Method for producing methane by anaerobic digestion of domestic sewage

Abstract

The invention provides a method for producing methane by anaerobic digestion of domestic sewage, and belongs to the technical field of environmental protection. The invention provides a method for producing methane by anaerobic digestion of domestic sewage, which comprises the following steps: pretreating black water by using a complex microbial inoculant, and mixing the pretreated black water with an inoculum to obtain a fermentation substrate; and mixing grey water containing a surfactant into the fermentation substrate, and then carrying out anaerobic digestion, wherein the generated gas contains methane. The invention also constructs a kit beneficial to methane production by anaerobic digestion of domestic sewage, and the kit contains an inoculum, a complex microbial inoculant and a protective agent. The method and the kit are suitable for ecological toilets, and the anaerobic digestion methane production capability of the ecological toilets on domestic sewage produced by the ecological toilets can be enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental protection, and particularly relates to a method for producing methane by anaerobic digestion of domestic sewage. Background Art

[0002] An eco-toilet is an environmentally friendly facility that uses microorganisms to decompose feces. Through biodegradation technology, feces and urine are converted into harmless, energy-efficient, and resource-efficient substances, achieving environmental protection, energy conservation, and wastewater resource recycling. Eco-toilets are divided into various functional types, among which eco-toilets that integrate bathing and anaerobic digestion and methane production systems (hereinafter referred to as eco-toilets) are widely accepted and trusted in rural areas, becoming a hot direction for industrialization.

[0003] Ecological toilets utilize surfactant-rich bathing wastewater (hereafter referred to as graywater) to flush human feces and urine (hereafter referred to as blackwater) into an anaerobic digestion system (graywater and blackwater together constitute domestic sewage). By adding inoculum to the system and creating an anaerobic environment, the anaerobic digestion of domestic sewage and the production of methane are achieved. The methane produced by anaerobic digestion in eco-toilets is a clean energy source that not only significantly reduces environmental pollution caused by domestic sewage discharge but also effectively mitigates greenhouse gas emissions caused by the discharge and accumulation of blackwater.

[0004] China has published patents such as CN114811710A (a combined heat and power and biogas eco-toilet system), CN217559946U (a combined heat and power and biogas eco-toilet device), and CN116651079A (a solid-liquid separation device in an eco-toilet). These have developed complete technical solutions for the structure, function, and system architecture of eco-toilets and have achieved industrial application. These eco-toilets integrate a solar thermal power energy system, a bathing system, a blackwater solid-liquid separation system, and an anaerobic digestion system for blackwater and graywater methane production, effectively utilizing solar energy, blackwater, and graywater resources. However, these systems currently suffer from unstable blackwater anaerobic digestion reactions and low methane production efficiency. In particular, the introduction of graywater causes a significant decrease in methane production from anaerobic digestion, or even cessation of methane production. This key technical issue urgently needs to be analyzed and resolved. Summary of the Invention

[0005] The present invention provides a method for producing methane by anaerobic digestion of domestic sewage. The method is a highly efficient and stable anaerobic digestion method for producing methane in domestic sewage from ecological toilets, and can significantly improve the ability of anaerobic digestion of domestic pollution to produce methane.

[0006] The present invention provides a method for producing methane by anaerobic digestion of domestic sewage, comprising the following steps:

[0007] (1) mixing black water from domestic sewage with a composite bacterial agent T1 to obtain pretreated black water; the composite bacterial agent T1 comprises Pseudomonas fluorescens CBS5, Pseudomonas alcaligenes CBS7, Pseudomonas psychrophilus CBSB, and Zoogloea ramificans CBS4;

[0008] (2) mixing the pretreated black water of step (1) with the inoculum to obtain a fermentation substrate;

[0009] (3) The fermentation substrate of step (2) is mixed with gray water in domestic sewage and then subjected to anaerobic digestion, whereby the generated gas contains methane; the gray water in the domestic sewage contains a surfactant, wherein the types of the surfactant include sodium stearate and / or sodium lauryl sulfate, and after the mixing, the content of sodium stearate in the mixed solution is not higher than 1200 mg / L, and the content of sodium lauryl sulfate is not higher than 600 mg / L.

[0010] In a preferred embodiment of the present invention, the added mass of the composite bacterial agent T1 is 0.01% to 12% based on the volume of the black water in the domestic sewage.

[0011] In a preferred embodiment of the present invention, in the composite bacterial agent T1, the addition amount of Pseudomonas fluorescens CBS5 is 1-3%, the addition amount of Pseudomonas alcaligenes CBS7 is 1-3%, the addition amount of Pseudomonas psychrophilus CBSB is 1-3%, and the addition amount of Zoogloeosporium branching CBS4 is 1-3%.

[0012] In a preferred embodiment of the present invention, the pretreatment time using the composite bacterial agent T1 is 12 to 24 hours.

[0013] In a preferred embodiment of the present invention, the ratio of the volatile solid content of the black water in the domestic sewage in step (1) to the volatile solid content of the inoculum in step (2) is (4:1) to (1:4).

[0014] In a preferred embodiment of the present invention, the mixing in step (3) further includes adding a protective agent.

[0015] In a preferred embodiment of the present invention, the protective agent includes betaine.

[0016] In a preferred embodiment of the present invention, based on the total solid content of the fermentation matrix in step (2), the amount of betaine added is 0 to 1.16 g / g of the total solids in the fermentation matrix.

