Method for relieving ammonia inhibition effect in anaerobic digestion process of nitrogen-rich organic waste and increasing methane yield based on microbial stress regulation

Abstract

The invention discloses a method for relieving an ammonia inhibition effect in a nitrogen-rich organic waste anaerobic digestion process and increasing methane yield based on microbial stress regulation. The method comprises the following steps that an active oxygen scavenger solution is added into an anaerobic reactor for producing methane, the ammonia inhibition effect is recovered, the methane yield is increased, and the active oxygen scavenger is selected from one or more of superoxide dismutase, catalase and vitamin C. According to the method, the active oxygen scavenger solution is precisely added into the fermentation system, so that the active oxygen scavenger solution plays a role in the intracellular or extracellular environment of the microorganisms, the microorganisms can still keep high metabolic activity in the high-ammonia-nitrogen environment, finally, the ammonia inhibition phenomenon is substantially recovered, and the methane yield of the system is remarkably recovered and increased.

Description

Technical Field

[0001] This invention relates to the field of anaerobic digestion treatment technology for nitrogen-rich organic waste, and in particular to a method for relieving the ammonia inhibition effect and increasing methane production in the anaerobic digestion process of nitrogen-rich organic waste based on microbial stress regulation. Background Technology

[0002] With the rapid development of intensive livestock and poultry farming in my country, nearly 4 billion tons of livestock and poultry manure are generated annually. If not treated promptly and properly, this manure will harm the atmosphere, water bodies, soil, and human health. Chicken manure is rich in organic matter and has high energy conversion potential. Anaerobic digestion technology is the mainstream technology for the harmless treatment of various organic pollutants while also recovering energy, and therefore it is widely promoted and applied worldwide. However, anaerobic digestion of chicken manure still faces the technical bottleneck of low methane production. The main reason is that chicken manure is rich in urea and protein, which decompose during hydrolysis and acidification to produce free ammonia and ammonium ions. Ammonia inhibits microbial activity; compared to bacteria, methanogens are less tolerant of ammonia, which is also the main reason for the decline in methane production. When the organic load of chicken manure is 2.5 gVS / L / Day, the ammonia concentration can be as high as 5-6 g / L. Research reports indicate that when the total ammonia concentration exceeds 1.5 g / L, volatile fatty acids begin to accumulate in the anaerobic system, and methane production capacity decreases; when the ammonia concentration directly rises to 4.5 g / L, methane production capacity loss can reach over 30%. In summary, the anaerobic digestion of chicken manure suffers from severe ammonia inhibition, causing significant economic losses to large-scale biogas projects. Therefore, there is an urgent need to develop a method to alleviate ammonia inhibition and increase methane production through anaerobic digestion.

[0003] Common control methods primarily aim to alleviate the microbial ammonia inhibition effect by reducing ammonia concentration in situ, such as physical dilution, air stripping, membrane separation, and chemical precipitation. Dilution is the simplest and most direct method, diluting the feed with water or low-nitrogen wastewater. However, it reduces the operating load and methane production capacity. Air stripping, membrane separation, and chemical precipitation are all costly. Another control approach is based on improving the ammonia tolerance and absolute biomass of the microorganisms within the system, such as through acclimatization and bioaugmentation, to increase methane production. Acclimatization methods involve long cycles, while bioaugmentation involves high initial investment costs and risks of colonization failure or incompatibility with the microbial agents. None of the above control methods fundamentally eliminate the microbial ammonia inhibition effect, and their high cost and complex operation necessitate new control methods. Summary of the Invention

[0004] The purpose of this invention is to provide a method for relieving the ammonia inhibition effect and increasing methane production in the anaerobic digestion process of nitrogen-rich organic waste based on microbial stress regulation. This method is based on the addition of reactive oxygen species scavengers to remove reactive oxygen species generated in the anaerobic microorganisms under ammonia stress, thereby restoring the activity of the microorganisms and increasing their methane production activity.

[0005] This invention is achieved through the following technical solutions:

[0006] This invention protects a method for relieving the ammonia inhibition effect and increasing methane production in the anaerobic digestion process of nitrogen-rich organic waste based on microbial stress regulation, comprising the following steps: restoring the ammonia inhibition effect and increasing methane production by adding a reactive oxygen species scavenger solution to the methanogenic anaerobic reactor, wherein the reactive oxygen species scavenger is selected from one or more of superoxide dismutase (SOD), catalase (CAT), and vitamin C.

