Method for evaluating influence of humus on toxicity of phosphorus-accumulating bacteria of nano-materials in sewage

Abstract

The invention relates to a method for evaluating the composite stress toxicity of amino acid and a nano material in sewage biochemical treatment by taking phosphorus-accumulating bacteria as model microorganisms, and belongs to the field of emerging pollutant toxicity in sewage treatment. The method mainly comprises the following steps: constructing an artificial simulation A / O process device, simulating a sewage system in a sewage treatment plant, and calculating the composite stress toxicity of the amino acid and the nano material; comprising the following steps: preparing a trace element solution, artificial sewage, an nZnO suspension, an aspartic acid solution and a concentrated phosphorus-accumulating bacteria solution, and calculating the phosphorus removal rate and the phosphorus removal inhibition rate of the phosphorus-accumulating bacteria to evaluate the antagonism of amino acid in the sewage on a new pollutant nZnO in a microbial water pollution treatment process.

Description

(I) Technical field

[0001] The invention belongs to the field of toxicity of emerging pollutants in sewage treatment, and in particular relates to a method for evaluating the composite stress toxicity of amino acids and nanomaterials in sewage biochemical treatment using polyphosphate bacteria as model microorganisms. (II) Background technology

[0002] Nitrogen and phosphorus are essential elements for many plants and animals, but they are also common pollutants in the environment. For water bodies, the most serious problem is that excessive nitrogen and phosphorus lead to eutrophication of water bodies, aquatic plants and algae multiply, resulting in decreased water transparency, reduced dissolved oxygen, changes in water quality, and mass deaths of fish and other organisms, thereby destroying the stability of the aquatic ecosystem and even causing the entire aquatic ecosystem to be on the verge of extinction. The activated sludge process is a general term for various methods of biological treatment of wastewater with microorganisms suspended in water. The activated sludge process can effectively remove nitrogen and phosphorus nutrients in sewage. Polyphosphate bacteria can transform and remove phosphorus in water through their own metabolic processes and are one of the very important microorganisms in the activated sludge process.

[0003] Metal nanomaterials include nano-metal elements and nano-metal oxides, which can achieve antibacterial functions in medicine, play a conductive role in the electronics and energy fields, and can also play a role in environmental governance such as nano-zero-valent iron. With the use, loss and abandonment of various nanomaterials, these nanomaterials will enter the atmospheric environment, soil environment and water environment. At present, a large number of studies have reported the detection of various metal nanomaterials in environmental water bodies, including nano-silver (Ag), nano-zinc oxide (ZnO) and nano-titanium dioxide (TiO2). These nanomaterials can produce reactive oxygen species (ROS) to cause cellular oxidative damage to microorganisms such as polyphosphate bacteria.

[0004] Dissolved organic matter (DOM) is mainly derived from the slow microbial decomposition of animal remains, plants and microorganisms, and is ubiquitous in the natural environment. The concentration of DOM in aquatic ecosystems depends on biogeochemical conditions and climate, but is generally between 0.1 and 10 mg / LDOC (dissolved organic carbon). It is not a single compound, but a collection of abundant substances with low molecular weight (MW, less than 2,000 Da) and high molecular weight (up to 10,000 Da), and contains many functional groups such as thiols, phenol-OH, quinones, aldehydes, ketones, carboxyls and methoxyls. Amino acids, as a type of DOM, are very important for biodegradation activity in some cases. When glutamic acid and aspartic acid are dosed in the EBPR system, 3.10 mg P.gVSS is produced. -1 and 6.81mgP.gVSS -1 The amount of phosphorus released affects the phosphorus removal efficiency of polyphosphate bacteria in sewage biochemical treatment.