[0017] The present invention also provides application of the above method in increasing the volume of methane produced by anaerobic digestion of domestic sewage produced by an ecological toilet device.

[0018] The present invention also provides a kit capable of increasing the volume of methane produced by anaerobic digestion of domestic sewage produced by an ecological toilet device, comprising independently packaged inoculum, a composite bacterial agent T1 and a protective agent; the composite bacterial agent comprises at least one of the following bacterial species: the composite bacterial agent T1 comprises Pseudomonas fluorescens CBS5, Pseudomonas alcaligenes CBS7, Pseudomonas psychrophilus CBSB and Zoogloea ramificans CBS4.

[0019] Beneficial Effects: The present invention provides a method for producing methane through anaerobic digestion of domestic sewage, comprising pre-treating black water with a composite bacterial agent T1, mixing the pre-treated black water with an inoculum to obtain a fermentation substrate, and then mixing the fermentation substrate with gray water for anaerobic digestion, whereby the generated gas contains methane. The present invention also provides a kit for increasing the volume of methane produced by anaerobic digestion of domestic sewage, comprising an inoculum, composite bacterial agent T1, and a protective agent.

[0020] In the examples of the present invention, by comparing the VS ratio of domestic sewage and inoculum, the content of sodium stearate and sodium lauryl sulfate, and the methane production capacity of composite inoculants composed of different bacterial species, it was found that cell and enzyme protectants such as betaine can synergize with surfactants and composite inoculants to enhance the methane production capacity of anaerobic digestion of domestic sewage generated in eco-toilets. Under the optimal process parameters of the present invention, the cumulative methane volume on day 8 was 12.22 times that of the control group. Furthermore, under the conditions of the eco-toilet device, the cumulative methane volume measured after gas production ceased was 2.98 times that of the control group. This demonstrates that the method of the present invention is an efficient and stable anaerobic digestion method for methane production of domestic sewage in eco-toilets, significantly improving the methane production capacity of anaerobic digestion of domestic sewage, thereby promoting the organic connection between the rural toilet revolution and the treatment of domestic sewage in eco-toilets. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the result graph showing the effect of the VS ratio of black water and inoculum on the cumulative methane volume on the 8th day;

[0022] Figure 2 This is the result of screening the appropriate content of SS or SDS in the fermentation matrix;

[0023] Figure 3 The effect of SS, SDS and betaine (B) on the cumulative methane volume on the 8th day;

[0024] Figure 4This is a composite bacterial agent screening diagram. Figure A: Effect of betaine addition on the cumulative methane volume on the 8th day; B: Effect of different composite bacterial agents and their pretreatment time on the cumulative methane volume on the 8th day; C: Composite bacterial agent screening diagram. The LSD method of SPSS software (version: 22.0) was used to perform a one-way ANOVA on the cumulative methane production of the latter and former groups on the 8th day. The characters above the bar graph of each group are the difference test results, "**" indicates an extremely significant difference (P < 0.01), and "*" indicates a significant difference (P < 0.05).

[0025] Figure 5 Response value plot for the optimized fitting model; coding level is the same as Table 1;

[0026] Figure 6 This is the optimization diagram of the black water pretreatment time of the composite bacterial agent T1-OPT; the asterisk above the bar graph means T1-OPT 12 The results of one-way ANOVA using the LSD method between the group and other groups were analyzed. "**" indicates significant differences at the 0.01 level.

[0027] Figure 7 This is the internal structure diagram of the ecological toilet device;

[0028] Figure 8 This is an application diagram of an ecological toilet device. DETAILED DESCRIPTION

[0029] The present invention provides a method for increasing the volume of methane produced by anaerobic digestion of domestic sewage, comprising the following steps:

[0030] (1) mixing black water from domestic sewage with a composite bacterial agent T1 to obtain pretreated black water; the composite bacterial agent T1 comprises Pseudomonas fluorescens CBS5, Pseudomonas alcaligenes CBS7, Pseudomonas psychrophilus CBSB, and Zoogloea ramificans CBS4;

[0031] (2) mixing the pretreated black water of step (1) with the inoculum to obtain a fermentation substrate;

[0032] (3) The fermentation substrate of step (2) is mixed with gray water in domestic sewage and then subjected to anaerobic digestion, whereby the generated gas contains methane; the gray water in the domestic sewage contains a surfactant, wherein the types of the surfactant include sodium stearate and / or sodium lauryl sulfate, and after the mixing, the content of sodium stearate in the mixed solution is not higher than 1200 mg / L, and the content of sodium lauryl sulfate is not higher than 600 mg / L.

[0033] The present invention does not specifically limit the specific type of the ecological toilet. For example, the ecological toilets disclosed in the patents CN114811710A (a thermoelectric biogas combined ecological toilet system), CN217559946U (a thermoelectric biogas combined ecological toilet device) and CN116651079A (a solid-liquid separation device in an ecological toilet) are not particularly limited. In one embodiment, the ecological toilet has Figure 5 The internal structure shown, and Figure 6 The application diagram of ecological toilet is shown.

[0034] The domestic sewage of the present invention includes grey water and black water, wherein the grey water includes bathing wastewater and the black water includes a mixture of human feces and urine.

