Method for treating acid manganese-containing wastewater by free microalgae and synergistically immobilized manganese-oxidizing bacteria

Abstract

The application relates to a method for treating acid manganese-containing wastewater by free microalgae and cooperatively immobilized manganese-oxidizing bacteria, comprising the following steps: (1) adding microalgae into an acid manganese-containing solution and domesticating and culturing for 4 days; (2) preparing a sodium alginate solution with a mass fraction of 2-4%, stirring uniformly manganese-oxidizing bacteria liquid into the sodium alginate solution, preparing mixed bacteria liquid by taking the volume ratio of the manganese-oxidizing bacteria liquid to the sodium alginate solution as 1:2.5-1:4.0, slowly dripping the mixed bacteria liquid into a CaCl2 solution with a mass fraction of 2-3% to prepare microspheres, taking out the microspheres after hardening at room temperature, and washing the microspheres with deionized water for 3-5 times to prepare immobilized particles; (3) adding the immobilized particles prepared in the step two into a dynamic test reactor, then adding the domesticated microalgae to form a bacteria-algae symbiotic system; adding manganese-containing wastewater into the dynamic test reactor, operating in a room temperature environment, and regularly adding bacteria and algae nutrients into the reactor.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of wastewater biological treatment, and particularly relates to a method for treating acid manganese-containing wastewater by using free microalgae in cooperation with immobilized manganese-oxidizing bacteria. The present application is a continuation application of the application No. CN202210864900.5 filed on July 21, 2022, and claims priority thereto, the entire contents of which are hereby incorporated by reference. BACKGROUND

[0002] Manganese is an important non-ferrous metal mineral resource, and plays an important role in the development of national economy. China is rich in manganese resources, ranking fourth in the world, and most of them are distributed in the southern region. The extensive application leads to a large amount of manganese-containing wastewater generated in the process of mining, smelting and processing. Most of the manganese-containing wastewater is acidic, and manganese exists in the form of Mn 2+ ion. If the manganese-containing wastewater is not properly treated, the manganese in the polluted water body and soil can be enriched in the human body through the food chain, causing biochemical changes and necrosis of organs such as liver, and also leading to many birth defects and the occurrence of malignant tumors. Therefore, it is crucial to develop a cheap, efficient and rapid method for removing Mn 2+ from wastewater to improve the water environment quality in China.

[0003] At present, it has been reported in the literature that microorganisms can oxidize Mn 2+ in solution to high-valence manganese, producing manganese oxide precipitate, thereby removing Mn 2+ from water body. Compared with adsorption method and chemical precipitation method, the biological oxidation manganese removal method has the characteristics of low cost and no secondary pollution, but the optimum pH of most manganese-oxidizing bacteria is neutral, and the acid environment will affect their Mn 2+ oxidation activity. Our research group has screened an acid-tolerant manganese-oxidizing bacteria Klebsiella sp. M3 from the environment, which has been preserved in China Center for Type Culture Collection on March 24, 2021, with the preservation number of CCTCC NO: M 2021261. The strain can grow in acidic conditions and oxidize high-concentration Mn 2+ ion to produce manganese oxide precipitate, but compared with neutral conditions, the strain still has poor removal capacity and low rate for Mn 2+ in acidic water body, and the greater the solution acidity, the worse the removal effect. In addition, the microbial oxidation manganese removal process is an aerobic process, and if the concentration of dissolved oxygen in wastewater can be improved, it will greatly benefit the biological oxidation manganese removal.

[0004] Microalgae are common organisms in acidic aquatic environments. Through photosynthesis, they can raise the pH of water and release oxygen, which can act as an electron acceptor for aerobic bacteria to degrade pollutants. Simultaneously, the carbon dioxide produced by bacteria can also be used for photosynthesis by microalgae. Current literature reports on algal-bacterial symbiotic systems for treating nitrogen, phosphorus, and organic pollutants in wastewater, achieving satisfactory results through the synergistic effect of bacteria and algae. Patent CN202111084994.6 discloses a method for enhancing the manganese oxidation function of bacteria using algae, introducing algae into wastewater containing low concentrations of Mn... 2+ Adding manganese-oxidizing bacteria (Pseudomonas putida QJX-1) and algae (Microcystis aeruginosa) to a solution containing diclofenac ions or algae, or adding the supernatant from a co-culture of bacteria and algae, revealed that the exudates produced during the coexistence of algae and bacteria could enhance the biological manganese oxidation process. However, the research group found that directly contacting the screened acid-tolerant manganese-oxidizing bacteria with microalgae for the treatment of manganese-containing wastewater was not conducive to the microbial oxidation and removal of Mn. 2+ On the contrary, Klebsiella sp. M3 The oxidation activity of Mn in acidic wastewater was inhibited. Therefore, how to utilize microalgae in synergy with manganese-oxidizing bacteria to improve the oxidation activity of Mn in acidic wastewater is a key issue. 2+ Its removal capacity accelerates the biological oxidation removal of high concentrations of Mn. 2+ The efficiency of ion exchange is key to the treatment of manganese-containing wastewater by biological oxidation. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned technical problems and provide a method for treating acidic manganese-containing wastewater by combining free microalgae with immobilized manganese-oxidizing bacteria.

