Mesophilic and thermophilic anaerobic bacteria for enhancing methane production and alleviating ammonia inhibition and their applications

Abstract

The present invention relates to the field of environmental biotechnology, and particularly to mesophilic and thermophilic anaerobic bacteria for enhancing methane production and alleviating ammonia inhibition. The present invention provides Anaerobic_zm02 with the strain number CGMCC No. 46069, which can significantly improve the organic matter degradation efficiency and methane production in the anaerobic digestion system, and can effectively alleviate ammonia inhibition. It can be used in the fermentation treatment of waste, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of environmental biotechnology, and in particular to medium- and high-temperature anaerobic bacteria capable of increasing methane production and alleviating ammonia inhibition, and applications thereof. Background Art

[0002] Food waste refers to waste generated from daily life, food processing, catering services, and workplace catering. It is complex and easily perishable. Incineration and landfill disposal not only have limited environmental capacity, but also produce harmful gases such as dioxins, sulfur dioxide, and nitrogen oxides, causing secondary air pollution, and leachate, contaminating soil and groundwater. However, food waste also has high water content and organic matter content, with organic matter comprising over 80% of its dry matter. This means that, in addition to its harmful properties, food waste also possesses resource properties. Therefore, sustainable resource utilization technologies and waste-to-resource conversion are crucial for food waste management. Currently, anaerobic digestion is the primary method for resource utilization of food waste, which recovers energy and achieves clean, green, and low-carbon treatment.

[0003] Due to the complex composition of food waste, anaerobic digestion often faces challenges such as acidification and ammonia inhibition, resulting in low methane production. This severely impacts resource recovery efficiency and significantly increases industrial costs. Therefore, improving system stability and methane production efficiency is a pressing issue in anaerobic digestion of food waste. Anaerobic digestion is a multiphase, multi-stage, and interconnected biochemical process driven by diverse microorganisms. Its stability relies on multiple assimilation interactions among different bacterial communities, and system instability is essentially the result of microbial metabolic perturbations. Bioaugmentation is a method that improves reactor performance by incorporating specialized microorganisms into a reactor system to enhance specific functions. Bioaugmentation technology has significant advantages due to its significant facilitation of anaerobic digestion, simple operation, and low cost, making it a promising technology. By adding functional microbial strains to anaerobic fermentation systems, the activity and performance of the system can be enhanced, ultimately alleviating ammonia inhibition and improving anaerobic digestion efficiency. Therefore, identifying strains that can enhance methane production and alleviate ammonia inhibition is of great significance and application value. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide medium- and high-temperature anaerobic bacteria and their applications that can increase methane production and alleviate ammonia inhibition.

[0005] The present invention provides Anaerobic_zm02 with a deposit number of CGMCC No.46069.

[0006] The present invention provides a bacterial flora comprising the Anaerobic_zm02 described in 1 of the present invention.

[0007] The present invention provides a bacterial agent, which includes Anaerobic_zm02 described in the present invention or the bacterial flora described in the present invention and excipients.

[0008] Furthermore, the auxiliary materials include anti-minerals, vitamins, antibiotics, culture media, stabilizers, anti-flocculants, antioxidants and / or solid media.

[0009] Furthermore, the solid medium includes sawdust, peat soil, cottonseed hulls, bran, gypsum, sand and / or sucrose.

[0010] In the present invention, the bacterial agent includes a liquid bacterial agent and / or a solid bacterial agent, which is not limited in the present invention; the liquid bacterial agent includes the Anaerobic_zm02 described in the present invention and a liquid or liquid-soluble auxiliary material, wherein the auxiliary material can be a mineral, a vitamin, an antibiotic, a culture medium, a stabilizer, a deflocculant and / or an antioxidant that assists the reproduction or function of the Anaerobic_zm02; in a specific embodiment of the present invention, the bacterial agent is a liquid bacterial agent, which is obtained by expanding the Anaerobic_zm02 in a culture medium, and the concentration of the liquid bacterial agent is at least 1×10 8 CFU / mL.

[0011] The Anaerobic_zm02 described in the present invention is obtained through sludge enrichment. Compared with other enriched bacterial species, it has a stronger ability to promote organic matter degradation and increase biogas production (the Anaerobic_zm02 described has no independent methane production ability, but plays an auxiliary role in enhancing methanogens). It also shows the ability to effectively alleviate ammonia inhibition.

