Method for treating aquaculture wastewater by using compound bacteria

Abstract

The invention discloses a method for treating aquaculture wastewater by using compound bacteria. The method comprises the following steps: putting a compound bacteria agent into the aquaculture wastewater, and stirring; the preparation method of the complex microbial inoculant comprises the following steps: respectively inoculating enterococcus faecalis, pseudomonas pseudoalcaligenes and klebsiella into a liquid culture medium to obtain bacterial solutions of the enterococcus faecalis, the pseudomonas pseudoalcaligenes and the klebsiella; and adding porous carbon and bacterial liquid into the new liquid culture medium, and loading to obtain the complex microbial inoculant. During treatment, loss of enzymes and antibacterial peptides generated by microorganisms can be prevented, so that the removal rate of ammonia nitrogen in the aquaculture wastewater is increased, part of nickel is released in the degradation process, and the activity of the microorganisms is improved; the DL-methionine is mixed with the carbon precursor, so that the nickel species are well dispersed, the specific surface area of the porous carbon is also increased, the content of nitrogen and sulfur elements is increased, the interaction between the porous carbon and microorganisms is enhanced, and the removal rate of ammonia nitrogen is remarkably increased.

Description

Technical Field

[0001] The invention relates to the technical field of wastewater treatment by microorganisms, and in particular to a method for treating aquaculture wastewater by using composite bacteria. Background Art

[0002] With the increasing economic development and urbanization level, while it brings convenience to human life, it also brings some disadvantages. At present, the problem of water pollution is becoming more and more serious with the development of the economy. The red tide and other environmental pollution problems caused by the discharge of sewage that cannot be effectively treated also seriously endanger people's daily life. Reasonable and effective solution to water pollution can not only effectively alleviate the problem of environmental pollution, but also solve the current problem of water shortage to a certain extent. Existing sewage treatment mainly relies on sewage treatment facilities such as sewage treatment plants. Seeking suitable methods to improve the treatment effect of sewage treatment plants is one of the most economical and effective ways to solve water pollution.

[0003] The immobilized microorganism method is developed from the immobilized enzyme method. It is a biological method that fixes free microorganisms to a carrier, limits them to a specific space, and the microorganisms rely on the carrier to grow and maintain activity, and reproduce rapidly and in large quantities under suitable conditions. Compared with traditional sewage treatment technology, the immobilized microorganism technology can separate the fixed microorganisms from the sewage, so that the microorganisms maintain a certain concentration and activity and can be used repeatedly. CN114317516A discloses a composite bacterial agent for wastewater treatment and a preparation method thereof. The composite bacterial agent includes Shewanella putrefaciens, Pseudomonas pseudoalcaligenes and modified zeolite. By introducing chitosan to the zeolite, the adsorption performance of the zeolite is improved, and then a composite modification is performed to introduce iron phosphate to the zeolite. Iron ions can be used as coagulants, and chitosan synergistically acts to flocculate and settle ammonia nitrogen substances in sewage, greatly improving the biological nitrification performance of the composite bacterial agent. However, the preparation method of the modified zeolite in this bacterial agent needs to be acid treated, calcined, chitosan cross-linked and adsorbed iron ions, and the steps are lengthy and complicated. CN115838717A discloses a white rot fungus-biochar composite bacterial pellet and its preparation method and application. After preparing biochar, a nutrient solution, a white rot fungus suspension and a sodium alginate aqueous solution are added, and the obtained mixed solution is shaken and freeze-dried to obtain a white rot fungus-biochar composite bacterial pellet. This method has the advantages of simple process, easy recovery of bacterial pellets, and high treatment capacity for heavy metals. However, the bacterial pellets mainly utilize adsorption, and additional treatment is required after the bacterial pellets are collected.

