A method for increasing the concentration and activity of methanogens in anaerobic digestion inoculum

Abstract

The present invention relates to a concentration method for improving the concentration and activity of methanogens in anaerobic digestion inoculum. The concentration method of the present invention comprises the following steps: providing a flocculant; adding an inoculant to a reactor, adding a flocculant, aerating with an inactive gas, and sealing the reactor; allowing the obtained sealed reactor to stand for stratification, removing the supernatant, and obtaining a bottom microbial flocculent precipitate; inoculating the microbial flocculent precipitate into an anaerobic bottle, adding an anaerobic culture medium and an organic carbon source, and aerating with an inactive gas; adding an oxidant and a complex vitamin to the anaerobic bottle, and culturing with an intermittent stirring method; adding an organic carbon source to the anaerobic bottle at intervals of 7-10 days, maintaining the culture temperature and stirring speed unchanged, stimulating the rapid proliferation of the restored active microorganisms, and obtaining a high-activity and high-concentration methanogen group. The present invention can improve the absolute number and methanogenic activity of subsequent methanogens by regulating and controlling some technical parameters during the concentration process.

Description

Technical Field

[0001] The invention relates to the technical field of water pollution control and sewage treatment, as well as anaerobic treatment of organic solid waste, and in particular to a concentration method for increasing the concentration and activity of methanogens in anaerobic digestion inoculum. Background Art

[0002] Anaerobic digestion is one of the main treatment technologies for recycling, reducing and harmless treatment of organic waste. That is, under anaerobic conditions, organic waste is converted into biogas through the action of microorganisms. A certain amount of active anaerobic microorganisms needs to be inoculated in the initial stage of a new large-scale anaerobic digestion project. The initial inoculation amount is generally 10-60% of the reactor volume. For an anaerobic digester in an actual project, this means that 1000 m 3 The above inoculation amount. The biogas slurry in the existing stably operating anaerobic digestion biogas digesters is rich in a large number of functional microorganisms and is often used as the initial inoculum for new large-scale anaerobic digestion projects. It is the main source of active inoculum. However, storing and transporting the inoculum in the form of raw biogas slurry will increase additional costs and will also be restricted by geographical location. At the same time, new biogas projects often face problems such as slow start-up and low initial gas production efficiency, causing certain economic losses to large-scale biogas projects. Therefore, it is urgent to invent an efficient concentration method for anaerobic digestion microorganisms that improves the activity of methanogens, while reducing the transportation volume of the inoculum and maintaining or even improving the activity of methanogens.

[0003] Common microbial concentration technologies include centrifugation, freeze drying and thermal drying. Centrifugation has the problem of high energy consumption, and high-speed centrifugation has a damaging effect on microorganisms. Although thermal drying technology can utilize waste heat from biogas plants and reduce energy input, thermal shock can cause irreversible damage to microbial activity. Freeze drying technology is beneficial for the dehydration of anaerobic digestion inoculants, but high energy consumption and the additional cost of cryoprotectants are the main challenges limiting the widespread use of this technology in biogas plants. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a concentration method for increasing the concentration and activity of methanogens in anaerobic digestion inoculum. The present invention uses chemical flocculants to concentrate anaerobic microorganisms, avoiding the high energy consumption and damage to microbial activity of existing concentration technologies. By regulating some technical parameters during the concentration process, the absolute number and methanogenic activity of subsequent methanogens can be increased.

[0005] The present invention is directed to a method for concentrating anaerobic digestion microorganisms to improve the activity of methanogens, comprising the following steps:

[0006] providing a flocculant solution;

[0007] Adding inoculum and the flocculant into the reactor, aerating with inactive gas for at least one minute, and sealing the reactor; allowing the obtained sealed reactor to stand for stratification, removing the supernatant, and obtaining microbial flocculent sediment at the bottom;

[0008] Inoculating the microbial flocculent precipitate into an anaerobic bottle, adding anaerobic digestion microbial nutrient solution and volatile solid carbon source, and aerating with inactive gas for at least five minutes; adding oxidants and multivitamins into the anaerobic bottle, and culturing by intermittent stirring method;

[0009] Add volatile solid carbon source substances to the anaerobic bottle at intervals of 7-10 days, maintain the culture temperature and stirring speed unchanged, stimulate the rapid proliferation of reactivated microorganisms, repeat several times, and you can obtain a highly active and high-concentration methanogenic bacteria group.

[0010] In some embodiments of the present invention, the flocculant solution is selected from one or more of a polyaluminium chloride solution, a cationic polyacrylamide solution and a polyferric sulfate solution; it needs to be used within four hours. The flocculant is generally a polymer, and it is easy to degrade and cause poor performance if stored for too long after preparation. Preparation and use immediately can ensure its efficiency.

