Method for evaluating influence of composite stress of humus and nanoparticles in sewage on phosphorus-accumulating bacteria

Abstract

The invention relates to a method for evaluating the influence of composite stress of humus and nano-particles in sewage on phosphorus-accumulating bacteria, and belongs to the field of soil and water environment ecological restoration, the method mainly comprises the following steps: nZnO and humus (fulvic acid and humic acid) exist in a composite manner, the biotoxicity of single nZnO on the phosphorus-accumulating bacteria is relieved, and the phosphorus removal inhibition rate is reduced. Wherein the fulvic acid is added, so that the phosphorus-accumulating bacterium toxicity of the nZnO can be relieved, the fulvic acid has a more obvious detoxification effect on the high-concentration nZnO, and the antagonism rate (detoxification rate) of the fulvic acid on 2.5 mg / L nZnO can reach 86.4% at most. For humic acid, the detoxification effect is higher than that of fulvic acid when the concentration is lower than or equal to 8 mg / L, and the detoxification effect is lower than that of fulvic acid when the concentration is high. Through ultraviolet spectrum and infrared spectrum analysis, the humus and the nZnO have obvious interaction and are compounded to form a coordination compound. The toxicity of the nano-zinc oxide is reduced through regulation and control, and the impact resistance of sewage treatment is improved.

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] In recent years, nano zinc oxide has attracted the attention of all sectors of society due to its high biological toxicity and high detection rate. The negative environmental impact caused by nZnO toxicity affects microbial cells in many ways. They enter cells through broken cell walls and inhibit energy metabolism when NPs are internalized by bacteria. The membrane dysfunction caused by nZnO nanoparticles leads to the internalization of nanoparticles into cells. The internalization of nZnO is usually determined by particle size, defects, surface, functionalization and chemical properties.

[0004] Nitrogen and phosphorus are basic elements that constitute many organisms, but both 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. Certain specific functional microorganisms in activated sludge, such as nitrifying bacteria, denitrifying bacteria, and polyphosphate bacteria, can use metabolic processes to transform and remove nitrogen, phosphorus, etc. in water. The coexistence of a variety of soluble organic matter in sewage has a certain impact on the microbial toxicity and regulatory effect of nanomaterials. Research on the impact of nanoparticles on phosphorus removal in sewage is also an emerging field. (III) Summary of the invention

[0005] The purpose of the present invention is to provide a method for evaluating the effect of combined stress of humus and nanoparticles in sewage on polyphosphate-accumulating bacteria. 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] (IV) Beneficial effects

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] (1) The present invention innovatively studies the effect of humus in dissolved organic matter on the polyphosphate toxicity of nZnO, which helps to regulate and reduce the toxicity of nZnO and improve the impact resistance of sewage treatment.

[0013] (2) The present invention innovatively studies the interaction mechanism between humus and nZnO 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

[0014] Figure 1 is the antagonistic rate of humic acid and nZnO compound on phosphorus removal inhibition, Figure 2 This is the infrared spectrum of the composite system of humic acid (HA) and nZnO. (VI) Specific implementation methods

[0015] The present invention is further described below through specific implementation cases. The embodiments of the present invention are intended to enable those skilled in the art to better understand the present invention, and do not limit the present invention in any way.

[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 and 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-accumulating bacteria in the presence of nZnO and humus. 3- The changes in the ability and the detoxification effect of humus on nZnO.

[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) First, nitrogen was introduced into the reaction bottle for half an hour by connecting the nitrogen bottle and the sealed glass air guide tube through a nitrogen separator. After the air was blown out, the nitrogen was maintained to flow in. The reaction bottle was incubated anaerobically in a water bath shaker at 140 rpm and 30°C for 2 hours in the dark. At this stage, the anaerobic process was simulated, and the polyphosphate bacteria released 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) First, prepare LB solid medium and LB liquid medium, dissolve them 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 them at 121°C for 20 min in an autoclave. After sterilization, pour the plates into solid medium in time, and refrigerate the liquid medium at 4°C for later use.

