A method for preparing magnetic biochar for immobilizing ammonia-nitrogen-tolerant anaerobic microorganisms and a method for alleviating ammonia-nitrogen inhibition

Abstract

The present invention relates to the field of anaerobic digestion, and specifically to a method for preparing magnetic biochar for immobilizing ammonia nitrogen-tolerant anaerobic microorganisms and a method for alleviating ammonia nitrogen inhibition. The present invention first domesticates anaerobic microorganisms with high tolerance to ammonia nitrogen under ammonia stress conditions, and the microbial structure and function are more complete, the adaptability is strong, and the microorganisms have general tolerance to high ammonia nitrogen systems. Then, the microorganisms with high tolerance to ammonia nitrogen are immobilized on magnetic biochar materials to construct a microhabitat system rich in ammonia nitrogen-tolerant anaerobic microorganisms, thereby ensuring efficient anaerobic digestion under high ammonia nitrogen conditions.

Description

Technical Field

[0001] The invention relates to the field of anaerobic digestion, and in particular to a method for preparing magnetic biochar for immobilizing ammonia nitrogen-tolerant anaerobic microorganisms and a method for alleviating ammonia nitrogen inhibition. Background Art

[0002] Anaerobic digestion (AD) is an environmentally friendly method for treating organic waste that can utilize renewable energy and can be used for livestock manure treatment. However, as a nitrogen-rich organic waste, manure AD processes always face the problem of ammonia inhibition. Ammonia toxicity, especially free ammonia (NH3) toxicity, can cause more than 30% methane production (MP) losses and even cause anaerobic digester collapse. Bioaugmentation refers to the addition of specific strains or bacterial communities to the system to enhance the desired activity. The advantage of successful bioaugmentation is that it can provide long-term efficiency with only a single application (i.e., adding a bioaugmentation inoculant), without the need for alternating reactor operations or diluting ammonia-rich substrates, and without the need for additional infrastructure and chemicals.

[0003] At present, the phenomenon of ammonia nitrogen inhibiting anaerobic digestion is mainly addressed through bioaugmentation, that is, by cultivating a bacterial community with specific functions that are resistant to high ammonia to restore the digestion capacity of the AD system. However, most of the existing bioaugmentation methods focus on pure strain bioaugmentation. However, the use of pure strains as bioaugmentation bacterial communities is costly, requires sterile conditions, special growth media, etc., is technically difficult, and bioaugmentation in high ammonia environments is more difficult. It is easy to lose or weaken in complex anaerobic digestion systems, making it difficult to effectively alleviate ammonia nitrogen inhibition, and higher ammonia nitrogen concentrations make it more difficult to cultivate bioaugmentation bacteria.

[0004] Chinese patent document CN110980945A (201911367717.9) discloses a method for relieving ammonia inhibition in anaerobic digestion reactions, by adding activated carbon and bio-enhancing agents to the anaerobic system. The effect of this patent in alleviating ammonia nitrogen inhibition is mainly due to the adsorption of ammonia nitrogen by activated carbon, thereby reducing the ammonia nitrogen concentration in the fermentation liquid; its bacterial agent is a methanogenic microorganism that produces methane using acetic acid or propionic acid as a substrate. It is not cultured under ammonia stress conditions and has no direct tolerance to ammonia nitrogen. Instead, it increases the concentration of methanogenic microorganisms, thereby ensuring the production of methanogens from the perspective of increasing methanogenic microorganisms. However, the bacterial community structure in this patent is relatively simple, and the doubling time of methanogens is short, which is easily disturbed, lost or weakened in a complex anaerobic microbial system. Summary of the invention

[0005] In order to solve the ammonia nitrogen inhibition phenomenon occurring in an anaerobic digestion system, the present invention provides a method for preparing magnetic biochar for immobilizing ammonia nitrogen tolerant anaerobic microorganisms and a method for alleviating ammonia nitrogen inhibition.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for preparing magnetic biochar for immobilizing ammonia-nitrogen-tolerant anaerobic microorganisms comprises the following steps:

[0008] (1) Preparation method of magnetic biochar:

[0009] The corn stalks are heated to 530-560°C in a nitrogen atmosphere at 3-8°C / min and maintained for 1-3 hours to obtain biochar; the biochar is added to a ferric chloride solution, soaked for 1-3 hours, and then dried, wherein the mass ratio of the biochar to the ferric chloride is 1:7-9;