[0011] The concentration of the polyaluminium chloride solution is 25.0-50.0 g / L;

[0012] The concentration of the cationic polyacrylamide solution is 1.0-2.0 g / L;

[0013] The concentration of the polyferric sulfate solution is 25.0-50.0 g / L.

[0014] In some embodiments of the present invention, the mass ratio of the inoculant to the flocculant solution is 125:1-10.

[0015] Further, the mass ratio of the inoculum to the polyaluminium chloride solution is 300 g: 6-18 mg;

[0016] The mass ratio of the inoculum to the polyferric sulfate solution is 300 g: 300-600 mg;

[0017] The mass ratio of the inoculum to the cationic polyacrylamide solution is 300 g:120-300 mg;

[0018] In some embodiments of the present invention, the inoculum is raw anaerobic digestion slurry, which contains anaerobic digestion slurry with active methanogenic microbial flora and comes from large and medium-sized biogas projects, such as livestock and poultry manure slurry, restaurant waste slurry, agricultural and forestry waste slurry, etc.; here, anaerobic digestion slurry from actual biogas projects that treat chicken manure is preferred.

[0019] In some embodiments of the present invention, the standing and stratifying time is 30-40 minutes.

[0020] In some embodiments of the present invention, the inert gas is nitrogen and / or argon;

[0021] The inert gas flow rate was 3.0-6.0 L gas / min / L inoculum.

[0022] In some embodiments of the present invention, the organic carbon source is an organic matter containing volatile solids, preferably livestock and poultry manure, such as chicken manure, duck manure, etc. "Volatile solids" is a professional term in the field. The test method is to burn the waste in a muffle furnace at 550-750°C for 3 hours, and the solid matter that evaporates is the "volatile solid, VS".

[0023] In some embodiments of the present invention, the oxidizing agent is selected from sodium sulfide and / or sulfite.

[0024] In some embodiments of the present invention, the amount of the oxidant is 5-10 mL / L relative to the inoculum, and the concentration of the oxidant is 25-50 g / L.

[0025] In some embodiments of the present invention, the amount of the multivitamin is 1-2 mL / L relative to the inoculum. The present invention uses multivitamins to provide nutrients, and the reason why they are not added to the anaerobic digestion microbial nutrient solution is that if multivitamins are added in advance, they are easily oxidized, so they are added later.

[0026] In some embodiments of the present invention, the intermittent stirring method conditions are: stirring for 1-2 hours every 3-5 hours, and the stirring speed is 200-300 rpm; the intermittent stirring method is used for culturing for 30-40 days.

[0027] Furthermore, the method for concentrating anaerobic digestion microorganisms to improve the activity of methanogens is specifically as follows:

[0028] 1. Preparation of flocculant mother solution (prepare and use within 4 hours):

[0029] 25.0-50.0g / L polyaluminium chloride solution:

[0030] 1.0-2.0g / L cationic polyacrylamide solution:

[0031] 25.0-50.0g / L polyferric sulfate solution:

[0032] 2. Flocculation process:

[0033] 2.1 Weigh 300g of inoculum and add a certain amount of flocculant mother solution: if polyaluminium chloride solution is used, add 5-12mL; if cationic polyacrylamide solution is used, add 6-18mL; if polyferric sulfate solution is used, add 12-24mL;

[0034] 2.2 After adding the flocculant, aerate with high-purity nitrogen at a flow rate of 3.0-6.0 L gas / min / L inoculum for at least 1 minute, and seal after aeration;

[0035] 2.3 After standing naturally at room temperature for 30-40 minutes, obvious stratification is observed. Remove the supernatant in time and retain the flocculent precipitate at the bottom.

[0036] 3. Flocculation microorganism activity recovery period:

[0037] 3.1 Inoculate the separated microbial flocculent precipitate into the anaerobic bottle in time, then add the anaerobic digestion microbial nutrient solution and organic carbon source, and aerate with inactive gas at an airflow rate of 3.0-6.0 L gas / min / L inoculum for at least 5 minutes.

[0038] 3.2 Add oxidant (Na 2 S solution) and multivitamins.

[0039] 3.3 Place the anaerobic bottle in a 37°C incubator, set the stirring speed to 200-300 rpm, and use intermittent stirring. It is recommended to stir for 1 hour every 3 hours and culture for about 30-40 days.

[0040] 4. Rapid growth period of flocculating microorganisms:

[0041] 4.1 Add 2-3g of organic carbon source to the anaerobic bottle in step 3 every 7-10 days, maintain the culture temperature and stirring speed unchanged, and stimulate the rapid proliferation of reactivated microorganisms.