[0004] It is still necessary to provide composite bacteria with simple preparation method, strong processing capacity and not easy to lose. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a method for treating aquaculture wastewater using composite bacteria, comprising:

[0006] Adding the composite bacterial agent into the aquaculture wastewater for stirring treatment;

[0007] The preparation method of the composite bacterial agent comprises:

[0008] Enterococcus faecalis, Pseudomonas pseudoalcaligenes, and Klebsiella are inoculated into liquid culture medium respectively to obtain bacterial liquids of Enterococcus faecalis, Pseudomonas pseudoalcaligenes, and Klebsiella;

[0009] Porous carbon and bacterial liquids of Enterococcus faecalis, Pseudomonas pseudoalcaligenes and Klebsiella are added into a new liquid culture medium to load a composite bacterial agent.

[0010] Compared with a single bacterial species, the joint action of multiple microorganisms can often achieve a more ideal effect. The microorganisms selected in the present invention are Enterococcus faecalis, Pseudomonas pseudoalcaligenes, and Klebsiella. Enterococcus faecalis is a facultative anaerobic Gram-positive lactic acid bacterium with a spherical or chain-shaped bacterial body, a small diameter, no capsule, no spores, strong adaptability and resistance to the environment, and no strict requirements for growth conditions; Pseudomonas pseudoalcaligenes is a Gram-negative bacterium that moves with a polar single flagellum. The colony is round, with neat edges, raised, smooth and moist surfaces. Its optimal growth temperature is 35°C, and it is obligately aerobic; Klebsiella is a Gram-negative bacterium, a relatively short and thick rod, arranged alone, in pairs or in short chains, without spores, without flagella, with a thicker capsule, and most have pili.

[0011] The material used for the immobilized microorganisms of the present invention is nickel-doped porous carbon prepared from biomass, which has stable physical and chemical properties, and the microorganisms can be well immobilized thereon. During the treatment, the loss of enzymes and antimicrobial peptides produced by the microorganisms can be prevented, thereby improving the degradation rate of ammonia nitrogen in the aquaculture wastewater. Part of the nickel is released during the degradation process, which improves the activity of the microorganisms. The mixing of DL-methionine and the carbon precursor allows the nickel species to be well dispersed, and also improves the specific surface area of ​​the porous carbon, increases the content of nitrogen and sulfur elements, and enhances the interaction between the porous carbon and the microorganisms.

[0012] Furthermore, the method for preparing the porous carbon comprises:

[0013] The biomass was immersed in a nickel salt solution for adsorption, and the insoluble matter was subsequently collected;

[0014] The insoluble matter is mixed with DL-methionine and then calcined under a protective gas atmosphere to obtain porous carbon.

[0015] In the present invention, the type of biomass does not need to be strictly limited, and can be straw, bamboo, bamboo leaves, tree leaves, etc. After the biomass is collected, it needs to be washed, dried and crushed and then passed through a 300-mesh sieve.

[0016] Furthermore, the mass volume ratio of the biomass to the nickel salt solution is 10-20 g: 100-150 mL;

[0017] The mass ratio of the insoluble matter to DL-methionine is 5-15:1-3.

[0018] More preferably, when the insoluble matter is mixed with DL-methionine, di-o-tolylthiourea in an amount of 0.05 to 0.3 times the mass of the insoluble matter is also added.

[0019] Furthermore, the concentration of the nickel salt solution is 0.05-0.1 mol / L.

[0020] Furthermore, the number of viable bacteria in the bacterial suspensions of Enterococcus faecalis, Pseudomonas pseudoalcaligenes, and Klebsiella pneumoniae was greater than 1.0×10 8 cfu / mL.

[0021] Furthermore, the mass volume ratio of the porous carbon to the new liquid culture medium is 0.2-1 g:1L.

[0022] Furthermore, the volume ratio of the bacterial liquid of Enterococcus faecalis, Pseudomonas pseudoalcaligenes and Klebsiella to the new liquid culture medium is 2-5:3-10:2-5:2000-5000.

[0023] Furthermore, the liquid culture medium is LB culture medium. The components of LB culture medium are: 10g peptone, 5g yeast extract, 5g NaCl, and 1L distilled water.