[0010] (2) Bio-augmented bacterial flora enrichment culture method:

[0011] After the anaerobic culture medium is sterilized, the anaerobic bacterial community liquid is added, and then ammonium chloride is added to adjust the ammonia nitrogen concentration to 1.5 g / L, and 0.6 g / L of sodium propionate is added every two days; the propionic acid concentration is measured every two days until all the propionic acid can be consumed within two days, and it is maintained for one week and then subcultured; the culture liquid of the previous generation is taken for enrichment culture, and the ammonia nitrogen concentration is increased by 0.5 g / L each time until it is adjusted to 4.5 g / L, to obtain a bio-fortified bacterial community solution;

[0012] (3) Preparation of magnetic biochar immobilized with ammonia-tolerant anaerobic microorganisms:

[0013] The magnetic biochar is first soaked in a dilute solution containing the substrate for 1 to 3 hours, taken out and filtered, and then soaked in a bio-enhanced bacterial community solution for 70 to 74 hours.

[0014] Preferably, in step (1), the corn stalks are heated to 550° C. at 5° C. / min in a nitrogen atmosphere and maintained for 2 hours to obtain biochar.

[0015] Preferably, the concentration of the ferric chloride solution in step (1) is 3 g / L, the soaking time is 2 h, and the biochar and ferric chloride are soaked in a mass ratio of 1:8.

[0016] Preferably, in step (2), the volume ratio of the anaerobic culture medium to the anaerobic bacterial liquid is 9:1, and the culture condition is 37°C.

[0017] Preferably, in step (3), the immersion time of the magnetic biochar in the dilution solution containing the substrate is 2 hours, and the immersion time of the magnetic biochar in the bio-augmentation bacterial community solution is 72 hours.

[0018] Preferably, in step (3), the substrate-containing dilution solution is 1 g propionic acid / 150 mL water.

[0019] Preferably, in step (3), the substrate is an anaerobic fermentation substrate, which is a mixture of rice and vegetables in a mass ratio of 2:1 and then crushed and homogenized, with TS (solid content) = 30% and organic matter content VS = 70%.

[0020] A method for alleviating ammonia nitrogen inhibition by combining magnetic biochar with bioaugmentation technology, wherein magnetic biochar and bioaugmentation bacterial liquid are added to an anaerobic digestion system, or magnetic biochar immobilized with ammonia nitrogen resistant anaerobic microorganisms is added to the anaerobic digestion system.

[0021] Preferably, the amount of magnetic biochar for immobilizing ammonia-tolerant anaerobic microorganisms in the anaerobic digestion system is 8 to 12 g / L, and more preferably 10 g / L.

[0022] The amount of magnetic biochar added to the anaerobic digestion system was 10 g / L, and the amount of bio-augmented bacterial liquid added was 1 / 40 (V / V).

[0023] Beneficial effects of the present invention:

[0024] (1) The magnetic biochar in the present invention can enrich ammonia-resistant bacteria. The combined use of magnetic biochar and ammonia-resistant bacteria can adapt to higher ammonia nitrogen concentrations compared to the method of bioaugmentation alone, reducing the time and cost of enriching high-ammonia-resistant bacteria; and the magnetic biochar itself will also have an impact on the bacterial community and promote the interspecies electron transfer process of the bacterial community.

[0025] (2) The present invention first domesticates anaerobic microorganisms with high tolerance to ammonia nitrogen under ammonia stress conditions. The microbial structure and function are more complete, the adaptability is strong, and the microorganisms have general tolerance to high ammonia nitrogen systems. The microorganisms with high tolerance to ammonia nitrogen are then immobilized on magnetic biochar materials to construct a microhabitat system rich in ammonia nitrogen-resistant anaerobic microorganisms, thereby ensuring efficient anaerobic digestion under high ammonia nitrogen conditions. The combination of activated carbon and bio-enhancing bacterial agents can not only better improve the effect of ammonia inhibition, but also overcome the problems of using either alone, so that they can be better applied to a large number of objects to be treated, further improving the efficiency of use and saving costs.

