Method for adjusting toxicity of phosphorus-accumulating bacteria based on composite stress of glucose and nano-zinc oxide particles

Abstract

The invention relates to a method for adjusting toxicity of phosphorus-accumulating bacteria based on composite stress of glucose and nano-zinc oxide particles, and belongs to the field of soil and water environment ecological restoration. The method mainly comprises the following steps: when nano-zinc oxide and glucose exist in a composite manner, glucose has a remarkable detoxification effect on biotoxicity of nano-zinc oxide, and when the nano-zinc oxide and glucose exist in a composite manner, glucose has a remarkable detoxification effect on biotoxicity of nano-zinc oxide; and the detoxification effect is improved and then reduced along with the increase of the glucose concentration. The relieving effect of glucose on the phosphorus removal inhibition rate of 2.50 mg / L nano-zinc oxide is obviously higher than that of 1.80 mg / L nano-zinc oxide, and the detoxification effect generated by compounding glucose and 1.80 mg / L nano-zinc oxide shows obvious difference (p is less than 0.05) under different nano-zinc oxide concentrations, namely the detoxification effect on high-concentration nano-zinc oxide is stronger. Ultraviolet and infrared spectroscopic analysis shows that the detoxification effect of the glucose may be that functional groups contained in the glucose and Zn < 2 + > released by the nano-zinc oxide are subjected to a coordination reaction, and the coordination site is at the aldehyde group CHO position.

Description

(I) Technical field

[0001] The invention belongs to the field of soil and water environment ecological restoration, and in particular relates to the microbial toxicity and regulatory effect of humus in sewage on nanoparticle materials. (II) Background technology

[0002] With the rapid development of nanotechnology, nanomaterials are widely used in daily life and industrial production processes. Wastewater treatment plants are an important sink for nanomaterials. The latest research also found that nanomaterials have a significant inhibitory effect on the biochemical metabolism of nitrogen and phosphorus removal in wastewater treatment. Nanotoxicology has become a new field of toxicology, mainly involving understanding the interaction between nanoparticles and the environment. Due to the increase in exposure to nanoparticles in the environment, there is a need to better assess nanotoxicity, adopt more efficient methods to utilize nanoparticles, and adopt appropriate disposal methods to help reduce adverse effects.

[0003] Nitrogen and phosphorus are the basic elements of many organisms, but they are also common pollutants. Excessive nutrients such as nitrogen and phosphorus can cause eutrophication of water bodies, leading to a rapid increase in algae or plankton in the water body, significantly reducing the dissolved oxygen content in the water body, and a large number of fish and many aquatic organisms will die due to lack of oxygen, thereby destroying the stability of the water ecosystem and even causing the entire aquatic ecosystem to be on the verge of extinction. The activated sludge method is a general term for various methods of biological treatment of wastewater with microorganisms suspended in water. Polyphosphate bacteria is a general term for a class of bacteria in the activated sludge system. Under aerobic conditions, this type of bacteria can excessively absorb phosphorus in sewage, and convert external phosphorus into polyphosphates through biological metabolic processes to achieve the purpose of polyphosphate. In biological phosphorus removal technology, the anaerobic and aerobic alternation method is generally adopted. After the polyphosphate bacteria absorb a large amount of phosphorus in the sewage, the purpose of phosphorus removal is achieved through the discharge of sludge.

[0004] In recent years, nano zinc oxide has attracted attention from all walks of life due to its high biological toxicity and high detection rate and the negative environmental impact it brings. Nano zinc oxide toxicity affects microbial cells in many ways. They enter cells through broken cell walls and inhibit energy metabolism when NPs are internalized by bacteria. Membrane dysfunction caused by nano zinc oxide nanoparticles leads to the internalization of nanoparticles into cells. The internalization of nano zinc oxide is usually determined by particle size, defects, surface, functionalization and chemical properties. Nanoparticles may interact with dissolved organic matter (DOM) in the environment, which will greatly affect the final fate, transport, transformation and biological effects of nanoparticles. DOM mainly comes from the slow microbial decomposition of animal remains, plants and microorganisms, and is ubiquitous in the natural environment. (III) Summary of the invention

[0005] The purpose of the present invention is to provide a method for regulating the toxicity of polyphosphate bacteria based on the combined stress of dissolved organic matter and nanoparticles. The method can reduce the toxicity of nano zinc oxide by regulation and improve the impact resistance of sewage treatment.

[0006] The technical solution of the present invention is: a method for evaluating the effect of combined stress of humus and nanoparticles in sewage on polyphosphate-accumulating bacteria, comprising the following steps:

[0007] (1) LB liquid culture was centrifuged at 4°C, 8000 rpm, 5 min, resuspended with sterile ultrapure water, and the bacterial concentration was adjusted. The stability of the inoculated bacterial amount was ensured by measuring OD600 (1.1-1.2).

[0008] (2) Take 12 conical flasks, add 12 mL of bacterial solution, 5 mL of artificial sewage, and pollutants and humus of different concentrations into the conical flasks, and add the same volume of ultrapure water to the control group, and finally maintain the total volume of the system at 120 mL.