[0035] The present invention mixes blackwater from domestic sewage with a composite bacterial agent T1, which includes Pseudomonas fluorescens CBS5 (MW981369.1), Pseudomonas alcaligenes CBS7 (MW981370.1), Pseudomonas psychrophilus CBSB (MW981371.1), and Zoogloea ramosissima CBS4 (MW981368.1). This compound produces digestive enzymes, including at least one of protease, amylase, lipase, cellulase, and lignocellulose. The addition of this compound accelerates the degradation of extracellular polymers in domestic sewage, thereby enhancing the anaerobic digestion and methanogenesis process.

[0036] In the examples of the present invention, three different composite bacterial agents were used for comparison, including composite bacterial agent T1, composite bacterial agent T2, and silage agent GSSW; composite bacterial agent T2 was a combination of Pseudomonas fluorescens CBS5, Pseudomonas alcaligenes CBS7, and Pseudomonas psychrophilus CBSB; silage agent GSSW included lactic acid bacteria MXLZ-1, MXLZ-2, and MXLZ-4, as well as cellulase-producing Bacillus Velezella K1 (GSICC 42323) and amylase-producing Bacillus subtilis J1 (GSICC 78393). The addition of the composite bacterial agent of the present invention, composite bacterial agent T1, significantly promoted the cumulative methane volume of anaerobic digestion of domestic sewage. The composite bacterial agent T1 and composite bacterial agent T2 described in the present invention have been disclosed in the article (Yang Tao, Yao Yangyang, et al. Composite bacterial agent regulates the nutrients in the rhizosphere soil of continuously cropped Angelica sinensis and its effect on yield. Bulletin of Microbiology. 2022); the silage bacterial agent GSSW was also disclosed in the article (Mao Ting, Niu Yongyan, et al. Effect of bacterial agent on the fermentation quality and microbial community of alfalfa silage. Bulletin of Biotechnology. 2021).

[0037] In the present invention, the addition amount of the composite bacterial agent T1 is 1-3% for Pseudomonas fluorescens CBS5, 1-3% for Pseudomonas alcaligenes CBS7, 1-3% for Pseudomonas psychrophilus CBSB, and 1-3% for Zoogloeosporium ramifieds CBS4. In a preferred embodiment of the present invention, the addition amount of the composite bacterial agent T1 is 0.01% to 12% of the black water volume, and the black water pretreatment time is 12 to 24 hours. The addition amount herein refers to the mass concentration percentage of the added agent.

[0038] The present invention mixes pretreated black water with an inoculum to prepare a fermentation matrix. The inoculum is composed of an anaerobic digestion bacterial community, a methanogenic archaeal community, and organic matter, including methane-producing anaerobic bacteria, such as anaerobic bacteria and / or anaerobic archaea. The present invention adds a mixture of bacterial communities rich in organic matter-degrading and methanogenic archaeal communities, along with organic matter that serves as nutrients for the communities, to the eco-toilets to increase gas production. In one embodiment, the inoculum was obtained from the Western Energy and Environmental Research Center of Lanzhou University of Technology. The inoculum was maintained at 37°C for a long period of time. Samples were taken when the methane volume fraction reached a stable level of 50% or higher, and large solid impurities were filtered out before use. This inoculum has been previously disclosed in the article (Fast treatment and recycling method of large-scale vegetable wastes, DOI: 10.1016 / j.scitotenv.2023.164308).

[0039] The present invention uses a VS ratio to characterize the mixing ratio of blackwater and the inoculum in an eco-toilet, where VS is short for volatile solids. The VS ratio of blackwater to the inoculum in the domestic sewage of the present invention can be (4:1) to (1:4), such as 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, or 1:4. Of course, it can also be a decimal ratio between any two ratios. In one embodiment, blackwater is mixed with the inoculum. As the VS ratio of blackwater to inoculum decreases, the cumulative methane production volume on the eighth day exhibits an inverted U-shaped change pattern, with the cumulative methane production volume reaching its highest level at a VS ratio of 1:3.

[0040] The present invention mixes the fermentation substrate with gray water from domestic sewage, wherein the gray water contains residues of cleaning and bathing substances, such as soap, hand sanitizer, or shower gel, and these ingredients contain surfactants (SF), and the types of SF mainly include sodium stearate (SS) and / or sodium dodecyl sulfate (SDS). In the anaerobic digestion system of the present invention, the SF has a promoting effect on anaerobic digestion and methane production, so the concentration of the SF content needs to be controlled. Generally, the maximum concentrations of SS and SDS in domestic sewage are required to be 1200 mg / L and 600 mg / L, respectively. The present invention does not specifically limit the method for determining the content of the SF, and the method known in the art for determining the corresponding SS and / or SDS can be used for determination.

[0041] The present invention uses black water with varying SS and / or SDS contents as the basis for anaerobic digestion, based on black water containing less than 1200 mg / L SS and less than 600 mg / L SDS. The highest cumulative methane volume was achieved on day 8 when the SS content was 82.3 mg / g TS and the SDS content was 13.7 mg / g TS.

[0042] In a preferred embodiment of the present invention, a protective agent may be added when mixing the fermentation substrate and the gray water. The protective agent may protect cells and enzymes in high-salt and other adverse environments, increase the survival rate of bacterial cells, and enhance the stress resistance of enzymes. In one embodiment, betaine is used as an example for illustration, but it cannot be considered the only protection scope of the present invention. In the present invention, general bath products (such as facial masks, skin care and skin moisturizing products) may contain betaine, but the betaine content in bath products is low and does not affect the amount of betaine added according to the present invention. In one embodiment, the betaine is added in an amount of 0 to 1.16 g / g TS relative to TS in the fermentation substrate.