[0012] The present invention provides the use of at least one of the following A) to C) in waste fermentation:

[0013] A), Anaerobic_zm02 of the present invention;

[0014] B), the bacterial flora of the present invention;

[0015] C) the bacterial agent of the present invention.

[0016] The present invention provides a method for waste fermentation, which comprises fermenting the waste using at least one of the following a) to c):

[0017] a) Anaerobic_zm02 of the present invention;

[0018] b) the bacterial flora of the present invention;

[0019] c) The bacterial agent of the present invention.

[0020] Furthermore, the waste includes livestock and poultry manure or scraps, agricultural waste and / or restaurant waste, which is not limited in the present invention. The bacterial strains described in the present invention participate in the treatment of the above-mentioned waste under anaerobic conditions; in a specific embodiment of the present invention, the waste is restaurant waste, and the restaurant waste and activated sludge are degraded by the action of the liquid bacterial agent to produce biogas during the degradation process, and ammonia inhibition can be relieved to increase treatment efficiency.

[0021] In a specific embodiment of the present invention, the food waste and activated sludge are uniformly mixed in a VS ratio of 2:1, and then the liquid bacterial agent of the present invention is added for anaerobic fermentation, or the liquid bacterial agent of the present invention is added during the continuous anaerobic fermentation process to achieve the effect of strengthening anaerobic digestion and relieving ammonia inhibition. The bacterial concentration in the bacterial agent used is not less than 1×10 8 CFU / mL, in the specific embodiment of the present invention, 1×10 8 CFU / mL; the amount of the bacterial agent added is 15-20% of the total volume of food waste and activated sludge.

[0022] The present invention provides the use of at least one of the following I) to III) in degrading organic matter, assisting in increasing methane production and / or relieving ammonia inhibition:

[0023] 1), Anaerobic_zm02 of the present invention;

[0024] II), the bacterial flora of the present invention;

[0025] III), the bacterial agent of the present invention.

[0026] The present invention provides a product for degrading organic matter, assisting in increasing methane production and / or relieving ammonia inhibition. The raw materials thereof include at least one of the following substances i) to iii) and other functional substances that assist in methane production and / or relieve ammonia inhibition.

[0027] i) Anaerobic_zm02 of the present invention;

[0028] ii), the bacterial flora of the present invention;

[0029] iii) the bacterial agent of the present invention.

[0030] Further functional substances include but are not limited to compounds, nucleic acids and / or enzymes.

[0031] The present invention provides a method for degrading organic matter, increasing methane production and / or relieving ammonia inhibition, which comprises utilizing at least one of the following i) to iv):

[0032] i) Anaerobic_zm02 of the present invention;

[0033] ii), the bacterial flora of the present invention;

[0034] ii), the bacterial agent of the present invention;

[0035] iv) The product of the present invention.

[0036] The present invention provides Anaerobic_zm02, strain numbered CGMCC No. 46069, which can significantly improve the organic matter degradation efficiency and methane production of anaerobic digestion systems, and can effectively alleviate ammonia inhibition. It can be used in the fermentation treatment of waste and has good application prospects.

[0037] Biological Deposit Description

[0038] The biological material Anaerobic_zm02, taxonomic name: Schnuerera sp., was deposited on July 29, 2024 at the General Microbiology Center of China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.46069. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is a scanning electron micrograph of strain Anaerobic_zm02;

[0040] Figure 2 This is the neighbor-joining phylogenetic tree of strain Anaerobic_zm02;

[0041] Figure 3 The gas production and organic matter conversion rate of anaerobic digestion of food waste by strain Anaerobic_zm02 are shown in Figure 1, where a is the change in gas production during anaerobic digestion; b is the improvement in gas production and organic matter conversion rate (VS removal efficiency);

[0042] Figure 4 The figure shows the effect of strain Anaerobic_zm02 on the ammonia inhibition (external ammonia nitrogen concentration of 2000 and 5000 mg / L) in the anaerobic digestion of food waste. a is the cumulative gas production; b is the remaining VS after the reaction is completed;