[0005] Dissolved organic matter (DOM) is commonly found in water environments and usually contains a variety of complex organic compounds, including amino acids, humic acid, polysaccharides, lipids, etc., which can interact with nanoparticles, change their surface reactivity, colloidal stability, cellular uptake capacity and bioaccumulation, which will greatly affect the final fate, transport, transformation and biological effects of the nanoparticles. Therefore, it is important to understand the effects of amino acids on the polyphosphate toxicity of nanomaterials. The present invention selects aspartic acid and provides a method for evaluating the effects of amino acids on the polyphosphate toxicity of nZnO in sewage, which has significant practical application significance for exploring the toxic effects of emerging pollutants in sewage treatment. (III) Summary of the invention

[0006] The purpose of the present invention is to provide a method for evaluating the toxic effects of amino acids on polyphosphate-accumulating bacteria of nanomaterials in sewage, so as to provide assistance for exploring the toxic effects of emerging pollutants in sewage treatment.

[0007] The technical solution of the present invention is:

[0008] A method for evaluating the effect of amino acids on the toxicity of nZnO to polyphosphate bacteria in sewage, comprising the following steps:

[0009] (1) Build an artificial simulation A / O process device;

[0010] (2) Simulating the sewage system in a sewage treatment plant, including the configuration of trace element solution, artificial sewage, nZnO suspension, and aspartic acid solution;

[0011] (3) Preparation of concentrated polyphosphate bacteria solution;

[0012] (4) Artificial simulation of A / O process: Add different concentrations of nZnO suspension, aspartic acid solution, artificial sewage, concentrated bacterial solution and deionized water into a conical flask, and then put it into a water bath shaker at 140 rpm and 30°C. First, introduce nitrogen (N2) into the reaction flask to ensure an anaerobic environment. After anaerobic culture for 2 hours, stop introducing N2, seal the conical flask with a sterile breathable membrane, and incubate the conical flask in a water bath shaker at 140 rpm and 30°C for 4 hours in the dark. Sampling is then performed after the end.

[0013] (5) After the sample is centrifuged (4°C, 8000 rpm, 5 min), the supernatant is drawn 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. The total phosphorus in the water sample is measured using the molybdenum antimony spectrophotometry.

[0014] (IV) Beneficial effects

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention evaluates the antagonistic effect of amino acids in sewage on the emerging pollutant nZnO in the process of microbial water pollution treatment by calculating the phosphorus removal rate and phosphorus removal inhibition rate of polyphosphate bacteria. Currently, there are few related schemes studied, which have practical application value. (V) Description of the drawings

[0017] Figure 1 Effect of different concentrations of aspartic acid on the inhibition of phosphorus removal by nZnO. (VI) Specific implementation methods

[0018] The present invention is further described below through specific implementation examples. The implementation examples of the present invention are intended to enable those skilled in the art to better understand the present invention, and do not impose any limitation on the present invention.

[0019] In this implementation case, a polyphosphate-accumulating bacteria isolated from the enhanced biological phosphorus removal activated sludge system (EBPR) was selected as the research object, and nZnO was selected as a typical nanometal oxide to evaluate the effect of the combined stress of amino acids and emerging pollutants on polyphosphate-accumulating bacteria.

[0020] (1) Build a simulated sewage treatment plant A / O process system;

[0021] (2) Simulate the sewage system in the sewage treatment plant; including the preparation of trace element solution, artificial sewage, aspartic acid solution, nZnO suspension and concentrated polyphosphate bacteria liquid. The polyphosphate bacteria liquid culture was centrifuged at 4°C, 8000rpm, 5min, resuspended with sterile ultrapure water, and the concentration of the liquid was adjusted. The stability of the inoculated bacteria was ensured by measuring OD600 (1.1-1.2).

[0022] (3) After the simulation system was built, 12 conical flasks were taken, and 12 mL of bacterial solution, 5 mL of artificial sewage, and pollutants and aspartic acid of different concentrations were added to the conical flasks. The same volume of ultrapure water was added to the control group, and the total volume of the system was finally maintained at 120 mL. In the experiment, each treatment was repeated twice in parallel.

[0023] (4) 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 2 hours 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 4 hours 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 total phosphorus in the water sample is measured using the national standard GB11893-891 ammonium molybdate spectrophotometry method.