[0043] The present invention performs anaerobic digestion on the anaerobic digestion system constructed as described above. In one embodiment, the anaerobic digestion is performed in a laboratory simulation, for example, at 37° C., 120 rpm, and in a 500 mL serum bottle with a working volume of 400 mL, nitrogen is passed through a catheter into the serum bottle for 2 minutes before initiating the anaerobic digestion reaction.

[0044] In the present invention, the composite bacterial agent can secrete a variety of digestive enzymes (including proteases and amylases) that promote the degradation of organic matter in black water, which is beneficial to the conversion of organic matter in black water into small molecules and is beneficial to the anaerobic digestion and methane production process of domestic sewage in ecological toilets; the surfactant promotes the dissolution of solid organic matter in the polymer in the fermentation matrix through emulsification and releases the microorganisms and enzymes in the polymer, thereby promoting the degradation of organic matter in the fermentation matrix, but the surfactant has a certain toxicity to microbial cells and enzymes, can destroy cell membranes and enzyme structures, and reduce the volume of methane produced by anaerobic digestion of the fermentation matrix; betaine can enhance the resistance of microbial cells to adverse environments, including high-salt environments. Black water, gray water and surfactants carry more sodium ions. Therefore, the fermentation matrix of the anaerobic digestion system of the ecological toilet is enriched with a high content of sodium ions. The addition of betaine can help microbial cells and their enzymes resist the high sodium salt environment, thereby promoting the volume of methane produced by anaerobic digestion of domestic sewage. At the same time, the surfactant adsorbs to the cell membrane proteins and phospholipid molecules and the active centers of the enzymes through the hydrophobic ends, and disturbs the hydrophilic ends located in the aqueous phase, causing the membrane proteins and phospholipids to move out of their original positions, forming cell membrane cavities, destroying enzyme activity, and causing cell damage. This is completely different from the cell damage mechanism caused by high salt osmotic pressure. However, even in these two adversities, the present invention found that betaine can still cooperate with surfactants to significantly promote the volume of methane production.

[0045] The present invention also provides application of the above method in increasing the volume of methane produced by anaerobic digestion of domestic sewage produced by an ecological toilet device.

[0046] The present invention also provides a kit capable of increasing the volume of methane produced by anaerobic digestion of domestic sewage produced by an ecological toilet device, comprising independently packaged inoculum, a composite bacterial agent T1 and a protective agent; the composite bacterial agent comprises at least one of the following bacterial species: the composite bacterial agent T1 comprises Pseudomonas fluorescens CBS5, Pseudomonas alcaligenes CBS7, Pseudomonas psychrophilus CBSB and Zoogloea ramificans CBS4.

[0047] In a preferred embodiment of the present invention, the protective agent includes betaine. Figure 5 and Figure 6 The structure shown.

[0048] The present invention also provides the use of the kit in increasing the volume of methane produced by anaerobic digestion of domestic sewage produced by an ecological toilet device.

[0049] In one embodiment of the present invention, under the optimal parameters of a simulated environment, the kit of the present invention was used to treat domestic sewage in an ecological toilet device. The cumulative methane volume on the 8th day was 12.22 times that of the control group. Further, under the conditions of the ecological toilet device, the cumulative methane volume measured after stopping gas production was 2.98 times that of the control group.

[0050] To further illustrate the present invention, a method for producing methane by anaerobic digestion of domestic sewage provided by the present invention is described in detail below with reference to examples, but these examples should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] 1. Experimental Materials

[0053] 1.1 Inoculum

[0054] The inoculum was obtained from the Western Energy and Environmental Research Center of Lanzhou University of Technology. The inoculum was maintained at 37°C for a long period of time. Samples were taken when the methane volume fraction reached a stable level of 50% or higher. Large solid impurities were filtered out and then set aside. The inoculum had the same composition and efficacy as that disclosed in the article (Fast treatment and recycling method of large-scale vegetable wastes, DOI: 10.1016 / j.scitotenv.2023.164308). The inoculum was fed with starch and a small amount of black water and cow dung to maintain bacterial activity.

[0055] 1.2 Blackwater

[0056] Black water samples were collected from the sewage outlet of the library restroom at the Langongping campus of Lanzhou University of Technology. The restrooms were not equipped with any detergents containing surfactants. To verify the absence of SS and SDS, two experiments were conducted: a) Phosphoric acid was added to the black water to fully convert SS into stearic acid. The stearic acid was then esterified using the same method described in the literature (Kinetic study on anaerobic digestion of long-chain fatty acid enhanced by activated carbon adsorption and direct interspecies electron transfer, DOI: 10.1016 / j.biortech.2024.130902). The esterification product was then analyzed by gas chromatography and the stearic acid content was calculated. b) The black water was concentrated 100-fold and SDS content was determined using the SDS Residue Detection Kit (Cat. No. C500055-0100) from Sangon Biotech (Shanghai) Co., Ltd. The results showed that neither SS nor SDS was detected in the black water samples, indicating that the black water samples did not contain surfactants. The black water samples were stored at 4°C until further use.

[0057] 1.3 Composite microbial agents T1, T2 and GSSW

[0058] It is deposited in the Gansu Branch of the China Industrial Microbial Culture Collection Center and has been published in articles (Yang Tao, Yao Yangyang, et al. Effects of composite microbial agents on rhizosphere soil nutrients and yield of continuously cropped Angelica sinensis. Bulletin of Microbiology. 2022; Mao Ting, Niu Yongyan, et al. Effects of microbial agents on fermentation quality and microbial community of alfalfa silage. Bulletin of Biotechnology. 2021).