[0043] Figure 5The figure shows the effect of strain Anaerobic_zm02 on the ammonia inhibition in the anaerobic digestion of food waste (with added ammonia nitrogen (TAN) concentrations of 1000 and 3000 mg / L), where a is the cumulative gas production; b is the VS remaining after the reaction is completed;

[0044] Figure 6 The figure shows the effect of strain Anaerobic_zm02 on the alleviation of ammonia inhibition in the anaerobic digestion of food waste at medium temperature, medium-high temperature and high temperature. Among them, a is the alleviation of ammonia inhibition in the anaerobic digestion of food waste at medium temperature; b is the alleviation of ammonia inhibition in the anaerobic digestion of food waste at medium and high temperature; c is the alleviation of ammonia inhibition in the anaerobic digestion of food waste at high temperature. DETAILED DESCRIPTION

[0045] The present invention provides medium- and high-temperature anaerobic bacteria and their applications that increase methane production and alleviate ammonia inhibition. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve this. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0046] The present invention provides a strain of bacteria that enhances the anaerobic digestion of food waste and produces methane. The strain, identified as Schnuererasp., has been deposited with the China General Microbiological Culture Collection (CGMCC) under the name Anaerobic_zm02 and the accession number CGMCC No. 46069. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Anaerobic_zm02 is a Gram-positive, strictly anaerobic bacterium.

[0047]

[0048] In the present invention, the Anaerobic_zm02 is separated and screened from the activated sludge of a continuously running kitchen waste anaerobic reactor (Hangzhou Energy and Environmental Engineering Co., Ltd.).

[0049] In some laboratory experiments, anaerobic activated sludge and food waste were evenly mixed at a VS ratio of 2:1 and placed in a 500 mL anaerobic flask with a working volume of 200 mL. Anaerobic fermentation was performed by inoculating 10% of the total volume with a bacterial inoculum (15% of the system's working volume was taken, centrifuged, and the pellet was resuspended in sterile water with 1 / 10 the volume of the bacterial solution. This bacterial suspension was then inoculated into the anaerobic fermentation system and incubated at 45°C until the system stopped producing gas. This increased the organic matter conversion rate by 14.56% and the cumulative gas production by 17.63%.

[0050] Other laboratory experiments examined the effectiveness of the strain Anaerobic_zm02 in mitigating ammonia inhibition during anaerobic fermentation of wet food waste at medium- and high-temperature conditions (45°C). By adding NH₄Cl to ammonia nitrogen concentrations of 1000 mg / L, 2000 mg / L, 3000 mg / L, and 5000 mg / L, the inoculum was added at 15% of the system's working volume. At an organic loading of 5 g VS / L₂, the experimental groups increased their cumulative methane production by 29.08%, 16.89%, 13.00%, and 20.26%, respectively, compared to the control group.

[0051] The effectiveness of the strain Anaerobic_zm02 in mitigating ammonia inhibition during anaerobic methane production from food waste was evaluated in a continuously operating 30-L anaerobic fermenter at varying temperatures: moderate, moderately high, and high. By adding NH₄Cl to the reactor to achieve ammonia inhibition (methane production was reduced by over 20% compared to the blank condition), the inoculum was then added at 20% of the system's working volume. At moderate, moderately high, and high temperatures, the addition of the inoculum increased average methane production by 24.08%, 28.58%, and 17.66%, respectively, compared to the inhibition state.

[0052] The high-efficiency anaerobic methanogenic agent provided by the present invention can improve organic matter removal rates, increase methane production, and effectively alleviate the effects of ammonia inhibition on gas production. This saves equipment and investment and operating costs, resulting in good economic and environmental benefits.