[0042] 4.2 Repeat 4.1 several times to obtain a high-activity and high-concentration methanogenic bacteria group.

[0043] The above technical solution of the present invention has the following advantages compared with the prior art:

[0044] The present invention uses polyaluminium chloride solution, cationic polyacrylamide solution and polyferric sulfate solution as flocculants. The positive charge of the flocculant can neutralize the negative charge of the microorganisms in the anaerobic digestion liquid, thereby reducing the electrostatic repulsion between the microorganisms and making it easy for the microorganisms to aggregate. At the same time, the high molecular weight long chain structure of the flocculant can connect the microorganisms with the long chain molecules to form a larger floc, so that the microorganisms in the anaerobic digestion liquid are flocculated and precipitated, and the anaerobic digestion microorganisms are separated from the biogas slurry with a high water content. At the same time, the microorganisms can also play a role in reducing the toxicity of the accumulation of intermediate products contained in the anaerobic digestion liquid after flocculation, such as ammonia nitrogen, volatile fatty acids, etc.

[0045] The present invention uses inert gas (such as N) during flocculation and microbial culture. 2 ) Aeration and addition of oxidants (such as sodium sulfide, etc.) create an anaerobic environment that is more suitable for the growth of anaerobic microorganisms.

[0046] The microbial cultivation process of the present invention is divided into a relatively long activity recovery period and a relatively short rapid growth period, which can enable the microorganisms to quickly adapt to the new environment after being impacted by the flocculant. The intermittent stirring method can allow the mutualistic methanogenic bacteria to rapidly proliferate and form a mutualistic community with an appropriate distance during the period when stirring is stopped.

[0047] In general, the present invention overcomes the problems of high energy consumption and high cost of traditional concentration technology, realizes efficient concentration of anaerobic digestion microorganisms at room temperature, reduces the transportation cost of inoculum in large-scale anaerobic digestion projects, and increases the absolute number and methanogenic activity of subsequent methanogens. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0049] The culture medium used in the present invention is: 500 mL of culture medium, and the formula of each 1 L of culture medium is as follows: the following chemicals are dissolved in 1 L of deionized water, 1 g of NH 4 Cl, 0.1 g NaCl, 0.1 g MgCl 2 6H 2 O, 0.05 g CaCl 2 ·2H 2 O, 0.4 g K 2 HPO 4 ·3H 2 O, 0.5 mg Resazurin, 2 mg FeCl 2 ·4H 2 O, 0.05 mg H 3 BO3 , 0.05mg ZnCl 2 , 0.03 mg CuCl 2 , 0.05 mg MnCl 2 ·4H 2 O, 0.05 mg (NH 4 )6Mo 7 O 24 ·4H 2 O, 0.05 mg AlCl 3 , 0.05 mg CoCl 2 6H 2 O, 0.05 mg NiCl 2 , 0.5 mg EDTA, 0.049 mg H 2 SeO 3 , 2.6 g NaHCO 3 .

[0050] The formula of sodium sulfide solution is as follows: 25 g Na 2 S·9H 2 O was dissolved in 1 L of deionized water.

[0051] Embodiment 1:

[0052] 1. Add 6.25g polyaluminium chloride to 250mL ultrapure water to prepare 25.0g / L polyaluminium chloride flocculant mother solution for standby use; use it within 4 hours after preparation.

[0053] 2. Weigh 300 g of anaerobic digested biogas slurry from an actual chicken manure biogas project into an anaerobic bottle, add 4.8 mL of the polyaluminium chloride flocculant mother liquor obtained in step 1, shake to mix evenly after adding the polyaluminium chloride flocculant mother liquor, and aerate with high-purity nitrogen at a gas flow rate of 3.0 L gas / min / L inoculum for 1 minute. After aeration, seal the lid and let it stand naturally at room temperature for 30 minutes, remove the supernatant in time, and retain the flocculent sediment at the bottom.

[0054] 3. Add 500 mL of anaerobic culture medium and 7.5 g of chicken manure with a volatile solid content of 30% to the anaerobic bottle that retains the flocculent precipitate in step 2. Use chicken manure as the carbon source, mix well, and aerate with high-purity nitrogen at a flow rate of 3.0 L gas / min / L inoculum for 5 minutes, then add 1 mL of Na2O3 with a concentration of 25 g / L. 2 S solution and 0.8 mL of multivitamin.

[0055] 4. Place the anaerobic bottle obtained in step 3 in a 37°C incubator, set the stirring speed to 220 rpm, and use an intermittent stirring method, specifically stirring for 1 hour every 3 hours, and culture for 35 days.