[0024] (5) The phosphorus removal rate and phosphorus removal inhibition rate of polyphosphate bacteria were calculated according to formula (1) and formula (2) respectively.

[0025]

[0026] The calculation formula of phosphorus removal inhibition rate of polyphosphate bacteria is:

[0027]

[0028] IR: inhibition rate of phosphorus removal by polyphosphate bacteria in the presence of different concentrations of nZnO and aspartic acid (%);

[0029] Ccnot: phosphorus removal rate of polyphosphate bacteria in the blank control group (%);

[0030] Cexp: phosphorus removal rate (%) of polyphosphate bacteria in the presence of different concentrations of nZnO and aspartic acid.

[0031] (8) According to the evaluation scheme of the present invention, under the pollution condition of nZnO, the phosphorus removal rate of polyphosphate bacteria will be inhibited to varying degrees, and aspartic acid will affect the inhibitory efficiency of nZnO on the phosphorus removal of polyphosphate bacteria. This result is consistent with the literature records, proving that it is feasible to use polyphosphate bacteria as a model microorganism to evaluate the combined toxicity of amino acids and nanomaterials, and proving the scientific nature of this method.

[0032] Most importantly, when nZnO and aspartic acid coexist, low concentrations of aspartic acid show a lower detoxification effect on nZnO (<15%), and high concentrations show a certain toxicity enhancement effect. Under the same aspartic acid concentration, the reduction in phosphorus removal inhibition rate of 2.50 mg / L nZnO is significantly higher than that of 1.80 mg / L nZnO, indicating that aspartic acid has a detoxification effect on high concentrations of nZnO.

[0033] 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. A method for evaluating the toxicity of amino acids to polyphosphate-accumulating bacteria of nanomaterials in sewage, characterized in that The following steps are involved: 1) Build a simulated sewage treatment plant A / O process system; 2) preparing the solution required for the simulated sewage system; The method includes the preparation of trace element solution, artificial sewage, aspartic acid solution, nZnO suspension and concentrated polyphosphate bacteria liquid. The polyphosphate bacteria liquid culture liquid is centrifuged at 4°C, 8000rpm, 5min, resuspended with sterilized ultrapure water, the concentration of the liquid is adjusted, and the stability of the inoculated bacteria amount is ensured by measuring OD600 (1.1-1.2). 3) Simulate the sewage system in the sewage treatment plant; after the simulation system is built, take 12 conical flasks, add 12mL of bacterial solution, 5mL of artificial sewage, and pollutants and aspartic acid of different concentrations into the conical flasks, and add the same volume of ultrapure water to the control group, and finally keep the total volume of the system at 120mL. In the experiment, each treatment was repeated twice in parallel. 6) Conduct experiments with different concentrations of aspartic acid and nZnO: The conical flask is equipped with a rubber stopper with a glass tube 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 (anaerobic). Then remove the ventilation device, seal it with a sterile breathable membrane, and culture it at 140rpm and 30℃ for 2h (aerobic). Samples are taken after 4h of aerobic culture. After centrifugation (4℃, 8000rpm, 5min), the supernatant is drawn with a 1mL sterile syringe to pass through a 0.22μm polyethersulfone filter membrane for measuring phosphate concentration. The total phosphorus in the water sample is measured using the national standard GB11893-891 ammonium molybdate spectrophotometry. 7) The phosphorus removal rate and phosphorus removal inhibition rate of polyphosphate bacteria were calculated according to formula (1) and formula (2) respectively. The calculation formula of phosphorus removal inhibition rate of polyphosphate bacteria is: IR: inhibition rate of phosphorus removal by polyphosphate bacteria in the presence of different concentrations of nZnO and aspartic acid (%); Ccnot: phosphorus removal rate of polyphosphate bacteria in the blank control group (%); Cexp: phosphorus removal rate (%) of polyphosphate bacteria in the presence of different concentrations of nZnO and aspartic acid.

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