[0053] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below with reference to the following examples:

[0054] Example 1 Isolation and screening of anaerobic digestion functional bacteria

[0055] 1. The formula of Modified Wolin medium used in the example is as follows:

[0056] Modified Wolin's mineral solution 1 (MWL1, Modified Wolin basal medium): KCl 0.34 g / L, MgCl2·6H2O 4.00 g / L, MgSO4·7H2O 3.45 g / L, NH4Cl 0.25 g / L, CaCl2·2H2O 0.14 g / L, K2HPO4 0.14 g / L, NaCl 18.00 g / L, Modified Wolin's mineral solution10.00 mL, Fe(NH4)2(SO4)2·6H2O (0.1% w / v) 2.00 mL, Na-acetate 1.00 g / L, Yeastextract 2.00 g / L, Trypticase peptone 2.00 g / L, Sodium resazurin (0.1% w / v) 1mL, NaHCO3 5.00 g / L, Wolin's vitamin solution 10.00 mL, L-Cysteine ​​HCl·H2O 0.50g / L, Na2S·9H2O 0.50 g / L, Distilled water 1000.00 mL.

[0057] Wolin's vitamin solution (Wolfe's vitamin solution): Biotin 2.00 mg / L, Folicacid 2.00 mg / L, Pyridoxine hydrochloride 10.00 mg / L, Thiamine HCl 5.00 mg / L, Riboflavin 5.00 mg / L, Nicotinic acid 5.00 mg / L, Calcium D-(+)-pantothenate 5.00 mg / L, Vitamin B12 0.10 mg / L, p-Aminobenzoic acid 5.00 mg / L, (DL)-alpha-Lipoic acid 5.00 mg / L, Distilled water 1000.00 mL.

[0058] Modified Wolin's mineral solution: Nitrilotriacetic acid 1.50 g / L, MgSO4·7H2O 3.00 g / L, MnSO4 ·H2O 0.50 g / L, NaCl1.00 g / L, FeSO4·7 H2O 0.10 g / L, CoSO4·7H2O 0.18 g / L, CaCl2·2H2O 0.10 g / L, ZnSO4·7H2O 0.18 g / L, CuSO4·5H2O 0.01 g / L, AlK(SO4)2·12H2O 0.02 g / L, H3BO3 0.01g / L, Na2MoO4·2H2O 0.01 g / L, NiCl2·6H2O 0.03 g / L, Na2SeO3·5H2O 0.30 mg / L, Na2WO4·2H2O 0.40 mg / L, Distilled water 1000.00 mL.

[0059] Modified Wolin basal medium was added with modified Wolfe's mineral solution (10 mL / L Modified Wolin basal medium) and sterilized by high-pressure steam at 121°C for 20 min. Then, filter-sterilized Wolfe's vitamin solution (10 mL / L Modified Wolin basal medium) was added to obtain Modified Wolin medium for later use.

[0060] The anaerobic sludge used to isolate functional bacteria was collected from the long-term anaerobic digestion reactor of Hangzhou Energy and Environmental Engineering Co., Ltd. in Hangzhou, Zhejiang Province. 10 mL of anaerobic sludge was inoculated into 200 mL of Modified Wolin medium and enriched at 45°C and 200 rpm for 7 days. Another 10 mL of culture medium was transferred to a new Modified Wolin medium and enriched for a second round at 45°C and 200 rpm. After 7 days, the enriched culture medium was serially diluted to a dilution of 10. -3 , 10 -4 , 10 -5 and 10 -6The enriched culture fluid was evenly spread onto a solid plate of Modified Wolin medium (15-20g of agar added to Modified Wolin medium). After 48-96 hours of constant temperature incubation at 44°C, a single colony was picked. The single colony was inoculated into Modified Wolin liquid medium and cultured, streaked, purified, and passaged three times. The strain was identified by 16S rRNA sequencing. The strain was found to have a 97.34% similarity to Schnuerera ultunensis (basal synonym Clostridium ultunense). The scanning electron micrograph is shown below. Figure 1 , phylogenetic tree such as Figure 2 The bacterial suspension was mixed with 50% glycerol at a ratio of 1:1 and stored at -80°C.

[0061] Example 2 Preparation of bacterial agent

[0062] Inoculate pure strains of Anaerobic_zm02 into 15 mL of Modified Wolin medium and culture anaerobically at 45°C and 200 rpm for 24–48 h. Then, inoculate the strain into the next level of Modified Wolin medium at an inoculum volume of 1% of the volume of the Modified Wolin medium. Continue to expand the culture under the same conditions to obtain a liquid inoculum of the strain.