[0059] 2 Experimental methods

[0060] 2.1 Experimental system

[0061] The experiment was initiated at 37°C, 120 rpm, and a serum bottle (500 mL) containing 400 mL of fermentation substrate (a mixture of black water and inoculum) was flowed with nitrogen gas via a tube for 2 minutes. Preliminary experiments revealed that anaerobic digestion ceased to produce gas after 8 days. Therefore, the accumulated biogas over the 8-day period was collected using a gas collection bag. The methane volume fraction of the accumulated biogas was measured using a Biogas 5000, and the accumulated biogas volume was determined using the water displacement method. Cumulative methane volume = cumulative biogas volume × cumulative methane volume fraction.

[0062] 2.2 Optimizing the VS ratio of black water to inoculum

[0063] The 8-day cumulative methane volume was used as a positive indicator to optimize the black water to inoculum VS ratio within the gradient range (4:1, 3:1, 2:1, 1:1, 1:2, 1:3 and 1:4).

[0064] 2.3 Simulation of the maximum content of SS or SDS in domestic sewage

[0065] Taking soap with the highest SF content (calculated as 100% sodium stearate content) as an example, the water consumption for each hand washing is about 1 L and the maximum amount of soap used is about 1.2 g. Therefore, the maximum concentration of SS in gray water is about 1200 mg / L. The content of SDS in bath products is relatively low, and the maximum concentration of SDS in gray water is about 600 mg / L. The volume of gray water used for flushing the toilet each time is 1 to 6 L. If the volume change caused by complete homogenization of feces into gray water is not considered, and the volume of a single urination is about 0.2 L, the volume of domestic sewage discharged each time is 1.2 to 6.2 L. The maximum mass concentrations of SS and SDS in sewage range from 1000 to 1161 mg / L and 500 to 581 mg / L, respectively. Considering that this maximum concentration is close to the maximum concentration of SS and SDS in gray water, the maximum concentrations of SS and SDS in domestic sewage are set at 1200 mg / L and 600 mg / L, respectively.

[0066] 2.4 Optimizing the concentration of SS or SDS in black water

[0067] The mixing ratio of black water and inoculum in the fermentation matrix was determined by optimizing the VS ratio in Section 2.2 of Example 1. SS (1200, 1000, 800, 600, 400, and 200 mg / L) or SDS (600, 500, 400, 300, 200, and 100 mg / L) of different concentration gradients were added to the black water. The fermentation matrix was then prepared according to the mixing ratio of black water and inoculum determined in this step to form each experimental group. The control group was set up with no addition of SS or SDS to the black water. The optimal ratio of SS or SDS to the total solids (TS) content of the fermentation matrix (respectively denoted as SS) was screened. max and SDS max ), so that the accumulated methane volume is the largest on the 8th day of anaerobic digestion.

[0068] 2.5 Betaine synergistically promotes methane production in anaerobic digestion with SS or SDS

[0069] Following the same method as in the previous step, 8 experimental groups were set up: group without SF and betaine (CK), group without SF and betaine (SS), ... max Group, SS min Group (SS min =SS max / 2) SDS max 、SDS min Group (SDS min =SDS max / 2)、Group B、Group SSB(SS max The fermentation medium of the group was supplemented with 1 g / L betaine, SDB group (SDS max Group added 1g / L betaine), SSDS min Group (SS min +SDS min ), SSDS med1 Group (SS max +SDS min ), SSDS med2 Group (SS min +SDS max ), SSDS max Group (SS max +SDS max )、SSDSB min Group (SSDS min Group supplemented with 1g / L betaine), SSDSB med1 Group (SSDS med1 Group supplemented with 1g / L betaine), SSDSB med2 Group (SSDS med2 Group supplemented with 1g / L betaine), SSDSB max Group (SSDS maxThe optimized process was selected based on the addition of 1 g / L betaine to each group (the group with the highest cumulative methane volume on day 8). The SS and / or SDS and betaine addition method was as follows: using the optimal ratio of SS or SDS to the TS of the fermentation substrate obtained in the previous step, SS and / or SDS were added to the fermentation substrate of the corresponding group, and then betaine was added to the fermentation substrate with or without SS and / or SDS, with a betaine concentration of 0.116 g / g TS relative to the fermentation substrate.

[0070] 2.6 Screening of pretreatment composite bacterial agents

[0071] Three batches of experiments were conducted to screen the process that maximized the cumulative methane volume on the 8th day: a) Based on the optimized process obtained in the previous step, the addition amount of betaine relative to the fermentation matrix was set to 0, 0.058, 0.116, 0.232, 0.348, 0.464, and 0.58 g / g. TS, screen an optimized process; b) according to the same method as the optimized process in step a) of this section, add composite bacterial agents T1, T2 and GSSW at an inoculum amount of 1% to black water without SS and SDS, respectively, and pretreat for 12, 24, and 36 hours, respectively, and screen the group with the largest cumulative methane volume on the 8th day of anaerobic digestion as the optimized process; c) according to the same method as the optimized process in step b) of this section, set the addition gradient of composite bacterial agents in black water without SS and SDS to 0.5, 1, 2, and 3%, and use the experiment with a black water and inoculum VS ratio of 1:1 in 2.2 of Example 1 and the experiment with a betaine addition amount of 0.116 g / g TS in step a) of this section as controls to screen a process that maximizes the cumulative methane volume on the 8th day.