[0006] This method is performed according to the following steps:

[0007] I. Acclimatization of microalgae: Add microalgae to an acidic manganese-containing solution and acclimatize them for 4 days;

[0008] II. Preparation of immobilized manganese oxidizing bacteria particles: Prepare a 2-4% sodium alginate solution, add the manganese oxidizing bacteria solution to the sodium alginate solution and stir evenly. The volume ratio of the manganese oxidizing bacteria solution to the sodium alginate solution is 1:2.5-1:4.0 to prepare a mixed bacterial solution. Slowly drop the mixed bacterial solution into a 2-3% CaCl2 solution to prepare microspheres. After the microspheres harden at room temperature, remove them and wash them with deionized water 3-5 times to prepare immobilized particles.

[0009] III. Treatment of acidic manganese-containing wastewater: The immobilized particles prepared in step II are added to a dynamic experimental reactor, and then domesticated microalgae are added to form a bacterial-algae symbiotic system; acidic manganese-containing wastewater is added to the dynamic experimental reactor, and the reactor is operated at room temperature, with bacterial and algal nutrients added to the reactor periodically.

[0010] Furthermore, the microalgae is *Scenedesmus tetracaudus*;

[0011] Further, the Mn 2+ concentration is 20-30 mg / L;

[0012] Further, the initial pH of the acidic manganese-containing solution is 4.0-5.0;

[0013] Further, the manganese-oxidizing bacteria are Klebsiella sp. M3 , and the accession number is CCTCC NO: M 2021261;

[0014] Further, the sodium alginate solution is autoclaved at 121°C for 30 minutes before being mixed with the manganese-oxidizing bacteria solution, and then naturally cooled to 35±5°C.

[0015] Further, the manganese-oxidizing bacteria in the manganese-oxidizing bacteria solution are in the logarithmic growth phase;

[0016] Further, the manganese-oxidizing bacteria solution is a manganese-oxidizing bacteria suspension, which is prepared by inoculating a cold-stored strain into JFM medium at a volume ratio of 3-5%, oscillating and culturing for 18-24 hours, collecting the bacteria solution, centrifuging, and resuspending in PYCM liquid medium;

[0017] Further, the volume ratio of the manganese-oxidizing bacteria suspension to the sodium alginate gel solution is 1:2.5-1:4.0;

[0018] Further, the bacterial density of the manganese-oxidizing bacteria in the dynamic test reactor in step three is 1-4×10 8 cfu / mL;

[0019] Further, the microalgae in step three are in a free state, and the algal cell concentration is 3-6×10 5 cell / mL;

[0020] Further, the light intensity in step three is 2000-3000 lux;

[0021] Further, the initial pH of the acidic manganese-containing wastewater pumped into the reactor in step three is 4.0-6.0;

[0022] Further, the Mn 2+ ion concentration in the influent in step three is 20-70 mg / L;

[0023] Further, the hydraulic retention time in step three is 24-48 hours;

[0024] Further, the PYCM nutrient solution is added to the reactor every 3-4 days in step three;

[0025] Further, the step three adds BG11 nutrient solution to the reactor every 7-10 days.

[0026] Further, the manganese-oxidizing bacteria are preserved in China Center for Type Culture Collection, Wuhan, China, on March 24, 2021;

[0027] The present application has the following advantages:

[0028] (1) The embedding and immobilization treatment of manganese-oxidizing bacteria can avoid the direct toxic effects of wastewater acidity and heavy metal ions on microorganisms;

[0029] (2) The concentration of free microalgae in the system is reasonably controlled, so that it can provide suitable oxygen and pH for the manganese oxidation of microorganisms in the particles, and also does not inhibit the activity of manganese-oxidizing bacteria;

[0030] (3) The microorganisms do not lose during the treatment of acidic manganese-containing wastewater by the present method, and no secondary pollution is generated;

[0031] (4) The present method is used for treating high-concentration manganese-containing wastewater, and has high efficiency and can meet the discharge standard;

[0032] (5) The system can be operated for a long time, is stable, has low operation cost, and is suitable for large-scale wastewater treatment. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 is a schematic diagram of a dynamic test reactor; DETAILED DESCRIPTION

[0034] The present application will be further described in detail by examples, so that those skilled in the art can implement it according to the description.