[0063] Example 3: Enhanced effect of microbial inoculants on gas production from wet anaerobic digestion of food waste

[0064] The food waste slurry was collected from the Xiaoshan Food Waste Treatment Plant. The total solids (TS) content of the slurry was 6.33%, and the VS (volatile solids) / TS ratio was 81.03%. Activated sludge was collected from a long-running anaerobic digester at Hangzhou Energy and Environmental Engineering Co., Ltd. in Hangzhou, Zhejiang Province. The TS content of the activated sludge was 10.754%, and the VS / TS ratio was 30.033%.

[0065] Food waste and activated sludge were evenly mixed at a VS ratio of 1:2 and placed in a 500 mL anaerobic flask with a working volume of 200 mL and an organic load of 7.5 g VS / L. Experimental and control groups were set up. The experimental group was inoculated with a liquid inoculum (at a concentration of at least 1 × 10 ) at 15% of the total volume of food waste and anaerobic activated sludge. 8CFU / mL), centrifuged, resuspended the bacterial pellet in sterile water (1 / 10 the volume of the bacterial solution), and inoculated the bacterial suspension into the anaerobic fermentation system. The control group added the same volume of sterile water instead of the bacterial agent, and the blank group was a single anaerobic activated sludge. Sterile water was added to all three groups to make the final working volume 200 mL. The temperature was controlled at 45°C, biogas production was tested daily, and VS in each system was measured before and after fermentation. The results showed (such as Figure 3 The experimental group's cumulative gas production reached 956.3 mL, a 17.63% increase compared to the control group's 813.0 mL. Furthermore, the organic matter conversion rate in the experimental group was 82.10%, a 14.56% increase compared to the control group's 71.67%. This indicates that strain Anaerobic_zm02 significantly enhances biogas production from anaerobic fermentation of food waste under medium- and high-temperature conditions.

[0066] Among them, organic matter conversion rate = [(VS (底物) )-(VS (1) -VS (污泥1) )] / (VS (0) -VS (污泥0) )×100%

[0067] VS (底物) : Volatile solid content of the food waste substrate added to the system (g);

[0068] VS (1) : Volatile solids content (g) at the end of fermentation in a fermentation system containing food waste substrate and anaerobic activated sludge;

[0069] VS (污泥1) : Volatile solid content (g) at the end of fermentation in the single anaerobic activated sludge fermentation system.

[0070] Example 4 Effect of microbial inoculants on alleviating ammonia stress in high-temperature wet anaerobic fermentation of food waste (with added ammonia nitrogen concentrations of 2000 and 5000 mg / L)

[0071] The food waste slurry was collected from the Xiaoshan Food Waste Treatment Plant. The TS content of the slurry was 6.33%, and the VS / TS ratio was 81.03%. Activated sludge was collected from a long-term anaerobic digester operated by Hangzhou Energy and Environmental Engineering Co., Ltd. in Hangzhou, Zhejiang Province. The TS content of the activated sludge was 8.764%, and the VS / TS ratio was 30.596%.

[0072] The food waste and activated sludge were mixed evenly at a VS ratio of 1:2. The working volume was 200 mL and the total organic load was 5 g VS / L. Two ammonia nitrogen concentrations were set by adding NH4Cl to simulate the ammonia inhibition encountered in the anaerobic digestion of food waste. The concentrations of added NH4Cl were: (1) 2000 mg / L; (2) 5000 mg / L. A control group and an experimental group were set for each concentration gradient. The experimental group was inoculated with liquid bacterial agent at 15% of the total volume of food waste and anaerobic activated sludge (the bacterial agent was taken at 15% of the total volume of the system, centrifuged, and the bacterial precipitate was resuspended with 1 / 10 volume of sterile water of the bacterial liquid, and the bacterial suspension was inoculated into the anaerobic fermentation system). The control group was added with the same volume of sterile water instead of the bacterial agent. Finally, sterile water was added to make the working volume of the experimental group and the control group 200 mL. Three replicates were set for each treatment. The temperature was controlled at 45 °C. The biogas production was tested every day. The VS in each system was measured before and after fermentation and the utilization of organic matter was calculated. The results showed that ( Figure 4 When the added ammonia nitrogen concentration was 2000 mg / L, the average cumulative gas production in the experimental group was 715.0 mL, a 16.89% increase compared to the control group's cumulative gas production of 611.7 mL. Furthermore, the amount of residual organic matter (measured in VS) decreased by 16.28% compared to the control group. When the added ammonia nitrogen concentration was 5000 mg / L, the cumulative gas production in the experimental group was 672.6 mL, a 20.26% increase compared to the control group's cumulative gas production of 559.3 mL. Furthermore, the amount of residual organic matter (measured in VS) decreased by 6.37% compared to the control group.