[0056] 5. Thereafter, add 7.5 g of chicken manure to the anaerobic bottle in step 3 every 8 days, maintain the culture temperature and stirring speed unchanged, repeat the addition method once, and take the microorganisms for high-throughput sequencing analysis before each addition of chicken manure.

[0057] Embodiment 2:

[0058] 1. Add 0.25g of cationic polyacrylamide to 250mL of ultrapure water to prepare 1.0g / L cationic polyacrylamide flocculant mother solution for use; use it within 4 hours after preparation.

[0059] 2. Weigh 300 g of anaerobic digested biogas slurry from an actual chicken manure biogas project into an anaerobic bottle, add 6 mL of the cationic polyacrylamide flocculant mother solution obtained in step 1, shake to mix evenly after adding the cationic polyacrylamide flocculant, and aerate with high-purity nitrogen at a gas flow rate of 3.0 L gas / min / L inoculum for 1 minute. After aeration, seal the lid and let it stand naturally at room temperature for 30 minutes. Remove the supernatant in time and retain the flocculent sediment at the bottom.

[0060] 3. Add 500mL of anaerobic culture medium and 7.5g of chicken manure with a volatile solid content of 30% to the anaerobic bottle with flocculent sediment in step 2, use chicken manure as carbon source, mix well, aerate with high-purity nitrogen at an airflow rate of 1.5L / min for 5 minutes, and then add 1mL of Na2O3 with a concentration of 25g / L. 2 S solution and 0.8mL multivitamin.

[0061] 4. Place the anaerobic bottle obtained in step 3 in a 37°C incubator, set the stirring speed to 220 rpm, and use an intermittent stirring method, specifically stirring for 1 hour every 3 hours, and culture for 35 days.

[0062] 5. Thereafter, add 7.5 g of chicken manure to the anaerobic bottle in step 3 every 8 days, maintain the culture temperature and stirring speed unchanged, repeat the addition method once, and take the microorganisms for high-throughput sequencing analysis before each addition of chicken manure.

[0063] Embodiment 3:

[0064] 1. Add 6.25g of polyferric sulfate to 250mL of ultrapure water to prepare 25.0g / L polyferric sulfate flocculant mother solution, which should be used within 4 hours after preparation.

[0065] 2. Weigh 300 g of anaerobic digested biogas slurry from an actual chicken manure biogas project into an anaerobic bottle, add 12 mL of the polyferric sulfate flocculant mother solution obtained in step 1, shake to mix evenly after adding the polyferric sulfate flocculant mother solution, and aerate with high-purity nitrogen at a gas flow rate of 3.0 L gas / min / L inoculum for 1 minute. After aeration, seal the lid and let it stand naturally at room temperature for 30 minutes, remove the supernatant in time, and retain the flocculent sediment at the bottom.

[0066] 3. Add 500 mL of anaerobic culture medium and 7.5 g of chicken manure with a volatile solid content of 30% as a carbon source to the anaerobic bottle that retains the flocculent precipitate in step 2. After mixing evenly, aerate with high-purity nitrogen at an airflow rate of 1.5 L / min for 5 minutes, and then add 1 mL of Na2O3 with a concentration of 25 g / L. 2 S solution and 0.8mL multivitamin.

[0067] 4. Place the anaerobic bottle obtained in step 3 in a 37°C incubator, set the stirring speed to 220 rpm, and use an intermittent stirring method, specifically stirring for 1 hour every 3 hours, and culture for 35 days.

[0068] 5. Thereafter, add 7.5 g of chicken manure to the anaerobic bottle in step 3 every 8 days, maintain the culture temperature and stirring speed unchanged, repeat the addition method once, and take the microorganisms for high-throughput sequencing analysis before each addition of chicken manure.

[0069] Comparative Example 1 (without flocculation and precipitation):

[0070] 1. Weigh 300g of anaerobic digested biogas slurry from an actual chicken manure biogas project into an anaerobic bottle, add 280mL of anaerobic culture medium and 7.5g of chicken manure with a volatile solid content of 30% as a carbon source, mix well, and aerate with high-purity nitrogen at a flow rate of 3.0 L gas / min / L inoculum for 5 minutes, then add 1mL of Na 2 S solution and 0.8mL multivitamin.

[0071] 2. Place the anaerobic bottle obtained in step 1 in a 37°C incubator, set the stirring speed to 220 rpm, and use an intermittent stirring method, specifically stirring for 1 hour every 3 hours, and culture for 35 days.