[0024] Furthermore, the composite bacterial agent is added into the aquaculture wastewater at a concentration of 1 to 10 g / L.

[0025] Furthermore, the stirring treatment is continued for 3 to 7 days at a temperature of 25 to 35° C. and a rotation speed of 150 to 300 rpm.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention uses Enterococcus faecalis, Pseudomonas pseudoalcaligenes and Klebsiella as active bacteria and uses nickel-doped porous carbon for immobilization. During the treatment, the loss of enzymes and antimicrobial peptides produced by the microorganisms can be prevented, thereby improving the removal rate of ammonia nitrogen in the aquaculture wastewater. Part of the nickel is released during the degradation process, which improves the activity of the microorganisms. The mixing of DL-methionine and the carbon precursor allows the nickel species to be well dispersed, and also increases the specific surface area of ​​the porous carbon, increases the content of nitrogen and sulfur elements, enhances the interaction between the porous carbon and the microorganisms, and significantly improves the removal rate of ammonia nitrogen. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 A flow chart showing the method for treating aquaculture wastewater using composite bacteria of the present invention;

[0030] Figure 2 shows a scanning electron microscope image of the porous carbon prepared in Example 1;

[0031] Figure 3 shows a scanning electron microscope image of the porous carbon prepared in Example 2;

[0032] Figure 4 shows a scanning electron microscope image of the porous carbon prepared in Example 3;

[0033] Figure 5 shows a scanning electron microscope image of the porous carbon prepared in Example 4;

[0034] Figure 6 The scanning electron microscope image of the porous carbon prepared in Comparative Example 3 is shown. DETAILED DESCRIPTION

[0035] The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in the present invention.

[0036] Some of the raw materials used in the examples and comparative examples of the present invention are described as follows:

[0037] Enterococcus faecalis, strain number CICC 25019, Klebsiella, strain number CICC 10870, were purchased from China Industrial Microbiological Culture Collection Administration Center; Pseudomonas pseudoalcaligenes, strain number CGMCC 1.10611, were purchased from China General Microbiological Culture Collection Administration Center;

[0038] The biomass was waste bamboo stalks and scraps collected from a timber factory. The waste bamboo stalks and scraps were washed with water and dried in a constant temperature oven at 120°C for 10 hours, and then crushed with a grinder and passed through a 300-mesh sieve to obtain 300-mesh biomass powder.

[0039] Other raw materials not mentioned are common raw materials in the art. The above content is only to help illustrate the present invention and should not be construed as a strict limitation of the present invention. Those skilled in the art can directly purchase or prepare the same / similar raw materials from the market. These contents will not be repeated in the examples.

[0040] The following will be combined with the specific embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Example 1

[0042] like Figure 1 As shown, a method for treating aquaculture wastewater using composite bacteria, the steps are as follows:

[0043] S1. Prepare LB medium according to the ratio of 10 g peptone, 5 g yeast extract, 5 g NaCl, and 1 L distilled water;

[0044] S2. Enterococcus faecalis, Pseudomonas pseudoalcaligenes, and Klebsiella were inoculated into LB medium at a volume ratio of 10%, transferred to a shaker, and cultured at a temperature of 25°C and a shaking speed of 150 rpm. The viable counts were 1.0×10 9 cfu / mL of Enterococcus faecalis, Pseudomonas pseudoalcaligenes, and Klebsiella spp.

[0045] S3, add 3g of porous carbon, 5mL of Enterococcus faecalis, 10mL of Pseudomonas pseudoalcaligenes, and 5mL of Klebsiella into 5L of new LB medium, transfer to a shaker, culture at 30°C and 150rpm for 24h to fully load, and then freeze-dry to obtain a composite bacterial agent;

[0046] S4. Add the composite bacterial agent at a concentration of 6 g / L into the simulated aquaculture wastewater at a temperature of 32°C and a rotation speed of 220 rpm for 5 days. The simulated aquaculture wastewater contains 260 mg / L ammonia nitrogen, 15 mg / L nitrate nitrogen, and 8 mg / L total phosphorus.