[0021] (5) Take the bacterial strain stored in the glycerol tube out of the -80°C ultra-low temperature freezer, thaw it quickly, and inoculate the bacterial strain onto a solid plate by the plate streak method.

[0022] (6) 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.

[0023] (7) 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).

[0024] (8) 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.

[0025] (9) 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.

[0026] (10) According to the test results of the present invention, the combined presence of nZnO and humus (fulvic acid, humic acid) alleviates the biological toxicity of single nZnO to polyphosphate bacteria, and the phosphorus removal inhibition rate is reduced. Figure 1 The figure shows the effect of the composite system of humic acid and nZnO on the antagonism rate of phosphorus removal inhibition. Among them, the pink, green and blue bar graphs represent 1.80, 2.25 and 2.50 mg / L nZnO respectively. When the humic acid concentration ranges from 3 to 50 mg / L, for three different concentrations of nZnO, they basically show an antagonistic effect on phosphorus removal inhibition, that is, the addition of humic acid can achieve the detoxification of the phosphorus removal toxicity of nZnO. For 1.80 and 2.25 mg / L nZnO, the phosphorus removal inhibition antagonism rate gradually increases with the addition of humic acid. Under the condition of 50 mg / L humic acid, the composite produces the highest antagonism rate, which is 43.4% and 67.7% respectively. For 2.50 mg / L nZnO, the antagonistic rates produced at 3-20 mg / L humic acid concentrations were significantly different (p < 0.05), which were 23.8%, 40.2%, 48.7%, 52.4% and 65.4%, respectively. Overall, the addition of humic acid is beneficial to alleviate the toxicity of nZnO, thereby promoting phosphorus removal by polyphosphate bacteria, and humic acid has a stronger detoxification effect on high-concentration nZnO. This may be due to the complexation reaction between humic acid and nZnO, thereby reducing the toxicity of nZnO.

[0027] Figure 2The infrared spectrum of the composite system of single humic acid and nZnO is shown. The red line is single humic acid, and the blue line is the infrared spectrum of the composite system of humic acid and nZnO. The free hydroxyl (OH) and hydrogen bonded hydroxyl (OH) stretching vibrations at 3616cm-1 and 3400cm-1 of single humic acid are the stretching vibrations of the hydroxyl (OH) associated with hydrogen bonds, the stretching vibrations of the amino group (-NH-) at 3230cm-1 are the vibration formation of the CH structure and -C=C- in the benzene ring, the stretching vibrations of the aliphatic CH at 2909cm-1 and the deformation vibrations of the aliphatic CH, the secondary stretching vibrations of C=O in the carboxyl group (-COOH) and C=O in the ketone group at 1711cm-1, the vibrations of the aromatic CC skeleton at 1600cm-1, the bending vibrations of the carboxyl anion (COO-) and the CH at the end of the aliphatic group at 1377cm-1, and the vibrations of C=O in -COOH at 1242cm-1. The infrared spectrum of the composite system product was compared with that of single humic acid. It was found that the absorption peak of hydroxyl group OH at 3616cm-1 disappeared, which may be due to the complexation reaction between Zn2+ released by nZnO and -OH of humic acid; the carbonyl group at 1708cm-1 shifted to 1621cm-1, and Zn2+ may have combined with the carbonyl bond (C=O) contained in the carboxyl group, and the absorption peak of carbon-oxygen single bond CO at 1025cm-1 disappeared.

[0028] It should be understood that the embodiments and examples discussed here are for illustrative purposes only and may be modified or altered by those skilled in the art, and all such modifications and alterations should fall within the scope of protection of the appended claims of the present invention.

Claims

1. Activation and cultivation of polyphosphate bacteria: Take out the activated plate, pick up a single colony with an inoculation needle and put it into LB liquid culture medium, seal it with a sterile breathable membrane, place it in a 30°C constant temperature water bath shaker, and culture it at 140rpm for 18h to complete the proliferation and cultivation of polyphosphate bacteria; centrifuge the LB liquid culture at 4°C, 8000rpm, 5min, resuspend it with sterile ultrapure water, adjust the bacterial concentration, and ensure the stability of the inoculated bacteria by measuring OD600 (1.1-1.2).

2. 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.

3. 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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