[0035] It should be understood that the terms such as "have", "contain" and "include" used herein do not exclude the presence or addition of one or more other elements or combinations thereof. Example 1

[0036] A 4.5 pH, 20 mg / L Mn 2+ solution was added to the four-tailed Scenedesmus domestication container, and domestication culture was carried out for 4 days. A 2% sodium alginate solution was prepared, then autoclaved at 121℃, and naturally cooled to about 35℃ to form a gel solution; the logarithmic growth phase of Klebsiella sp. M3 suspension was added to the above gel solution, stirred uniformly, slowly dripped into a 2% CaCl2 solution, and washed after hardening to prepare immobilized particles. The obtained immobilized manganese-oxidizing bacteria particles were added to a cylindrical reactor made of organic glass, as shown in Figure 1 , the initial bacterial density in the container was 2 ×10 8cfu / mL; then added the free Tetraselmis liquid after domestication culture, so that the initial algal cell concentration in the container was 3 x 10 5 cell / mL, forming a bacteria-algae symbiotic system; the light-dark cycle of the whole system was 12 hours of light and 12 hours of darkness, and the light intensity was 3000 lux; a peristaltic pump was used to pump the pH 4.5 Mn 2+ wastewater containing 30 mg / L Mn2+ solution from the bottom to the top, and the hydraulic retention time was controlled to be 24 hours; the PYCM nutrient solution was added to the reactor every 4 days, and the BG11 nutrient solution was added to the reactor every 10 days. The total manganese solubility in the effluent was determined every day, and the results showed that the whole reaction system could be stably operated for 79 days, the pH of the water body in the reactor was always maintained at about 6.0, the dissolved oxygen concentration was 7.5-8.0 mg / L, and the total manganese concentration in the effluent was lower than 0.5 mg / L, reaching the first level standard of the "Integrated Wastewater Discharge Standard" (GB8978-1996). Example 2

[0037] The pH 4.5 Mn 2+ solution containing 20 mg / L Mn2+ was added to the Tetraselmis domestication container, and domestication culture was carried out for 4 days. A 2% sodium alginate solution was prepared and then autoclaved at 121°C, and naturally cooled to about 35°C to form a gel solution; the Klebsiella sp. M3 suspension was added to the above gel solution, stirred uniformly, and then slowly dripped into a 2% CaCl2 solution, and after hardening, the immobilized particles were washed to prepare the immobilized manganese-oxidizing bacteria particles. The obtained immobilized manganese-oxidizing bacteria particles were put into a cylindrical reactor made of organic glass, and the initial bacterial density in the container was 3 x 10 8 cfu / mL; then added the free Tetraselmis liquid after domestication culture, so that the initial algal cell concentration in the container was 5 x 10 5 cell / mL, forming a bacteria-algae symbiotic system; the light-dark cycle of the whole system was 12 hours of light and 12 hours of darkness, and the light intensity was 3000 lux; a peristaltic pump was used to pump the pH 4.5 Mn 2+ wastewater containing 30 mg / L Mn2+ solution from the bottom to the top, and the hydraulic retention time was controlled to be 24 hours; the PYCM nutrient solution was added to the reactor every 4 days, and the BG11 nutrient solution was added to the reactor every 10 days. The total manganese solubility in the effluent was determined every day, and the results showed that the whole reaction system could be stably operated for 79 days, the pH of the water body in the reactor was always maintained at about 6.0, the dissolved oxygen concentration was 7.5-8.0 mg / L, and the total manganese concentration in the effluent was lower than 0.5 mg / L, reaching the first level standard of the "Integrated Wastewater Discharge Standard" (GB8978-1996). Example 3