[0047] The method for preparing porous carbon is as follows:

[0048] T1. Soak 15 g of biomass in 120 mL of 0.05 mol / L nickel chloride hexahydrate aqueous solution for 12 h for adsorption, filter and dry in a 120 °C constant temperature oven for 10 h to obtain a carbon precursor;

[0049] T2. After mixing 80 g of carbon precursor with 24 g of DL-methionine, transfer the mixture to a nitrogen atmosphere tube furnace, heat the mixture to 700 °C at a heating rate of 3 °C / min and keep the mixture for 1.5 h. After natural cooling, wash the black powder with water and ethanol three times each, filter it and transfer it to a 120 °C constant temperature oven for drying for 10 h to obtain porous carbon.

[0050] Example 2

[0051] A method for treating aquaculture wastewater using composite bacteria is basically the same as Example 1, except that in step T1 of the method for preparing porous carbon, the biomass is immersed in an aqueous solution of 0.075 mol / L nickel chloride hexahydrate.

[0052] Example 3

[0053] A method for treating aquaculture wastewater using composite bacteria is basically the same as Example 1, except that in step T1 of the method for preparing porous carbon, the biomass is immersed in a 0.1 mol / L aqueous solution of nickel chloride hexahydrate.

[0054] Example 4

[0055] A method for treating aquaculture wastewater using composite bacteria, the steps are as follows:

[0056] S1. Prepare LB medium according to the ratio of 10 g peptone, 5 g yeast extract, 5 g NaCl, and 1 L distilled water;

[0057] S2. Enterococcus faecalis, Pseudomonas pseudoalcaligenes, and Klebsiella were inoculated into LB medium at a volume ratio of 10%, transferred to a shaker, and cultured at a temperature of 25°C and a shaking speed of 150 rpm. The viable counts were 1.0×10 9 cfu / mL of Enterococcus faecalis, Pseudomonas pseudoalcaligenes, and Klebsiella spp.

[0058] S3, add 3g of porous carbon, 5mL of Enterococcus faecalis, 10mL of Pseudomonas pseudoalcaligenes, and 5mL of Klebsiella into 5L of new LB medium, transfer to a shaker, culture at 30°C and 150rpm for 24h to fully load, and then freeze-dry to obtain a composite bacterial agent;

[0059] S4. Add the composite bacterial agent at a concentration of 6 g / L into the simulated aquaculture wastewater at a temperature of 32°C and a rotation speed of 220 rpm for 5 days. The simulated aquaculture wastewater contains 260 mg / L ammonia nitrogen, 15 mg / L nitrate nitrogen, and 8 mg / L total phosphorus.

[0060] The method for preparing porous carbon is as follows:

[0061] T1. Soak 15 g of biomass in 120 mL of 0.075 mol / L nickel chloride hexahydrate aqueous solution for 12 h for adsorption, filter and dry in a 120 °C constant temperature oven for 10 h to obtain a carbon precursor;

[0062] T2. Mix 80g of carbon precursor, 15g of DL-methionine and 8g of di-o-tolylthiourea, transfer to a nitrogen atmosphere tubular furnace, heat to 700℃ at a heating rate of 3℃ / min and keep warm for 1.5h. After natural cooling, wash the black powder with water and ethanol three times each, filter and transfer to a 120℃ constant temperature oven for drying for 10h to obtain porous carbon.

[0063] Comparative Example 1

[0064] A method for treating aquaculture wastewater using composite bacteria is basically the same as Example 1, except that in step T1 of the method for preparing porous carbon, the biomass is immersed in a 0.15 mol / L aqueous solution of nickel chloride hexahydrate.

[0065] Comparative Example 2

[0066] A method for treating aquaculture wastewater using composite bacteria is basically the same as Example 2, except that DL-methionine is not introduced in step T2 of the method for preparing porous carbon.