[0072] 2.7 Screening the best process

[0073] 2.7.1 Central Composite Design: Preliminary experiments revealed that when the amount of bacterial species in composite agent T1 alone was higher than 3%, the cumulative methane volume decreased significantly. Therefore, the addition range of each bacterial species was set to 0-3%. In Design-Expert (version: 13.0.1), a central composite design (CCD) method was used to design a 4-factor (different bacterial species) 3-level (different addition amount) experiment. Pretreatment was performed for 24 hours, and the cumulative methane volume on the 8th day was used as the response value. By constructing a fitting model for the interaction of factors, the addition amount of each bacterial species in composite agent T1 (i.e., the compounding method) was optimized. This composite agent T1 with the optimized compounding method was used as composite agent T1-OPT for subsequent optimization of pretreatment time.

[0074] 2.7.2 Optimizing the pretreatment time of the composite bacterial agent T1-OPT: The T1-OPT was set according to the control group, B3, T11 in Section 2.6 of Example 1, and the same method as that of the B3 group in Section 2.6 of Example 1. 12 (i.e., composite bacterial agent T1-OPT pre-treated black water for 12 hours), T1-OPT 24 (Pretreatment of black water with composite bacterial agent T1-OPT for 24 hours), T1-OPT 36 (i.e., composite bacterial agent T1-OPT pre-treated black water for 36 hours), T1-OPT 48 The experiments were set up in comparison with the anaerobic enhancing bacteria (collectively referred to as compound bacteria agent LBY) produced by Shandong Liboyuan Environmental Protection Materials Co., Ltd. for increasing the biogas production of domestic sewage (i.e., pre-treatment of black water with compound bacteria agent T1-OPT for 48 hours). The compound bacteria agent LBY is composed of enterococci, yeast and nutrient complexes, with an effective live bacteria count of 5 billion / gram. The compound bacteria agent LBY was used according to the instructions.

[0075] 3. Results Analysis

[0076] 3.1 Optimizing the VS ratio of black water to inoculum and the addition amount of SS and SDS

[0077] The results are as follows Figure 1 As shown in the figure, as the VS ratio of blackwater to inoculum decreases, the cumulative methane production volume on day 8 shows an inverted U-shaped change pattern, with the highest cumulative methane production volume at a VS ratio of 1:3. Therefore, the VS ratio of blackwater to inoculum of 1:3 is collectively referred to as the optimized VS ratio.

[0078] The fermentation substrate was prepared according to the optimized VS ratio, and different addition amounts of SS or SDS were set to test the cumulative methane volume on the 8th day.

[0079] The results are as follows Figure 2 As shown in Figure 2, with the increase of SS or SDS addition, the cumulative methane volume on the 8th day showed a trend of first increasing and then decreasing. When the SS addition amount in black water was 600 mg / L or the SDS addition amount was 200 mg / L, the cumulative methane volume on the 8th day reached the maximum value. At this time, the addition concentrations of SS and SDS relative to the fermentation substrate were 82.3 and 27.4 mg / g TS, respectively, which were set as SS max (82.3 mg / g TS) and SDS max (27.4 mg / g TS).

[0080] 3.2 Betaine synergistically with SS or SDS to enhance methanogenesis in anaerobic digestion of blackwater

[0081] According to SS max (82.3 mg / g TS) and SDS max(27.4mg / g TS) value, set CK group, SS min Group, SS max Group, SDS min Group, SDS max Group, Group B, SSB Group, SDB Group, SSDS min Group (SS min +SDS min ), SSDS med1 Group (SS max +SDS min ), SSDS med2 Group (SS min +SDS max ), SSDS max Group (SS max +SDS max )、SSDSB min Group (SSDS min Group supplemented with 1g / L betaine), SSDSB med1 Group (SSDS med1 Group supplemented with 1g / L betaine), SSDSB med2 Group (SSDS med2 Group supplemented with 1g / L betaine), SSDSB max Group (SSDS max The cumulative methane volume after anaerobic digestion for 8 days was as follows: Figure 3 As shown in the figure, SS and SDS can promote the 8-day cumulative methane volume, and betaine can synergize with SS and / or SDS to significantly promote the 8-day cumulative methane volume. med1 The group with the largest cumulative methane volume was taken as the optimized condition in this step.

[0082] 3.3 Screening of pretreatment composite bacterial agents for methanogenesis in anaerobic digestion of domestic sewage