[0073] Example 5: Effect of microbial inoculants on alleviating ammonia stress in high-temperature wet anaerobic fermentation of food waste (with added ammonia nitrogen concentrations of 1000 and 3000 mg / L)

[0074] The food waste slurry was collected from the Xiaoshan Food Waste Treatment Plant. The TS content of the slurry was 5.071%, and the VS / TS ratio was 76.261%. Activated sludge was collected from a long-term anaerobic digester operated by Hangzhou Energy and Environmental Engineering Co., Ltd. in Hangzhou, Zhejiang Province. The TS content of the activated sludge was 7.817%, and the VS / TS ratio was 27.311%.

[0075] The food waste and activated sludge were mixed evenly at a VS ratio of 1:2. The working volume was 100 mL and the total organic load was 5 g VS / L. Two ammonia nitrogen concentrations were set by adding NH4Cl to simulate the ammonia inhibition encountered in the anaerobic digestion of food waste. The concentrations of added NH4Cl were: (1) 1000 mg / L; (2) 3000 mg / L. A control group and an experimental group were set for each concentration gradient. The experimental group was inoculated with liquid bacterial agent at 15% of the total volume of food waste and anaerobic activated sludge (the bacterial agent was taken at 15% of the total volume of the system, centrifuged, and the bacterial precipitate was resuspended with 1 / 10 volume of sterile water of the bacterial liquid, and the bacterial suspension was inoculated into the anaerobic fermentation system). The control group was added with the same volume of sterile water instead of the bacterial agent. Finally, sterile water was added to make the working volume of the experimental group and the control group 100 mL. Three replicates were set for each treatment. The temperature was controlled at 45 °C. The biogas production was tested every day. The VS in each system was measured before and after fermentation and the utilization of organic matter was calculated. The results showed that ( Figure 5 When the added ammonia nitrogen concentration was 1000 mg / L, the average cumulative gas production in the experimental group was 651.0 mL, a 29.08% increase compared to the control group's cumulative gas production of 504.3 mL. Furthermore, the amount of residual organic matter (measured in VS) decreased by 7.25% compared to the control group. When the added ammonia nitrogen concentration was 3000 mg / L, the cumulative gas production in the experimental group was 336.0 mL, a 13.00% increase compared to the control group's cumulative gas production of 297.3 mL. Furthermore, the amount of residual organic matter (measured in VS) decreased by 7.85% compared to the control group.

[0076] Example 6 Effect of bacterial agents on alleviating ammonia stress in medium-temperature, medium-high-temperature, and high-temperature wet anaerobic fermentation of food waste

[0077] Food waste was collected from the Xiaoshan Food Waste Treatment Plant as a food waste slurry; activated sludge was collected from an anaerobic digester operated by Hangzhou Energy and Environmental Engineering Co., Ltd. in Hangzhou, Zhejiang Province. The physical and chemical properties of the activated sludge and food waste used in the reactor are shown in Table 1.

[0078] Table 1. Physicochemical properties of food waste and activated sludge

[0079]

[0080] This experiment was conducted in a completely stirred anaerobic digestion reactor with a working volume of 30 L. Based on the VS of the food waste slurry, the organic loading rate of the reactor was controlled to be 2.1 g / (L·d) -1Feed and discharge were performed daily from the top feed port. The generated gas was discharged through the exhaust port, and the gas volume was measured using a gas flowmeter connected to the exhaust port. Three reactors (M, MH, and H) were set up for anaerobic fermentation at 37°C, 45°C, and 55°C, respectively. The generated gas volume was recorded daily. Biogas indicators such as methane content, pH, and ammonia nitrogen concentration were also monitored.