[0067] Comparative Example 3

[0068] A method for treating aquaculture wastewater using composite bacteria, the steps are as follows:

[0069] S1. Prepare LB medium according to the ratio of 10 g peptone, 5 g yeast extract, 5 g NaCl, and 1 L distilled water;

[0070] S2. Enterococcus faecalis, Pseudomonas pseudoalcaligenes, and Klebsiella were inoculated into LB medium at a volume ratio of 10%, transferred to a shaker, and cultured at a temperature of 25°C and a shaking speed of 150 rpm. The viable counts were 1.0×10 9 cfu / mL of Enterococcus faecalis, Pseudomonas pseudoalcaligenes, and Klebsiella spp.

[0071] S3, add 3g of porous carbon, 5mL of Enterococcus faecalis, 10mL of Pseudomonas pseudoalcaligenes, and 5mL of Klebsiella into 5L of new LB medium, transfer to a shaker, culture at 30°C and 150rpm for 24h to fully load, and then freeze-dry to obtain a composite bacterial agent;

[0072] S4. Add the composite bacterial agent at a concentration of 6 g / L into the simulated aquaculture wastewater at a temperature of 32°C and a rotation speed of 220 rpm for 5 days. The simulated aquaculture wastewater contains 260 mg / L ammonia nitrogen, 15 mg / L nitrate nitrogen, and 8 mg / L total phosphorus.

[0073] The method for preparing porous carbon is as follows:

[0074] After mixing 80 g of biomass with 24 g of DL-methionine, the mixture was transferred to a nitrogen atmosphere tube furnace, heated to 700 °C at a heating rate of 3 °C / min and kept warm for 1.5 h. After natural cooling, the black powder was washed three times with water and ethanol respectively, filtered and transferred to a 120 °C constant temperature oven for drying for 10 h to obtain porous carbon.

[0075] Comparative Example 4

[0076] The method is basically the same as Example 4, except that in step T2 of the method for preparing porous carbon, 80 g of carbon precursor and 24 g of di-o-tolylthiourea are mixed and then calcined.

[0077] Test Case

[0078] The microscopic morphology of the porous carbon materials of Examples 1 to 4 and Comparative Example 3 was observed using a scanning electron microscope. Figure 2 to Figure 6 As shown. Figure 6 It can be seen that the porous carbon prepared without immersion in nickel salt solution presents a lamellar structure with a large number of wrinkles on the surface. Such a structure is conducive to the enrichment of microorganisms on its surface. Figure 2 and Figure 4 It can be seen that the nickel species anchored on the porous carbon appear larger particles regardless of the concentration of nickel chloride aqueous solution. Figure 3 It can be seen that the appropriate nickel concentration can regulate the size of nickel species. Figures 2 to 4 The nickel species are concentrated in the wrinkles. In Example 4, di-o-tolylthiourea was added together with DL-methionine. Figure 5 As shown, nickel species are well dispersed on the surface of porous carbon without obvious agglomeration, and such a structure may be conducive to the interaction with microorganisms.

[0079] The specific surface area of ​​porous carbon was tested by nitrogen adsorption and desorption. The results are shown in Table 1.

[0080] Table 1 Specific surface area of ​​porous carbon

[0081]

[0082]

[0083] From the test results in Table 1, it can be seen that the specific surface area of ​​the porous carbon in Comparative Example 3 is the lowest, which is calcined after mixing biomass with DL-methionine. In other embodiments, nickel salts are soaked, and the nickel salts will decompose during calcination, and the formed carbon will be etched, thereby increasing the pore size. In Examples 1 to 3, the concentration of nickel salts gradually increases, and the specific surface area decreases successively, because the generated nickel species blocks the pore size; Compared with Example 2, Comparative Example 2 does not add DL-methionine, and the specific surface area also decreases significantly, indicating that the addition of DL-methionine enhances the etching effect. Example 4 has the largest specific surface area, which shows that DL-methionine and di-o-tolylthiourea enhance the dispersibility of nickel species on the porous carbon surface, thereby further increasing the specific surface area.