[0083] In order to screen the pretreatment composite bacterial agent for methanogenesis in anaerobic digestion of domestic sewage, based on the optimized conditions obtained in the previous step, the addition amount of betaine was optimized in batches ( Figure 4 A), screening of composite bacterial agents and their pretreatment time ( Figure 4 B), optimize the addition amount of the screened composite bacterial agent and explore the difference in the cumulative methane volume on the 8th day in the optimized process of each step above ( Figure 4 Middle C). Figure 4The results of Figure A showed that when the addition amount of betaine relative to TS of the fermentation substrate was in the range of 0-1.16 g / g TS, the cumulative methane volume on the 8th day continued to increase. When 0.232 g / g TS was added, the cumulative methane volume on the 8th day reached the maximum, so this was used as the optimized condition. When the addition amount of betaine was higher than 1.044 g / g TS, the cumulative methane volume on the 8th day was significantly lower than that of the control group, indicating that betaine inhibited methane production. Figure 4 The results in Figure B showed that the cumulative methane volume on the 8th day in the B3 group and the composite bacterial agent pretreatment group was significantly higher than that in the control group, and the composite bacterial agent pretreatment effect was better than that in the B3 group. Among them, the cumulative methane volume on the 8th day after 24 hours of pretreatment with composite bacterial agent T1 was significantly higher (P<0.01) than that in other groups, which was used as the optimization condition; 8 groups of experiments were set up according to the same method as the corresponding groups in the present invention, and the cumulative methane volume on the 8th day in each group was significantly higher than that in the CK group (P<0.01), indicating that each optimized process of the present invention can significantly promote the increase of the cumulative methane volume on the 8th day, but the degree of promotion of methane accumulation in different groups was different: (A) Betaine and surfactants (SS and SDS) had a significant synergistic effect (P<0.05); (B) The methane accumulation when the betaine addition amount was 0.232 g / g TS (i.e., group B3) was significantly higher than when the betaine addition amount was 0.116 g / g TS (P<0.05); (C) Based on the process of group B3, the process of pretreating black water with 0.5-3% composite bacterial agent T1 can significantly increase the methane accumulation, among which the 1% composite bacterial agent pretreatment process is significantly better than group B3 (P<0.01). Compared with group B3, the cumulative methane volume of group T11 on the 8th day achieved an extremely significant effect. At the same time, the cumulative methane volume of group T11 on the 8th day was 8.61 times that of group CK.

[0084] 3.4 Screening the best process

[0085] 3.4.1 Optimize the addition amount of each bacterial species in the T1 composite bacterial agent: Table 1 is the CCD test level table, Table 2 is the CCD test results, and construct a fitting model for the interaction effect of different addition amounts of each bacterial species in the composite bacterial agent T1 on the cumulative methane volume on the 8th day. The fitting model formula is shown in Formula 1, where Y represents the cumulative methane volume on the 8th day (mL). The results of variance analysis of the fitting model shown in Formula 1 are shown in Table 3. The results show that: 1) the model test is extremely significant, the lack-of-fit term is not significant, and the corrected coefficient of determination of the model is less than 0.01, indicating that the fitting model is reliable; 2) the variance test of each item of the fitting model shows that the interaction factors ABCD are extremely significant, the interaction factor AC is significant, and the single factors A, B, C, and D are extremely significant, indicating that the interaction factors ABCD and the single factors have extremely significant effects on the cumulative methane volume, and the interaction factor AC has a significant effect on the cumulative methane volume; 3) from the analysis of the fitting model shown in Formula 1, the coefficient of the interaction factor ABCD reaches 105.06, and the coefficient of the interaction factor AC is 30.31. However, the coefficients of the single factors A, B, C, and D are indeed negative, indicating that the interaction factors ABCD have an extremely significant promoting effect on the cumulative methane volume and are the key influencing factors for promoting the cumulative methane volume. The interaction factor AC is a secondary influencing factor for increasing the cumulative methane volume, but the single factors A, B, C, and D can extremely significantly reduce the cumulative methane volume. In summary, when CBS5, CBS7, CBSB, and CBS4 in the composite microbial agent T1 were added individually, they significantly reduced the cumulative methane volume. When CBS5 and CBSB were pretreated together, they significantly increased the cumulative methane volume. Only when the composite microbial agent T1 composed of CBS5, CBS7, CBSB, and CBS4 was pretreated together, could the cumulative methane volume be significantly increased. On this basis, the optimal solution of the fitting model was analyzed with the cumulative methane volume as the positive response value. The results are as follows: Figure 5 As shown in the figure: when the addition amount of CBS5, CBS7, CBSB and CBS4 in the composite bacterial agent T1 is 1.177%, 1.150%, 1.205% and 1.206%, the black water is pretreated with the composite bacterial agent T1 for 24 hours and then Figure 4 The B3 group of medium C conducted anaerobic digestion experiments, and the cumulative methane volume reached the maximum value. The composite bacterial agent T1 with this optimized compounding method was used as the composite bacterial agent T1-OPT for optimizing the pretreatment time.

[0086] Table 1 Factors and coding levels of CCD

[0087]

[0088] Note: Y represents the cumulative methane volume on day 8, in mL. A represents the amount of CBS5 added, in %. B represents the amount of CBSB added, in %. C represents the amount of CBS7 added, in %. D represents the amount of CBS4 added, in %. Identical abbreviations throughout the text have the same meanings as in the table notes.

[0089] Y=105.06×ABCD+22.44×ABC+8.19×ABD+6.69×ACD-12.3×BCD+17.31×AB+30.31×AC+13.8 1×AD-4.44×BC+9.56×BD-0.94×CD-231.67×A-197.28×B-152.06×C-131.33×D-564.19×A 2 -433.69×B 2 -398.69×C 2 -349.19×D 2 +2545.35, formula 1;