[0081] After the reactor gas production stabilizes (denoted as Blank), the ammonia nitrogen concentration of the system is adjusted by adding NH4Cl solids. A gradient increase in the added ammonia nitrogen concentration is adopted so that the anaerobic digestion systems at the three temperatures eventually reach an ammonia inhibition state. That is, the added ammonia nitrogen concentration is first increased to 1000 mg / L and maintained for 5 to 7 days. Pay attention to indicators such as methane production. If the methane production drops to 20% of the stable stage (Blank), stop increasing the added ammonia nitrogen concentration and enter the bio-augmentation stage. If the methane production does not decrease significantly, continue to add NH4Cl solids to increase the added ammonia nitrogen concentration in the system. Each gradient increases the added ammonia nitrogen concentration by 500 mg / L compared to the previous gradient. After entering the bio-augmentation stage, 20% of the total volume of food waste and anaerobic activated sludge is inoculated with liquid bacterial agent (at a concentration of at least 1×10 8 CFU / mL. Take 20% of the total system volume of the inoculum, centrifuge, and resuspend the bacterial pellet in sterile water (1 / 10 the volume of the bacterial solution). Inoculate the bacterial suspension into the reactor and pay attention to indicators such as methane production to verify the effect of the inoculum on alleviating ammonia inhibition in anaerobic digestion at different temperatures.

[0082] The results are as follows Figure 6 As shown, in the reactor operating at medium temperature, when the added ammonia nitrogen concentration was 2500 mg / L, the average methane production decreased by 28.07%, but after the addition of the bacterial agent, the average methane production increased by 24.08% compared with the inhibition state. In the reactor operating at medium-high temperature, when the added ammonia nitrogen concentration was 3000 mg / L, the average methane production decreased by 20.13%, but after the addition of the bacterial agent, the average methane production increased by 28.58% compared with the inhibition state. In the reactor operating at high temperature, when the added ammonia nitrogen concentration was 2500 mg / L, the average methane production decreased by 36.40%, but after the addition of the bacterial agent, the average methane production increased by 17.66% compared with the inhibition state. This shows that the bacterial agent has a significant effect in alleviating ammonia inhibition.

[0083] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A medium-temperature anaerobic bacterium Anaerobic_zm02 that increases methane production and alleviates ammonia inhibition, characterized in that: The strain is classified and named as Schnuerera sp., and its deposit number is CGMCC No.46069.

2. A flora, characterized in that Including Anaerobic_zm02 as described in claim 1.

3. A bacterial agent, characterized in that It comprises the Anaerobic_zm02 described in claim 1 or the flora and auxiliary materials described in claim 2.

4. The bacterial agent according to claim 3, characterized in that The auxiliary materials include minerals, vitamins, antibiotics, culture media, stabilizers, anti-flocculants, antioxidants and / or solid media.

5. Application of at least one of the following A) to C) in waste fermentation: A), Anaerobic_zm02 as described in claim 1; B) the bacterial colony according to claim 2; C) The bacterial agent according to claim 3 or 4.

6. The use according to claim 5, characterized in that: The waste includes food waste.

7. A method for waste fermentation, characterized in that: The method comprises fermenting waste using at least one of the following a) to c): a) Anaerobic_zm02 as described in claim 1; b) the bacterial flora according to claim 2; c) The bacterial agent according to claim 3 or 4.

8. Use of at least one of the following I) to III) in degrading organic matter, assisting in increasing methane production and / or relieving ammonia inhibition: 1) Anaerobic_zm02 according to claim 1; II), the bacterial colony according to claim 2; III), the bacterial agent according to claim 3 or 4.

9. A product for degrading organic matter, assisting in increasing methane production and / or relieving ammonia inhibition, characterized in that: The raw materials include at least one of the following i) to iii) and other functional substances that assist in the degradation of organic matter, increase methane production and / or relieve ammonia inhibition: i) Anaerobic_zm02 as described in claim 1; ii) the bacterial flora according to claim 2; iii) The bacterial agent according to claim 3 or 4.

10. A method for degrading organic matter, increasing methane production and / or relieving ammonia inhibition, characterized in that: Including using at least one of the following i) to iv): i) Anaerobic_zm02 as described in claim 1; ii) the bacterial flora according to claim 2; iii) the bacterial agent according to claim 3 or 4; iv) The product according to claim 9.

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