[0026] (3) The magnetic biochar immobilized with ammonia-tolerant anaerobic microorganisms provided by the present invention can stably and efficiently realize the microhabitat system of anaerobic digestion function under high ammonia-nitrogen conditions. At the same time, the magnetic biochar is easy to recycle on the one hand, and on the other hand, it can provide an electronic conductor material to promote electron transfer between microorganisms in the microhabitat, thereby more effectively performing anaerobic digestion. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the cumulative methane production graph under different anaerobic fermentation conditions;

[0028] Figure 2is a bacterial composition diagram of the bioaugmentation flora used in the present invention;

[0029] Figure 3 It is a diagram of the archaeal composition of the bioaugmentation flora used in the present invention. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the accompanying drawings.

[0031] Experimental Materials:

[0032] Inoculum (TS=2%, VS=50%): taken from a medium-temperature anaerobic digestion tank, temperature 37°C, carbon source is citric acid, 30 g is added every two days.

[0033] Substrate: Rice and vegetables were mixed in a mass ratio of 2:1 and then crushed into a homogenate, TS (solid content) = 30%, organic matter content VS = 70%.

[0034] Biochar: 15 g corn straw was heated to 550 °C at 5 °C / min in a nitrogen atmosphere and maintained for 2 h.

[0035] Preparation of magnetic biochar: 1 g of burned corn straw biochar was added into 3 g / L ferric chloride solution and soaked for 2 h. The biochar and ferric chloride were soaked in a mass ratio of 1:8.

[0036] The bio-enhanced bacterial community enrichment culture method used in this experiment is as follows: use a serum bottle with a working volume of 200mL, add 90mL of anaerobic culture medium, sterilize the culture medium in an autoclave at 121℃ for 15min, then add 10mL of anaerobic bacterial community liquid, add ammonium chloride to adjust the ammonia nitrogen concentration to 1.5g / L, add 0.06g of sodium propionate as a substrate every two days, and the culture condition is 37℃. Measure the propionic acid concentration every two days until all the propionic acid can be consumed within two days, and maintain it for about a week, then subculture, take 10mL of the previous generation for enrichment culture, and increase the ammonia nitrogen concentration by 0.5g / L each time until it is adjusted to 4.5g / L to obtain a bio-enhanced bacterial community solution.

[0037] Anaerobic media were prepared from the following stock solutions (chemicals given below are concentrations in g / l in distilled water).

[0038] Solution A: NH4Cl, 100; NaCl, 10; MgCl2·6H2O, 10; CaCl2·2H2O, 5;

[0039] Solution B: K2HPO4·3H2O, 200;

[0040] Solution C: Resazurin 0.5;

[0041] Solution D: trace metal and selenite solution: FeCl2·4H2O, 2; H3BO3, 0.05; zinc chloride, 0.05; cupric chloride dihydrate, 0.038; manganese chloride tetrahydrate, 0.05; (NH4)6Mo7O 24 ·4H2O, 0.05; AlCl3, 0.05; CoCl2·6H2O, 0.05; NiCl2·6H2O, 0.092; EDTA, 0.5; Concentrated hydrochloric acid, 1 ml; Na2SeO3·5H2O, 0.1;

[0042] Solution E: Vitamin mixture (ingredients are given in mg / L): biotin, 2; folic acid, 2; pyridoxine, 10; ridophen, 5; thiamine hydrochloride, 5; cyanocobalamin, 0.1; niacin, 5; p-aminobenzoic acid, 5; lipoic acid, 5; DL-pantothenic acid.

[0043] The anaerobic nutrient solution is: add 10 ml of the above stock solution (A); 2 ml of (B); 1 ml of (C); 1 ml of (D); and 1 ml of (E) to 985 ml of distilled water.

[0044] The anaerobic bacterial culture fluid comes from the medium-temperature anaerobic fermentation tank in the laboratory.

[0045] Preparation of magnetic biochar immobilized with ammonia-tolerant anaerobic microorganisms: The dried magnetic biochar was first soaked in a propionic acid aqueous solution (1 g of propionic acid was diluted with 150 mL of water) for 2 h, then taken out and filtered, and then soaked in a bio-enhanced bacterial solution for 72 h.

[0046] Anaerobic system: Use an anaerobic bottle with a total volume of 300 mL and a working volume of 200 mL, add 40 mL of inoculum and 1.9 g of substrate, add deionized water to 200 mL, aerate with argon for 3 minutes, seal, and place in a shaker at 37°C and 120 rpm.

[0047] The experiments were carried out under the following conditions, with 3 parallels for each experiment.

[0048] R1: Add 5 mL of bio-augmented bacteria to the above anaerobic system. Before adding the bio-augmented bacteria, centrifuge at 5000 rpm for 10 min and remove the supernatant before adding.

