *Vibrio fischeri*, with reduction rates of 98% and 35%, respectively.
[0110] Example 6
[0111] (1) CTAB was added to 89 mg / L of Microcystis aeruginosa algal solution to make the final CTAB concentrations 80, 100 and 115 mg / L, respectively. The algae were cultured for 6 days at a temperature of 35℃, a light intensity of 2000 lux and a humidity of 65% RH. The chlorophyll a (Chl a) content of Microcystis aeruginosa was determined.
[0112] The activity changes of *Microcystis aeruginosa* were assessed by measuring the chlorophyll a (Chl a) content using a UV-1900 UV-Vis spectrophotometer. In each experiment, 3 mL of algal solution was filtered through 0.22 μm glass fiber filter paper to obtain algal biomass, which was then transferred to a 2 mL cryopreservation tube along with 0.1 mm and 0.5 mm zirconium beads and 1.5 mL of 90% (v / v) acetone. The samples were then stored in the dark at 4 °C for 12 h and centrifuged at 10,000 rpm. The absorbance values of the samples at 630, 647, 664, and 750 nm were measured to assess the Chl a content and reflect changes in *Microcystis aeruginosa* activity. The Chl a content (CC) was calculated using Equation 1.
[0113]
[0114] (2) Add bacterial polysaccharide XG to the mixture of Microcystis aeruginosa and CTAB in step (1) so that the concentration of XG in the mixture reaches 5 mg / L, mix evenly to flocculate the bacterial polysaccharide, Microcystis aeruginosa and CTAB.
[0115] (3) Separate the flocs formed by the bacterial polysaccharide, Microcystis aeruginosa and CTAB in step (2) through a 30-mesh sieve.
[0116] (4) The CTAB content in the subnatus was tested using the Cyclocarya quaternary ammonium salt kit.
[0117] Preparation of CTAB quantitative standard curve. CTAB standard solutions were prepared at concentrations of 0 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, and 6 mg / L. CTAB was quantified using a quaternary ammonium salt reagent kit from Guangdong Huankai Biotechnology Co., Ltd. The reaction solution was prepared according to the instructions. After thorough mixing, the concentration at 535 nm (A) was measured using a UV-1900 spectrophotometer within 10 seconds. 535 Absorbance. All data are the average of three parallel measurements. Bacterial polysaccharides and Microcystis aeruginosa culture media did not interfere with CTAB quantification. Standard curves were obtained in CTAB solutions of known concentrations (0-6 mg / L). The CTAB quantification standard curve is shown below. Figure 1 As shown in 1-1. The calibration equation is shown in Equation 1.
[0118] A 535 =0.044×C CTAB R 2 =0.998, n=7 (2)
[0119] Since this kit can only test CTAB concentrations from 0 to 6 mg / L, the subnaphthalene is diluted, and the absorbance value of the diluted subnaphthalene is measured. Substituting this value into the calibration equation yields the CTAB concentration of the diluted subnaphthalene. Multiplying this by the dilution factor gives the CTAB concentration of the undiluted subnaphthalene.
[0120] (5) The inhibitory effect of the subsoil on Vibrio luminescence was tested using the Boluteng water quality test kit.
[0121] Inhibitory effect of CTAB on Vibrio fischeri. 1 mL of test sample, 0.1 mL of NaCl solution (21 wt%), and 0.5 mL of V. fischeri preparation were transferred to test tubes. The test sample for the experimental group was the subnavigation liquid, and the test sample for the control group was a mixture of Microcystis aeruginosa and CTAB prepared in step (1). After incubation at 24℃ for 15 min, the incubation time (S0) and time (S2) were measured. t The luminescence intensity of the samples was measured using a BLT Lux-T010 detector. Simultaneously, the luminescence values of bacteria in ultrapure water were measured as controls (C0 and C10). t Based on the activity of V. fischeri, its inhibition percentage (IP) was calculated as shown in Equation 2. vf ).
[0122]
[0123]
[0124] from Figure 1From 1-2, it was found that 80 mg / L CTAB could inactivate more than 98% of Microcystis aeruginosa after 72 hours. Figure 7 This is Example 6, showing the changes in CTAB content and inhibition rate against Vibrio fischeri activity before and after treatment with different concentrations. For CTAB at concentrations of 80, 100, and 115 mg / L, 5 mg / L XG could remove 36%, 45%, and 43%, respectively. Due to the high toxicity of CTAB at concentrations of 80, 100, and 115 mg / L, the removal treatment could not significantly reduce the inhibition rate of CTAB against Vibrio fischeri.