[0038] pH 4.5, containing 20 mg / L Mn 2+ solution, and incubated for 4 days. A 3% sodium alginate solution was prepared, then autoclaved at 121°C, and naturally cooled to about 35°C to form a gel solution. The suspension was added to the gel solution and stirred uniformly, and a 2% CaCl2 solution was slowly dripped into it. After hardening, the immobilized particles were washed to form immobilized manganese-oxidizing bacteria particles. Klebsiella sp. M3 The obtained immobilized manganese-oxidizing bacteria particles were added to a cylindrical reactor made of organic glass, and the initial bacterial density in the container was 4 x 10 8 cfu / mL. Then, free Tetraselmis sp. liquid after incubation was added to make the initial algal cell concentration in the container 6 x 10 5 cell / mL, forming a bacteria-algae symbiotic system. The light-dark cycle of the whole system was 12 hours of light and 12 hours of darkness, and the light intensity was 3000 lux. A peristaltic pump was used to pump the pH 6.0, 65 mg / L Mn 2+ wastewater from the bottom to the top, and the hydraulic retention time was controlled to be 48 hours. PYCM nutrient solution was added to the reactor every 3 days, and BG11 nutrient solution was added to the reactor every 7 days. The total manganese solubility in the effluent was measured every day, and the results showed that the whole reaction system could be stably operated for 77 days. The pH of the water in the reactor was maintained at about 7.2, the dissolved oxygen concentration was 7.6-8.2 mg / L, and the total manganese concentration in the effluent was less than 1.8 mg / L, reaching the first level standard of the "Integrated Wastewater Discharge Standard" (GB8978-1996). Example 4

[0039] pH 4.5, containing 20 mg / L Mn 2+ solution, and incubated for 4 days. A 3% sodium alginate solution was prepared, then autoclaved at 121°C, and naturally cooled to about 35°C to form a gel solution. The suspension was added to the gel solution and stirred uniformly, and a 2% CaCl2 solution was slowly dripped into it. After hardening, the immobilized particles were washed to form immobilized manganese-oxidizing bacteria particles. Klebsiella sp. M3 The obtained immobilized manganese-oxidizing bacteria particles were added to a cylindrical reactor made of organic glass, and the initial bacterial density in the container was 4 x 10 8 cfu / mL. Then, free Tetraselmis sp. liquid after incubation was added to make the initial algal cell concentration in the container 6 x 10 5 cell / mL, forming a bacteria-algae symbiotic system. The light-dark cycle of the whole system was 12 hours of light and 12 hours of darkness, and the light intensity was 3000 lux. A peristaltic pump was used to pump the pH 6.0, 65 mg / L Mn 2+The wastewater was added into the reactor at a speed to control the hydraulic retention time to be 24 hours, and the PYCM nutrient solution was added into the reactor every 4 days, and the BG11 nutrient solution was added into the reactor every 10 days; the total manganese solubility in the effluent was measured every day, and the results show that the whole reaction system can be stably operated for 72 days, the pH of the water in the reactor is always maintained at 6.6, the dissolved oxygen concentration is 7.3-7.7 mg / L, the total manganese concentration in the effluent is lower than 2.7 mg / L, and the second standard of the "Integrated Wastewater Discharge Standard" (GB8978-1996) is reached.

[0040] Comparative Example 1

[0041] The manganese-oxidizing bacteria suspension was directly added into a cylindrical reactor made of organic glass, the initial bacterial density in the container was 1 × 10 8 cfu / mL; the light and dark cycle was 12 hours of light and 12 hours of darkness, the light intensity was 2000 lux; a peristaltic pump was used to pump the pH 5.0 Mn 2+ The wastewater was added into the reactor at a speed to control the hydraulic retention time to be 24 hours, and the PYCM nutrient solution was added into the reactor every 4 days, and the BG11 nutrient solution was added into the reactor every 10 days; the total manganese solubility in the effluent was measured every day, and the results show that the whole reaction system can be stably operated for 72 days, the pH of the water in the reactor is always maintained at 6.6, the dissolved oxygen concentration is 7.3-7.7 mg / L, the total manganese concentration in the effluent is lower than 2.7 mg / L, and the second standard of the "Integrated Wastewater Discharge Standard" (GB8978-1996) is reached.

[0042] Comparative Example 2

[0043] The pH 4.5 Mn 2+ solution containing 20 mg / L Mn 8 was added into the domestication container containing Tetraselmis sp., and domestication culture was carried out for 4 days. The manganese-oxidizing bacteria suspension was directly added into a cylindrical reactor made of organic glass, the initial bacterial density in the container was 1 × 10 5 cfu / mL; then the domesticated free Tetraselmis sp. liquid was added to form a bacteria-algae direct mixing system with the initial algal cell concentration in the container being 3 × 10 2+Wastewater, control the water speed, the hydraulic retention time is 24 hours, every 4 days to the reactor is added PYCM nutrient solution, every 10 days to the reactor is added BG11 nutrient solution;Every day the total manganese solubility in effluent is determined, the result discovers: the first 3 days reactor water pH always maintains at 6.1-6.5, dissolved oxygen concentration is 6.7-7.2 mg / L, the total manganese concentration in effluent is about 5.5 mg / L, subsequently reactor water pH, dissolved oxygen variation although not big, but the effluent Mn concentration rises, the 7th day rises to 8.2 mg / L, then the reactor continues to run 40 days, the effluent Mn concentration is 8.8-9.4 mg / L, can not reach "Integrated Wastewater Discharge Standard" (GB8978-1996).