[0009] (3) The conical flask is equipped with a rubber stopper with a glass tube, which is used to connect the diverter and the nitrogen bottle. The anaerobic environment is maintained by continuously introducing N2, and the culture is shaken at 120rpm and 30℃ for 2h in a constant temperature and light-proof manner (anaerobic). Then the ventilation device is removed, and the flask is sealed with a sterile breathable membrane, and the culture is shaken at 140rpm and 30℃ for a constant temperature and light-proof manner (aerobic). Samples are taken after 4h of aerobic culture. After the samples are centrifuged (4℃, 8000rpm, 5min), the supernatant is aspirated with a 1mL sterile syringe and filtered through a 0.22μm polyethersulfone filter membrane for measuring phosphate concentration. The phosphate test method refers to the national standard GB11893-891 ammonium molybdate spectrophotometry.

[0010] (4) The product produced by the combination of organic matter (humus, glucose, amino acid) and nano zinc oxide was obtained by freeze drying. The obtained solid product was prepared by KBr tableting method and the infrared spectrometer of Shimadzu IRAffinity-1S was used to analyze the solid product at 4000 cm -1 ~400cm -1 The infrared spectra of organic matter in the coagulant and nano zinc oxide were scanned separately for comparative analysis.

[0011] (IV) Beneficial effects

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] (1) The present invention innovatively studies the effect of humus in dissolved organic matter on the polyphosphate bacteria toxicity of nano-zinc oxide, which helps to regulate and reduce the toxicity of nano-zinc oxide and improve the impact resistance of sewage treatment.

[0014] (2) The present invention innovatively studies the interaction mechanism between humus and nano zinc oxide in dissolved organic matter and analyzes the detoxification regulation mechanism, which can maximize the use of active components to treat pollutants. (V) Description of the drawings

[0015] Figure 1 is the antagonistic rate of glucose and nano zinc oxide compound on phosphorus removal inhibition, Figure 2 This is the infrared spectrum of the composite system of glucose (Glc) and nano-zinc oxide. (VI) Specific implementation methods

[0016] Dissolved organic matter is commonly found in water environments and usually contains a variety of complex organic compounds, including humus, sugars, amino acids, etc., which can interact with nanoparticles to change their surface reactivity, colloidal stability, cellular uptake capacity and bioaccumulation. Taking all the above into consideration, this study built a simulated anaerobic / aerobic (A / O) process reactor for wastewater treatment to study the removal of PO4 by polyphosphate bacteria in the presence of nano-zinc oxide and humus. 3- Changes in capacity, and the detoxification effect of humus on nano zinc oxide.

[0017] (1) The culture device for simulating the A / O process includes a reactor such as a conical flask, a glass air guide tube, a rubber stopper, an aeration stirring device such as a constant temperature water bath shaker, and an anaerobic control device such as a nitrogen diversion tube. This device mainly simulates the reaction process of the A / O process by controlling the nitrogen introduction rate and changing the speed of the water bath shaker. The reactor mainly includes artificial sewage, coagulant / dissolved organic matter, nano zinc oxide and polyphosphate bacteria system.

[0018] (2) Connect the nitrogen bottle and the sealed glass air guide tube through the nitrogen separator, introduce nitrogen into the reaction bottle for half an hour, blow out the air and maintain the nitrogen flow, and incubate the reaction bottle in a water bath shaker at 140 rpm and 30° C. in the dark for 2 hours. At this stage, the anaerobic process is simulated, and the polyphosphate bacteria release phosphorus anaerobically in a low-oxygen environment.

[0019] (3) After 2 hours, stop the nitrogen, remove the rubber stopper, and seal the conical flask with a sterile breathable membrane. The conical flask was incubated in a water bath shaker at 140 rpm and 30°C for 4 hours in the dark to simulate the aerobic process of the A / O process. The experimental device is easy to adjust parameters, simple to operate, and has good simulation effects, which can provide reliable experimental conditions for this study.

[0020] (4) Prepare LB solid culture medium and LB liquid culture medium, dissolve with ultrapure water, and adjust the volume to 1 L. During the process of adjusting the volume, adjust the pH to 7.0±0.1 with 1 mol / L NaOH and HCl, and then sterilize in an autoclave at 121°C for 20 min. After sterilization, pour the plate into a solid culture medium in time, and refrigerate the liquid culture medium at 4°C for use.

[0021] (5) After inoculation, invert the plate and incubate it in a 30°C constant temperature incubator for 24 hours to activate the bacteria. Take out the activated plate, pick a single colony with an inoculation needle and place it in LB liquid culture medium, seal it with a sterile breathable membrane, place it in a 30°C constant temperature water bath shaker, and incubate it at 140 rpm for 18 hours to complete the proliferation and cultivation of polyphosphate bacteria.