[0090] Table 2 CCD test results

[0091] test A B C D Cumulative methane volume (mL) 1 0 0 0 0 1666 2 0 0 0 3 1152 3 0 0 3 0 1135 4 0 0 3 3 1060 5 0 1.5 1.5 1.5 2202 6 0 3 0 0 1041 7 0 3 0 3 1002 8 0 3 3 0 872 9 0 3 3 3 333 10 1.5 0 1.5 1.5 2275 11 1.5 1.5 0 1.5 2311 12 1.5 1.5 1.5 0 2336 13 1.5 1.5 1.5 1.5 2559 14 1.5 1.5 1.5 1.5 2482 15 1.5 1.5 1.5 1.5 2509 16 1.5 1.5 1.5 3 2085 17 1.5 1.5 3 1.5 2011 18 1.5 3 1.5 1.5 1977 19 3 0 0 0 938 20 3 0 0 3 840 21 3 0 3 0 832 22 3 0 3 3 386 23 3 1.5 1.5 1.5 1789 24 3 3 0 0 680 25 3 3 0 3 282 26 3 3 3 0 275 27 3 3 3 3 271

[0092] Table 3 Analysis of variance table of fitting model

[0093]

[0094]

[0095] 3.4.2 Screening the best process for anaerobic digestion of domestic sewage: Figure 6 As shown, T1-OPT 12 The cumulative methane volume of the group was significantly higher than that of the other groups (P<0.01), indicating that after the black water was pretreated with the composite bacterial agent T1-OPT for 12 hours and then anaerobic digested in the same way as the B3 group, the cumulative methane volume on the 8th day was significantly increased. 12 The cumulative methane volume of the T1-OPT group on the 8th day was significantly higher than that of the T11 group (pretreated with the composite bacterial agent T1), the B3 group (not pretreated), and the CK group, which was 1.50 times that of the T11 group, 1.87 times that of the B3 group, and 12.22 times that of the CK group. 12 The cumulative methane volume of the group B3 on day 8 was significantly higher than that of the LBY group (P < 0.01). Therefore, pretreating blackwater with the composite bacterial agent T1-OPT for 12 hours, followed by anaerobic digestion of the pretreated blackwater using the same method as group B3, achieved the desired effect. This method was adopted as the optimal process of the present invention to conduct experiments on anaerobic digestion of domestic sewage for methane production in ecological toilets.

[0096] 3.5 Implementing best practices with eco-toilet installations

[0097] In such Figure 7 and Figure 8 Batch experiments were conducted in an eco-toilet device using fresh human feces and urine to create black water of a certain concentration. The eco-toilet device was configured according to the same parameters as the CK group and the optimal process described in Section 3.4.2 of Example 1. A G16 gas meter was connected in series to the biogas outlet. A certain amount of biogas was collected periodically using a gas collection bag. After gas production ended, the cumulative biogas volume was calculated by calculating the difference between the end and initial readings of the gas meter. Equal volumes of biogas collected at each stage were mixed, and methane concentration was measured using a portable biogas analyzer to determine the cumulative methane volume. The method and kit of the present invention were applied to the eco-toilet device. Results showed that after gas production ceased, the cumulative methane volume of the optimal process group reached 32,062 L, 2.98 times that of the CK group.

[0098] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for producing methane by anaerobic digestion of domestic sewage, characterized in that: The following steps are involved: (1) Mixing black water from domestic sewage with composite bacterial agent T1 to obtain pretreated black water; The composite bacterial agent T1 comprises Pseudomonas fluorescens CBS5, Pseudomonas alcaligenes CBS7, Pseudomonas psychrophilus CBSB and Zoogloea ramificans CBS4; (2) mixing the pretreated black water of step (1) with the inoculum to obtain a fermentation substrate; (3) The fermentation substrate of step (2) is mixed with gray water in domestic sewage and then subjected to anaerobic digestion, whereby the generated gas contains methane; the gray water in the domestic sewage contains a surfactant, and the types of the surfactant include sodium stearate and / or sodium lauryl sulfate; after the mixing, the content of sodium stearate in the mixed solution is not higher than 1200 mg / L, and the content of sodium lauryl sulfate is not higher than 600 mg / L.

2. The method according to claim 1, characterized in that The added mass of the composite bacterial agent T1 is 0.01% to 12% of the volume of the black water.

3. The method according to claim 1 or 2, characterized in that In the composite bacterial agent T1, the addition amount of Pseudomonas fluorescens CBS5 is 1-3%, the addition amount of Pseudomonas alcaligenes CBS7 is 1-3%, the addition amount of Pseudomonas psychrophilus CBSB is 1-3%, and the addition amount of Zoogloea ramificans CBS4 is 1-3%.

4. The method according to claim 3, characterized in that The pretreatment time using the composite bacterial agent T1 is 12 to 24 hours.

5. The method according to claim 1, characterized in that: The ratio of the volatile solid content of the black water in step (1) to the inoculum in step (2) is (4:1) to (1:4).

6. The method according to claim 1, characterized in that During the mixing in step (3), a protective agent is also added.

7. The method according to claim 6, characterized in that The protective agent includes betaine.

8. The method according to claim 7, characterized in that: Based on the total solid content in the fermentation matrix in step (2), the added amount of betaine is 0 to 1.16 g / g of the total solids in the fermentation matrix.

9. Use of the method according to any one of claims 1 to 8 in increasing the volume of methane produced by anaerobic digestion of domestic sewage produced by an ecological toilet device.

10. A method for producing methane by anaerobic digestion of domestic sewage, characterized in that: The invention comprises independently packaged inoculum, a composite bacterial agent T1 and a protective agent; the composite bacterial agent comprises at least one of the following bacterial species: the composite bacterial agent T1 comprises Pseudomonas fluorescens CBS5, Pseudomonas alcaligenes CBS7, Pseudomonas psychrophilus CBSB and Zoogloea ramificans CBS4.

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