[0084] The results of the removal rates of pollutants in the simulated aquaculture wastewater by the embodiments and comparative examples are shown in Table 2.

[0085] Table 2 Results of removal rate of pollutants in simulated aquaculture wastewater

[0086]

[0087] It can be seen from the test results in Table 2 that the embodiments of the present invention have good removal capabilities for ammonia nitrogen and nitrate nitrogen. Except for Example 4, which has good removal of phosphorus, the removal effects of other embodiments are not ideal. In the nickel-containing group, the removal rate of Comparative Example 1 is poor, which may be due to the higher nickel content, which makes the dissolved nickel ions larger, but inhibits the activity of microorganisms. This is because the larger specific surface area and good nickel species dispersion enhance the effect of porous carbon and microorganisms, making it difficult for the decomposition enzymes and antimicrobial peptides produced by microorganisms to be lost. Part of the nickel is released during the degradation process, which improves the activity of microorganisms. Therefore, Example 4 has good removal rates for ammonia nitrogen, nitrate nitrogen and phosphorus.

[0088] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for treating aquaculture wastewater using composite bacteria, characterized in that: include, Adding the composite bacterial agent into the aquaculture wastewater for stirring treatment; The preparation method of the composite bacterial agent comprises: Enterococcus faecalis, Pseudomonas pseudoalcaligenes, and Klebsiella are inoculated into liquid culture medium respectively to obtain bacterial liquids of Enterococcus faecalis, Pseudomonas pseudoalcaligenes, and Klebsiella; Porous carbon and bacterial liquids of Enterococcus faecalis, Pseudomonas pseudoalcaligenes and Klebsiella are added into a new liquid culture medium to load a composite bacterial agent.

2. The method for treating aquaculture wastewater using composite bacteria according to claim 1, characterized in that: The method for preparing the porous carbon comprises: The biomass was immersed in a nickel salt solution for adsorption, and the insoluble matter was subsequently collected; The insoluble matter is mixed with DL-methionine and then calcined under a protective gas atmosphere to obtain porous carbon.

3. The method for treating aquaculture wastewater using composite bacteria according to claim 2, characterized in that: The mass volume ratio of the biomass to the nickel salt solution is 10-20 g: 100-150 mL; The mass ratio of the insoluble matter to DL-methionine is 5-15:1-3.

4. The method for treating aquaculture wastewater using composite bacteria according to claim 2, characterized in that: The concentration of the nickel salt solution is 0.05-0.1 mol / L.

5. The method for treating aquaculture wastewater using composite bacteria according to claim 1, characterized in that: The number of viable bacteria in the bacterial suspension of Enterococcus faecalis, Pseudomonas pseudoalcaligenes, and Klebsiella was greater than 1.0×10 8 cfu / mL.

6. The method for treating aquaculture wastewater using composite bacteria according to claim 1, characterized in that: The mass volume ratio of the porous carbon to the new liquid culture medium is 0.2-1 g:1L.

7. The method for treating aquaculture wastewater using composite bacteria according to claim 1, characterized in that: The volume ratio of the bacterial liquid of Enterococcus faecalis, Pseudomonas pseudoalcaligenes and Klebsiella to the new liquid culture medium is 2-5:3-10:2-5:2000-5000.

8. The method for treating aquaculture wastewater using composite bacteria according to claim 1, characterized in that: The liquid culture medium is LB culture medium.

9. The method for treating aquaculture wastewater using composite bacteria according to claim 1, characterized in that: The composite bacterial agent is added into the aquaculture wastewater at a concentration of 1-10 g / L.

10. The method for treating aquaculture wastewater using composite bacteria according to claim 1, characterized in that: The stirring treatment is continued for 3 to 7 days at a temperature of 25 to 35° C. and a rotation speed of 150 to 300 rpm.

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