[0022] (6) The LB liquid culture was centrifuged at 4°C, 8000 rpm, for 5 min, resuspended in sterile ultrapure water, and the bacterial concentration was adjusted. The stability of the inoculated bacterial amount was ensured by measuring OD600 (1.1-1.2).

[0023] (7) Take 12 conical flasks, add 12 mL of bacterial solution, 5 mL of artificial sewage, and pollutants and humus of different concentrations into the conical flasks, and add the same volume of ultrapure water to the control group, so that the total volume of the system is finally maintained at 120 mL.

[0024] (8) The conical flask is equipped with a rubber stopper with a glass tube, which is used to connect the diverter and the nitrogen bottle. The anaerobic environment is maintained by continuously introducing N2, and the culture is shaken at 120rpm and 30℃ for 2h in a constant temperature and away from light (anaerobic). Then the ventilation device is removed, and the bottle is sealed with a sterile breathable membrane, and the culture is shaken at 140rpm and 30℃ for a constant temperature and away from light (aerobic). Samples are taken after 4h of aerobic culture. After the samples are centrifuged (4℃, 8000rpm, 5min), the supernatant is aspirated with a 1mL sterile syringe and filtered through a 0.22μm polyethersulfone filter membrane for measuring phosphate concentration. The phosphate test method refers to the national standard GB11893-891 ammonium molybdate spectrophotometry.

[0025] (9) According to the test results of the present invention, when nano zinc oxide and glucose are present in combination, as the concentration of glucose added increases, the phosphorus removal inhibition rate shows a trend of first decreasing and then increasing, and the inhibition mitigation effect of glucose on 2.50 mg / L nano zinc oxide is significantly higher than that of 1.80 mg / L nano zinc oxide. The detoxification effect of glucose on nano zinc oxide shows significant differences at different nano zinc oxide concentrations (p < 0.05), and the detoxification effect on high concentration nano zinc oxide is stronger. When nano zinc oxide and amino acids are present in combination, low concentrations of aspartic acid and glutamic acid show a lower detoxification effect on nano zinc oxide (< 15%), and a certain toxicity enhancement effect is shown at high concentrations. Under the same aspartic acid concentration, the reduction in the phosphorus removal inhibition rate of 2.50 mg / L nano zinc oxide is significantly higher than that of 1.80 mg / L nano zinc oxide, indicating that aspartic acid has a detoxification effect on high concentration nano zinc oxide.

[0026] UV full-wavelength analysis shows that under low-concentration nano-zinc oxide (10 mg / L) conditions, the absorbance of the composite product of glucose and nano-zinc oxide is significantly higher than the sum of the single systems; under high-concentration nano-zinc oxide (50 mg / L) conditions, the absorbance of the composite product is significantly lower than the sum of the single systems. This shows that glucose and nano-zinc oxide interact significantly, thereby achieving detoxification. Infrared spectral analysis verifies the above results. The infrared spectra of glucose and nano-zinc oxide change significantly compared to the single spectra, indicating that the Zn released by glucose and nano-zinc oxide in the water environment 2+ A coordination reaction occurred, and the coordination site was on the C=O of the glucuraldehyde group (-CHO). Compared with the single system, the ultraviolet characteristic peaks of the composite system of aspartic acid or glutamic acid and nano-zinc oxide were found to have a lower ultraviolet absorption at 376nm than the sum of the single system, that is, amino acids can interact with nano-zinc oxide. Infrared spectroscopy analysis verified the above results. The infrared spectrum of amino acids and nano-zinc oxide changed significantly compared with the single spectrum, indicating that the Zn released by amino acids and nano-zinc oxide in the water environment 2+ A coordination reaction occurs, and the coordination sites are on the C=O and OH of the amino acid carboxyl group (-COOH).

Claims

1. The conical flask is equipped with a rubber stopper with a glass tube, which is used to connect the diverter and the nitrogen bottle. The anaerobic environment is maintained by continuously introducing N2. The culture is shaken at 120 rpm and 30°C for 2 h (anaerobic). Then the ventilation device is removed and sealed with a sterile breathable membrane. The culture is shaken at 140 rpm and 30°C for 2 h (aerobic). Samples are taken after 4 h of aerobic culture. After the samples are centrifuged (4°C, 8000 rpm, 5 min), the supernatant is aspirated with a 1 mL sterile syringe and filtered through a 0.22 μm polyethersulfone filter membrane for measuring phosphate concentration. The phosphate test method refers to the national standard GB11893-891 ammonium molybdate spectrophotometry.

2. The phosphorus removal inhibition rate is calculated according to formula (1): The total phosphorus in water samples was measured by molybdenum antimony spectrophotometry. The toxic effect was expressed by the inhibition rate (IR), which was calculated as follows: IR: inhibition rate of phosphorus removal by polyphosphate bacteria in the presence of different concentrations of nZnO, coagulants or organic matter (%); C cnot : Phosphorus removal rate of polyphosphate bacteria in the blank control group (%); C exp : Phosphorus removal rate (%) of polyphosphate bacteria in the presence of different concentrations of nZnO, coagulants or organic matter.

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