[0007] This invention precisely adds a reactive oxygen species (ROS) scavenger solution to the fermentation system, allowing it to act within or outside the microbial cells. This captures free radicals such as O2·⁻ and ·OH generated under ammonia stress, blocking their chain reaction of oxidative damage. This effectively reduces the total intracellular ROS content, mitigating or eliminating oxidative stress damage to microbial cells (especially methanogenic archaea) at its source, thereby protecting their key metabolic enzyme systems and energy synthesis pathways. This enables the microorganisms to maintain high metabolic activity even in high ammonia nitrogen environments, ultimately achieving a substantial recovery from the "ammonia inhibition" phenomenon and significantly restoring and increasing methane production in the system.

[0008] Active oxygen scavenger solutions need to be prepared and used immediately, as they are generally easily oxidized. If stored for too long after preparation, they are prone to oxidation and will lose their performance. Preparing and using them immediately can ensure their effectiveness.

[0009] Preferably, the method specifically includes the following steps: adding inoculum containing active methanogenic microorganisms and nitrogen-rich organic waste into the anaerobic reactor, wherein the ratio of volatile solids in the inoculum to nitrogen-rich organic waste is 10%-30%, aerating with nitrogen (for more than 5 minutes) to make the anaerobic reactor meet the anaerobic reaction conditions, then adding an active oxygen scavenger solution into the anaerobic reactor, maintaining the reactor operating temperature at 37°C for constant temperature incubation, and the hydraulic retention time is 20-40 days to produce methane.

[0010] Preferably, the method specifically includes the following steps: adding inoculum and mixed substrate to an anaerobic reactor, wherein the inoculum contains active methanogenic microbial strains, and the mixed substrate includes nitrogen-rich organic waste and organic waste containing active oxygen scavengers, with the amount of active oxygen scavengers in the mixed substrate reaching the target addition amount; the ratio of volatile solids in the inoculum to the mixed substrate is 10%-30%, and the reactor is kept at a constant temperature of 37°C throughout the experiment, with a hydraulic retention time of 20-40 days, to prepare methane.

[0011] Preferably, the method specifically includes the following steps: adding inoculum and mixed substrate to an anaerobic reactor, wherein the inoculum contains active methanogenic microbial strains, and the mixed substrate includes nitrogen-rich organic waste and organic waste containing active oxygen scavengers; the ratio of volatile solids in the inoculum to the mixed substrate is 10%-30%; if the amount of active oxygen scavengers in the mixed substrate is less than the target addition amount, aeration with nitrogen gas is performed first (for more than 5 minutes) to ensure that the anaerobic reactor meets the anaerobic reaction conditions, and then active oxygen scavenger solution is added to the anaerobic reactor to supplement the target content; the entire experimental process maintains the reactor operating temperature at 37°C for constant temperature incubation, and the hydraulic retention time is 20-40 days to prepare methane.

[0012] The method proposed in this invention can be achieved by adding an active oxygen scavenger or by mixing organic waste containing an active oxygen scavenger during fermentation, as long as the amount of active oxygen scavenger added meets the actual needs.

[0013] Preferably, the concentration of the superoxide dismutase solution or catalase solution is 90-110 U / mL, and the concentration of the vitamin C solution is 0.008-0.012 g / L.

[0014] More preferably, the reactive oxygen species scavenger solution is a mixed solution of superoxide dismutase solution and catalase solution in a volume ratio of 1:1.

[0015] Preferably, the inoculum is a bacterial flora or pure bacteria containing active methanogenic bacteria.

[0016] Further preferably, the inoculum is chicken manure fermentation liquid, and the substrate is chicken manure. Specifically, the inoculum is chicken manure biogas fermentation liquid.

[0017] Preferably, the volume ratio of the inoculum to the reactive oxygen species scavenger solution is 450-2200:1.

[0018] Further preferably, the volume ratio of the inoculum to the reactive oxygen species scavenger solution is 1000:1.

[0019] This invention addresses the intracellular oxidative stress effect in chicken manure anaerobic fermentation systems where ammonia nitrogen levels above 1.5 g / L trigger microbial oxidative stress. During this process, the generation and scavenging of intracellular reactive oxygen species (ROS) (such as ·OH, H₂O₂, and O₂·⁻) become unbalanced, leading to ROS accumulation. ROS possess strong oxidizing properties and readily undergo chain-reaction oxidative damage reactions with microbial metabolites, even key enzymes and DNA, altering their biochemical properties and functions. This weakens microbial activity and reduces methane production. Therefore, under high ammonia concentrations, microbial stress leading to ROS production and intracellular / extracellular oxidative damage is the primary cause of ammonia inhibition. This inhibition mechanism has long been overlooked. Based on this mechanism, this invention addresses the inhibition effect by adding ROS scavengers to fundamentally alleviate ammonia nitrogen inhibition, restoring methanogenesis activity and methane production capacity. This avoids the shortcomings of existing technologies that indirectly alleviate ammonia inhibition by reducing reactor ammonia concentration or increasing microbial ammonia tolerance.