[0049] R2: Add 5 mL of bio-augmented bacteria to the above anaerobic system (before adding the bio-augmented bacteria, centrifuge at 5000 rpm for 10 min and remove the supernatant before adding), and add 3.44 g of ammonium chloride to adjust the ammonia nitrogen concentration to 4.5 gN / L.

[0050] R3: Add 5 mL of bio-augmented bacteria to the above anaerobic system (before adding the bio-augmented bacteria, centrifuge at 5000 rpm for 10 min and remove the supernatant before adding); add 2 g of biochar and adjust the ammonia nitrogen concentration to 4.5 gN / L.

[0051] R4: Add 5 mL of bio-augmented bacteria to the above anaerobic system (before adding the bio-augmented bacteria, centrifuge at 5000 rpm for 10 min and remove the supernatant before adding); add 2 g of magnetic biochar and adjust the ammonia nitrogen concentration to 4.5 gN / L.

[0052] R5: Add 2 g of magnetic biochar immobilized with ammonia-tolerant anaerobic microorganisms to the above anaerobic system and adjust the ammonia nitrogen concentration to 4.5 gN / L.

[0053] From the attached Figure 1 It can be seen that the use of magnetic biochar and bioaugmented flora (R4) has a shorter lag period and higher methane production than the addition of biochar + bioaugmented flora (R3), indicating a better ammonia nitrogen inhibition and mitigation effect. And the magnetic biochar immobilized with ammonia nitrogen-tolerant anaerobic microorganisms has a better effect than the two added separately.

[0054] Figure 2 and Figure 3 In the figure, BS0 represents the bacterial community composition after enrichment culture, and X0 represents the inoculum sludge used. It can be seen from the figure that X0 is the community composition of ordinary anaerobic digestion sludge, and BS0 is the community composition after enrichment. Bacteria such as Smithella and Syntrophobacter are bacteria with propionic acid degradation ability, which can be seen to be significantly improved after enrichment.

Claims

1. A method for alleviating ammonia nitrogen inhibition by combining magnetic biochar with bioaugmentation technology, characterized in that: Add 2g of magnetic biochar immobilized with ammonia-tolerant anaerobic microorganisms to the anaerobic system, and adjust the ammonia nitrogen concentration to 4.5gN / L; Anaerobic system: Use an anaerobic bottle with a total volume of 300mL and a working volume of 200mL, add 40mL of inoculum and 1.9g of substrate, add deionized water to 200mL, use argon aeration for 3min, seal, and place in a shaker at 37°C and 120rpm; The method for preparing the magnetic biochar immobilized with ammonia nitrogen resistant anaerobic microorganisms comprises the following steps: (1) Preparation method of magnetic biochar: The corn stalks were heated to 550°C at 5°C / min in a nitrogen atmosphere and maintained for 2 hours to obtain biochar; the biochar was added to a 3g / L ferric chloride solution and soaked for 2 hours and then dried, and the mass ratio of biochar to ferric chloride was 1:8; (2) Bioaugmented bacterial flora enrichment culture method: After the anaerobic culture medium is sterilized, the anaerobic bacterial community liquid is added, and then ammonium chloride is added to adjust the ammonia nitrogen concentration to 1.5g / L, and 0.6g / L of sodium propionate is added every two days; the propionic acid concentration is measured every two days until the propionic acid can be completely consumed within two days, and it is maintained for one week for subculture; the culture liquid of the previous generation is taken for enrichment culture, and the ammonia nitrogen concentration is increased by 0.5g / L each time until it is adjusted to 4.5g / L to obtain a bio-fortified bacterial community solution; the volume ratio of the anaerobic culture medium to the anaerobic bacterial community liquid is 9:1, and the culture condition is 37°C; (3) Preparation of magnetic biochar for immobilizing ammonia-tolerant anaerobic microorganisms: The magnetic biochar was first soaked in a dilution containing a substrate for 2 hours, taken out and filtered, and then soaked in a bio-enhanced bacterial solution for 72 hours. The concentration of the dilution containing the substrate was 1g substrate / 150mL water. The substrate was an anaerobic fermentation substrate. The substrate was made of rice and vegetables mixed in a mass ratio of 2:1 and then crushed into a homogenate. The solid content TS was 30% and the organic matter content VS was 70%.

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