[0072] 3. Thereafter, every 8 days, 7.5 g of chicken manure was added to the anaerobic bottle in step 3, the culture temperature and stirring speed were maintained unchanged, and the addition method was repeated once. Before each addition of chicken manure, microorganisms were collected for high-throughput sequencing analysis.

[0073] Test Case

[0074] The flocculation effect was characterized by removing the volume of the supernatant after flocculation. The biogas components of the anaerobic digestion flora during the rapid proliferation period were measured by gas chromatography (GC-7860 plus) equipped with a thermal conductivity detector (TCD), and the methane production was fitted using the modified Gompertz model to obtain the key parameter of methane production rate, which was used to characterize the activity intensity of the methanogenic microbial community. In addition, the absolute amount of methanogenic groups in the flora was quantitatively analyzed by high-throughput sequencing. The results showed that Examples 1-3 could flocculate the microorganisms in the anaerobic digestion liquid to between 20% and 25% of the original volume; the maximum methanogenic rate (methanogenic activity) of Examples 1-3 after the second addition of chicken manure was 125%, 114%, and 239% higher than that of Comparative Example 1, respectively; the maximum methanogenic rate (methanogenic activity) of Examples 1-3 after the third addition of chicken manure was 18%, 192%, and 130% higher than that of Comparative Example 1, respectively; at the same time, the methanogenic concentrations of Examples 1-3 after the second addition of chicken manure were 4.91×10 6 , 1.02×10 7 , 1.17×10 7 copies / mL bacterial solution, compared with Comparative Example 1 (the concentration of methanogens in the bacterial solution in Comparative Example 1 was 1.62×10 6 copies / mL bacterial solution) were 203%, 528%, and 622% higher than those in Examples 1-3. The concentrations of methanogens after the second addition of chicken manure were 6.31×10 6 , 5.53×10 6 ,8.72×10 6 copies / mL bacterial solution, compared with Comparative Example 1 (the concentration of methanogens in the bacterial solution in Comparative Example 1 was 6.64×10 5 copies / mL bacterial liquid) were 851%, 733% and 1214% higher.

[0075] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for increasing the concentration and activity of methanogens in anaerobic digestion inoculum, characterized in that: The following steps are involved: providing a flocculant solution; Adding inoculum and the flocculant into the reactor, aerating with inactive gas for at least one minute, and sealing the reactor; standing the obtained sealed reactor for stratification, removing the supernatant, and obtaining microbial flocculent sediment at the bottom; the inoculum is original anaerobic digested biogas slurry, and the original anaerobic digested biogas slurry contains anaerobic digested biogas slurry of active methanogenic microbial flora; The microbial flocculent sediment was inoculated into an anaerobic bottle, and the anaerobic digestion microbial culture medium and organic carbon source were added, and aerated with inactive gas for at least five minutes; adding an oxidant and a multivitamin into the anaerobic bottle, and culturing the culture by an intermittent stirring method; Add organic carbon sources to the anaerobic bottle at intervals of 7-10 days, maintain the culture temperature and stirring speed unchanged, stimulate the rapid proliferation of reactivated microorganisms, and repeat several times to obtain a highly active and high-concentration methanogenic bacteria group.

2. The concentration method according to claim 1, characterized in that: The flocculant solution is selected from one or more of a polyaluminium chloride solution, a cationic polyacrylamide solution and a polyferric sulfate solution; The concentration of the polyaluminium chloride solution is 25.0-50.0 g / L; The concentration of the cationic polyacrylamide solution is 1.0-2.0 g / L; The concentration of the polyferric sulfate solution is 25.0-50.0 g / L.

3. The concentration method according to claim 1, characterized in that: The mass ratio of the inoculum to the flocculant solution is 125:1-10.

4. The concentration method according to claim 1, characterized in that: The inert gas is nitrogen and / or argon; The inert gas flow rate was 3.0-6.0 L gas / min / L inoculum.

5. The concentration method according to claim 1, characterized in that: The organic carbon source is an organic matter containing volatile solids.

6. The concentration method according to claim 1, characterized in that: The oxidizing agent is selected from sodium sulfide and / or sulfite.

7. The concentration method according to claim 1, characterized in that: The amount of the oxidant used is 5-10 mL / L relative to the inoculum, and the concentration of the oxidant is 25-50 g / L.

8. The concentration method according to claim 1, characterized in that: The dosage of the multivitamin is 1-2 mL / L relative to the inoculum.

9. The concentration method according to claim 1, characterized in that: Conditions of the intermittent stirring method: stirring for 1-2 hours every 3-5 hours, and a stirring speed of 200-300 rpm; the cultivation time of the intermittent stirring method is 30-40 days.