[0020] The present invention also protects the application of the method in anaerobic digestion methanogenic systems.

[0021] Preferably, the total ammonia concentration in the anaerobic digestion system is ≥1.5 g NH4. + -N / L.

[0022] Further preferably, the total ammonia concentration in the anaerobic digestion system is ≥2.5 g NH4. + -N / L.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] This invention precisely adds a reactive oxygen species (ROS) scavenger solution to the fermentation system, allowing it to act within or outside the microbial cells to capture generated free radicals such as O2·⁻ and ·OH, thus blocking their chain reaction of oxidative damage. This effectively reduces the total intracellular ROS content, mitigating or eliminating oxidative stress damage to microbial cells (especially methanogenic archaea) at its source, thereby protecting their key metabolic enzyme systems and energy synthesis pathways. This enables the microorganisms to maintain high metabolic activity even in high ammonia nitrogen environments, ultimately achieving a substantial recovery from the "ammonia inhibition" phenomenon and significantly restoring and increasing methane production in the system.

[0025] Compared to traditional mitigation technologies, this invention, based on in-depth analysis of ammonia inhibition, essentially adds reactive oxygen species (ROS) scavengers directly to eliminate the stress products of ammonia inhibition, effectively restoring microbial methanogenesis activity and increasing methane production. While ensuring technical effectiveness, it achieves the application goal of "minimizing engineering modifications and maximizing operational convenience." This method does not rely on complex equipment or lengthy biological acclimatization processes; it only requires low-cost chemical integration or changes in feed composition at existing process nodes to quickly restore and improve the stability and energy efficiency of anaerobic fermentation systems under high ammonia nitrogen conditions. This economical, flexible, and easily scalable characteristic makes it particularly suitable for practical application in small and medium-sized biogas projects or large-scale livestock farm waste treatment systems with limited equipment investment and relatively weak operation and maintenance capabilities, providing a new and highly engineering-feasible path to solve the common problem of ammonia inhibition.

[0026] Overall, this invention overcomes the technical bottlenecks of high economic cost, high site construction requirements, and complex processes of traditional technologies, and achieves the goal of quickly and easily relieving the ammonia inhibition effect of anaerobic digestion, significantly restoring the activity of methanogenic bacteria and methane production. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions in the art or as recommended by the manufacturer; the raw materials and reagents used, unless otherwise specified, are considered to be commercially available materials and reagents that can be obtained through conventional markets and other commercial channels.

[0028] In the following embodiments, the biogas slurry of the chicken manure biogas project is taken from the reactor of the chicken manure biogas project undertaken by Shandong Minhe Animal Husbandry Co., Ltd.

[0029] Example 1

[0030] A method for enhancing ammonia inhibition of methane production in anaerobic digestion based on microbial oxidative stress regulation includes the following steps:

[0031] (1) Prepare 100 U / mL superoxide dismutase (SOD) solution, 100 U / mL catalase (CAT) solution, and 0.01 g / L vitamin C solution respectively;

[0032] (2) Take 12 anaerobic bottles (divided into six groups: A, B, C, D, E, and F, with two parallel samples in each group). Inoculate each group with 100 mL of pure bacterial suspension of *Methanococcus methanans* into the anaerobic bottle. Add additional ammonium chloride solution to 10 bottles (groups B, C, D, E, and F) to make the ammonia nitrogen concentration reach 4.5 g / L. Do not add ammonium chloride solution to the other 2 bottles as a control (group A).

[0033] (3) In step (2), group B was given 0.1 mL of superoxide dismutase (SOD) solution, group C was given 0.1 mL of catalase (CAT) solution, group D was given 0.1 mL of vitamin C solution, group E was given 0.1 mL of a mixture of superoxide dismutase (SOD) solution and catalase (CAT) solution with a volume ratio of 1:1, and group F was given no reactive oxygen species scavenger solution.

[0034] (4) Place the anaerobic bottle obtained in step (3) in a constant temperature incubator at 37°C and measure its methane production daily.