[0044] Although embodiments of the application have been disclosed in connection with the above specification and drawings it will be understood that they are not limited to the specific details described therein but rather can be applied to various fields of endeavor where the principles of the application are applicable. Additional modifications will readily occur to those skilled in the art. The application is therefore not limited to the exact construction and arrangement of parts illustrated and described nor limited the exact embodiments disclosed.

Claims

1. A method for treating acidic manganese-containing wastewater by co-treatment of free microalgae and immobilized manganese-oxidizing bacteria, characterized in that, include: (1) Acclimatization of microalgae: Add microalgae to an acidic manganese-containing solution and acclimatize them for 4 days; (2) Preparation of immobilized manganese oxidizing bacteria particles: Prepare a sodium alginate solution with a mass fraction of 2-4%, add the manganese oxidizing bacteria solution to the sodium alginate solution and stir evenly. The volume ratio of the manganese oxidizing bacteria solution to the sodium alginate solution is 1:2.5-1:4.0 to prepare a mixed bacterial solution. Slowly drip the mixed bacterial solution into a CaCl2 solution with a mass fraction of 2-3% to prepare microspheres. After the microspheres are hardened at room temperature, they are taken out and washed with deionized water 3-5 times to prepare immobilized particles. (3) Treatment of acidic manganese-containing wastewater: The immobilized particles prepared in step two are added to the dynamic test reactor, and then the domesticated microalgae are added to form a bacterial-algae symbiotic system; manganese-containing wastewater is added to the dynamic test reactor, and the reactor is operated at room temperature, and bacterial and algal nutrients are added to the reactor periodically; The manganese-oxidizing bacterium is Klebsiella sp. M3, with accession number CCTCC NO: M 2021261.

2. The method for treating acidic manganese-containing wastewater by synergistic treatment of free microalgae and immobilized manganese-oxidizing bacteria as described in claim 1, characterized in that, The microalgae mentioned is *Scenedesmus tetracaudus*.

3. The method for treating acidic manganese-containing wastewater by synergistic treatment of free microalgae and immobilized manganese-oxidizing bacteria as described in claim 1, characterized in that, The acidic manganese-containing solution contains Mn 2+ The concentration is 20-30 mg / L; the initial pH of the acidic manganese-containing solution is 4.0-5.

0.

4. The method for treating acidic manganese-containing wastewater by co-immobilized manganese-oxidizing bacteria with free microalgae as described in claim 1, characterized in that, Before mixing the sodium alginate solution with the manganese oxidizing bacteria solution, the sodium alginate solution is first autoclaved at 121°C for 30 minutes, and then naturally cooled to 35±5°C.

5. The method for treating acidic manganese-containing wastewater by synergistic treatment of free microalgae and immobilized manganese-oxidizing bacteria as described in claim 1, characterized in that, The manganese-oxidizing bacteria in the manganese-oxidizing bacterial solution are manganese-oxidizing bacteria in the logarithmic growth phase.

6. The method for treating acidic manganese-containing wastewater by synergistic treatment of free microalgae and immobilized manganese-oxidizing bacteria as described in claim 1, characterized in that, The manganese oxidizing bacteria suspension is prepared by inoculating the refrigerated strain into JFM medium at a volume ratio of 3-5%, shaking and culturing for 18-24 hours, collecting the bacterial solution, centrifuging, and resuspending it in PYCM liquid medium to prepare the bacterial suspension.

7. The method for treating acidic manganese-containing wastewater by synergistic treatment of free microalgae and immobilized manganese-oxidizing bacteria as described in claim 1, characterized in that, The volume ratio of manganese-oxidizing bacteria suspension to alginate gel solution is 1:2.5-1:4.

0.

8. The method for treating acidic manganese-containing wastewater by synergistic treatment of free microalgae and immobilized manganese-oxidizing bacteria as described in claim 1, characterized in that, The density of manganese-oxidizing bacteria in step (3) in the dynamic experimental reactor was 1-4 × 10⁻⁴. 8 cfu / mL.

9. The method for treating acidic manganese-containing wastewater by synergistic treatment of free microalgae and immobilized manganese-oxidizing bacteria as described in claim 1, characterized in that, The microalgae in step (3) are in a free state, and the concentration of algal cells in the dynamic experimental reactor is 3-6 × 10⁻⁶. 5 cell / mL.

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