[0125] Example 7
[0126] Normally developing zebrafish embryos (wild-type AB strain, age 4–128 cell cycles) were randomly selected and placed in 6-well plates, 30 embryos per well. Different volumes of culture medium were used to dissolve and dilute the samples, and a normal control group was also included, with 3 mL of culture medium per well. The embryos were incubated at 26°C in the dark in a constant temperature and humidity incubator for 96 h, and the mortality rate and LC50 concentration of the zebrafish embryos were determined. The zebrafish embryo culture medium (Ringer's liquid culture medium) consisted of: NaCl 116 mmol / L, KCl 2.9 mmol / L, CaCl2 1.8 mmol / L, MgCl2 1.4 mmol / L, HEPES (4-hydroxyethylpiperazine ethanethiolic acid) 10 mmol / L, pH 7.2–7.5. The sample processing methods for the acute toxicity test of zebrafish embryos are shown in Table 1.
[0127] Table 1
[0128]
[0129] Note: Ma represents the *Microcystis aeruginosa* algal solution used here, at a concentration of 70 mg / L.
[0130] Figure 8 The results of acute toxicity testing of low concentrations of CTAB (5.5 mg / L) in zebrafish were presented. 5.5 mg / L CTAB had a 100% lethality rate in zebrafish embryos. After flocculation treatment with AMP, XG, and ALP, the CTAB solution (initial concentration 5.5 mg / L, supernatant after flocculation) did not inhibit the growth of zebrafish embryos. This indicates that BEPS can remove the toxicity of low concentrations (≤5 mg / L) of CTAB, thereby reducing its harm to aquatic animals.
[0131] However, when the CTAB concentration reached 80 mg / L, bacterial polysaccharides alone could not completely neutralize their toxicity to zebrafish embryos, as shown in the following results. Figure 9 When AMP, XG, and ALP were added individually, the lethality of 80 mg / L CTAB in zebrafish embryos remained 100%, but the LC...50 There were some improvements, with Microcystis aeruginosa co-cultured with CTAB for 72 hours followed by the addition of AMP, and 80 mg / L CTAB showing an effect on the LC50 of zebrafish embryos. 50 The concentration was increased from 1.36 mg / L to 36.0 mg / L. ALP was the second most effective (28.4 mg / L), while XG was the least effective (10.1 mg / L).
[0132] The above embodiments are used to explain and illustrate the present invention. The implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the claims of the present invention.
Claims
1. A method for efficiently and rapidly removing Microcystis aeruginosa and CTAB using bacterial polysaccharides, characterized in that, Includes the following steps: (1) Mix CTAB with the water sample to be treated to obtain a mixed solution and co-culture the water sample to be treated, which contains Microcystis aeruginosa; the temperature of the co-culture is 25~35℃, the light intensity of the co-culture is 900~2000 lux, the humidity of the co-culture is 40~65RH% and the co-culture time is 3 days; (2) Add the bacterial polysaccharide to the mixture in step (1) and mix evenly so that the bacterial polysaccharide, Microcystis aeruginosa, and CTAB flocculate to form flocs; the bacterial polysaccharide is Agrobacterium. Agrobacterium sp. extracellular polysaccharide (AMP) and Alcaligenes Alcaligenes One or more of the following: sp. extracellular polysaccharides (ALP); (3) The bacterial polysaccharide described in step (2) is flocculated with Microcystis aeruginosa and CTAB to form flocs and then separated.
2. The method for efficiently and rapidly removing Microcystis aeruginosa and CTAB using bacterial polysaccharides according to claim 1, characterized in that, The co-cultivation temperature in step (1) is 28℃; the light intensity during co-cultivation is 1100 lux; and the humidity during co-cultivation is 50 RH.
3. The method for efficiently and rapidly removing Microcystis aeruginosa and CTAB using bacterial polysaccharides according to claim 1, characterized in that, The bacterial polysaccharide mentioned in step (2) is Alcaligenes. Alcaligenes extracellular polysaccharide (ALP); the concentration of the bacterial polysaccharide is 5-60 mg / L.
4. The method for efficiently and rapidly removing Microcystis aeruginosa and CTAB using bacterial polysaccharides according to claim 1, characterized in that, The concentration of the bacterial polysaccharide in step (2) is 40 mg / L.
5. The method for efficiently and rapidly removing Microcystis aeruginosa and CTAB using bacterial polysaccharides according to claim 1, characterized in that, The concentration of CTAB in step (1) is 1.25 mg / L to 115 mg / L.
6. The method for efficiently and rapidly removing Microcystis aeruginosa and CTAB using bacterial polysaccharides according to claim 5, characterized in that, The concentration of CTAB was 50 mg / L.
7. The method for efficiently and rapidly removing Microcystis aeruginosa and CTAB using bacterial polysaccharides according to claim 1, characterized in that, The concentration of Microcystis aeruginosa in the water sample to be treated in step (1) is 34 ~ 89 mg / L.