[0035] The final results showed that methane production was significantly increased in all groups (B (2380 mL / L), C (2210 mL / L), D (2180 mL / L), and E (2500 mL / L)) with the addition of reactive oxygen species scavenger solution. Group E had the highest production, followed by groups B, C, and D. There was no significant difference in methane production among groups A, B, C, and D. Group E's production was more than 8.6% higher than the control group A (no ammonia inhibition, 2300 mL / L), and the methane production of all regulated groups was 19.7%-37.3% higher than that of group F (ammonia inhibition, no reactive oxygen species scavenger, 1820 mL / L). When the same amount of reactive oxygen species scavenger was added, the methane production of the mixed solution of superoxide dismutase (SOD) and catalase (CAT) in group E was higher than that of superoxide dismutase (SOD) or catalase (CAT) alone, indicating that superoxide dismutase (SOD) and catalase (CAT) solutions had a synergistic effect.

[0036] Example 2

[0037] A method for enhancing ammonia inhibition of methane production in anaerobic digestion based on microbial oxidative stress regulation includes the following steps:

[0038] (1) Prepare 100 U / mL superoxide dismutase (SOD) solution, 100 U / mL catalase (CAT) solution, and 0.01 g / L vitamin C solution respectively;

[0039] (2) Take 100 mL of chicken manure biogas slurry from 10 groups (divided into five groups: A, B, C, D, and E) into anaerobic bottles, and add 1.80 g of VS chicken manure as substrate to make the ammonia nitrogen concentration in the reactor 2.5 g / L. Use nitrogen gas to aerate the anaerobic reactor to meet the anaerobic reaction conditions.

[0040] (3) In step (2), group A was given 0.1 mL of superoxide dismutase (SOD) solution, group B was given 0.1 mL of catalase (CAT) solution, group C was given 0.1 mL of vitamin C solution, and group D was given 0.1 mL of a mixture of superoxide dismutase (SOD) solution and catalase (CAT) solution with a volume ratio of 1:1. Group E was given as a control without reactive oxygen species scavenger solution.

[0041] (4) Place the anaerobic bottle obtained in step (3) in a constant temperature incubator at 37°C and measure its methane production daily.

[0042] The composition of biogas was determined by gas chromatography to obtain methane production, a key parameter used to characterize the activity of methanogenic bacteria. The results showed that the methane production of all groups (A (2115 mL / L), B (2100 mL / L), C (2055 mL / L), and D (2285 mL / L)) with added reactive oxygen species scavenger solution was significantly higher than that of group E (1875 mL / L) (without reactive oxygen species scavenger) by 9.6%-21.8%. Group D had the highest methane production, followed by groups A, B, and C. There was no significant difference in methane production among groups A, B, and C.

[0043] Example 3

[0044] A method for enhancing ammonia inhibition of methane production in anaerobic digestion based on microbial oxidative stress regulation includes the following steps:

[0045] (1) Prepare 90 U / mL superoxide dismutase (SOD) solution, 90 U / mL catalase (CAT) solution, and 0.008 g / L vitamin C solution respectively;

[0046] (2) Take 100 mL of chicken manure biogas slurry from 10 groups (divided into five groups: A, B, C, D, and E) into an anaerobic bottle, add 1.08 g of VS chicken manure to make the ammonia nitrogen concentration 1.5 g / L, and use nitrogen gas to aerate the anaerobic reactor to meet the anaerobic reaction conditions.

[0047] (3) In step (2), group A was given 0.22 mL of superoxide dismutase (SOD) solution, group B was given 0.22 mL of catalase (CAT) solution, group C was given 0.22 mL of vitamin C solution, and group D was given a mixture of superoxide dismutase (SOD) solution and catalase (CAT) solution with a volume ratio of 1:1. Group E was given as a control without reactive oxygen species scavenger solution.

[0048] (4) Place the anaerobic bottle obtained in step (3) in a constant temperature incubator at 37°C and measure its methane production daily.

[0049] The composition of biogas was determined by gas chromatography to obtain methane production, a key parameter used to characterize the activity of methanogenic bacteria. The results showed that the methane production of all groups (A, B, C, and D) with added antioxidant solutions was significantly higher than that of group E (without reactive oxygen species scavengers) by 10%-30%, with group B having the highest methane production.

[0050] Example 4

[0051] A method for enhancing ammonia inhibition of methane production in anaerobic digestion based on microbial oxidative stress regulation includes the following steps:

[0052] (1) Prepare 110 U / mL superoxide dismutase (SOD) solution, 110 U / mL catalase (CAT) solution, and 0.012 g / L vitamin C solution respectively;

[0053] (2) Take 100 mL of chicken manure biogas slurry from 10 groups (divided into five groups: A, B, C, D, and E) into an anaerobic bottle, add 3.24 g of VS chicken manure, and make the ammonia nitrogen concentration of the reaction system 4.5 g / L. Use nitrogen gas to aerate the anaerobic reactor to meet the anaerobic reaction conditions.

[0054] (3) In step (2), group A was given 0.05 mL of superoxide dismutase (SOD) solution, group B was given 0.05 mL of catalase (CAT) solution, group C was given 0.05 mL of vitamin C solution, and group D was given 0.05 mL of a mixture of superoxide dismutase (SOD) solution and catalase (CAT) solution with a volume ratio of 1:1. Group E was given as a control without reactive oxygen species scavenger solution.

[0055] (4) Place the anaerobic bottle obtained in step (3) in a constant temperature incubator at 37°C and measure its methane production daily.

[0056] Gas chromatography was used to determine the components of biogas, obtaining methane production as a key parameter to characterize the activity of methanogenic bacteria. The results showed that the methane production of all groups (A, B, C, and D) with added antioxidant stress agent solution was significantly higher than that of group E (without reactive oxygen species scavenger) by 50%, with group D having the highest methane production, followed by groups A, B, and C.

[0057] The above description of the embodiments is only for the purpose of helping to understand the technical solution and core idea of ​​the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for relieving ammonia inhibition and increasing methane production during the anaerobic digestion of nitrogen-rich organic waste based on microbial stress regulation, characterized in that, The process includes the following steps: by adding a reactive oxygen species scavenger solution to the methanogenic anaerobic reactor, the inhibitory effect of ammonia on methanogenic bacteria is relieved and the methane production is increased. The reactive oxygen species scavenger is selected from one or more of superoxide dismutase, catalase and vitamin C.

2. The method according to claim 1, characterized in that, The specific steps include: adding inoculum containing active methanogenic microorganisms and nitrogen-rich organic waste into the anaerobic reactor. The ratio of volatile solids in the inoculum to nitrogen-rich organic waste is 10%-30%. Nitrogen gas is used to aerate the anaerobic reactor to meet the anaerobic reaction conditions. Then, an active oxygen scavenger solution is added to the anaerobic reactor. The reactor is kept at a constant temperature of 37°C for incubation, and the hydraulic retention time is 20-40 days to produce methane.

3. The method according to claim 1, characterized in that, The specific steps include: adding inoculum and mixed substrate to the anaerobic reactor, wherein the inoculum contains active methanogenic microbial strains, and the mixed substrate includes nitrogen-rich organic waste and organic waste containing active oxygen scavengers, with the amount of active oxygen scavengers in the mixed substrate reaching the target addition amount; the ratio of volatile solids in the inoculum to the mixed substrate is 10%-30%, and the reactor is kept at a constant temperature of 37℃ throughout the experiment, with a hydraulic retention time of 20-40 days, to produce methane.

4. The method according to claim 1, characterized in that, The specific steps include: adding inoculum and mixed substrate to the anaerobic reactor, wherein the inoculum contains active methanogenic microorganisms, and the mixed substrate includes nitrogen-rich organic waste and organic waste containing active oxygen scavengers; the ratio of volatile solids in the inoculum to the mixed substrate is 10%-30%. If the amount of active oxygen scavengers in the mixed substrate is less than the target amount, the anaerobic reactor is first aerated with nitrogen to meet the anaerobic reaction conditions, and then an active oxygen scavenger solution is added to the anaerobic reactor to supplement the target content. Throughout the experiment, the reactor is kept at a constant temperature of 37°C for incubation, and the hydraulic retention time is 20-40 days to prepare methane.

5. The method according to claim 2, 3 or 4, characterized in that, The concentration of superoxide dismutase solution or catalase solution is 40-200 U / mL, and the concentration of vitamin C solution is 0.002-0.04 g / L.

6. The method according to claim 2, 3 or 4, characterized in that, The reactive oxygen species scavenger solution is a mixture of superoxide dismutase solution and catalase solution in a volume ratio of 1:

1.

7. The method according to claim 2, 3 or 4, characterized in that, The inoculum is a bacterial flora or pure bacteria containing live methanogens.

8. The method according to claim 2, 3 or 4, characterized in that, The volume ratio of the inoculum to the reactive oxygen species scavenger solution is 450-2200:

1.

9. The method according to any one of claims 1-8 is used in an anaerobic digestion methanogenic system.

10. The application according to claim 9, characterized in that, The total ammonia concentration in the anaerobic digestion system is ≥1.5 gNH4. + -N / L.