Method for enriching anaerobic fermentation acid-producing microbial community, flora and application

Abstract

The invention relates to a method for enriching anaerobic fermentation acid-producing microbial communities, a flora and application of the flora, belonging to the field of biological fermentation. The method comprises an F1 stage, an F2 stage and an F3 stage, and the organic load of the straw is gradually increased in the three stages until the straw is stably operated in the F3 stage. Wherein the stage F1 is a reactor starting stage, and the solid content and the load are low. After the acid production of the F2 is stable, the straw proportion in the material is increased, the C / N ratio is increased, and the material enters the F3 stage, so that the flora adapts to the straw material with the high C / N ratio. According to the method disclosed by the invention, the microbial flora capable of efficiently producing the SCCAs by utilizing the straw material is obtained. The medium-temperature flora and the high-temperature flora can generate SCCAs close to 20 g / L by utilizing the straw with the initial solid content of 5%, and the method has a good application prospect.

Description

Technical Field

[0001] The invention relates to the field of biological fermentation, and more particularly to a method for enriching anaerobic fermentation acid-producing microbial communities, a bacterial community and an application thereof. Background Art

[0002] Anaerobic digestion to produce biogas is a traditional way to utilize straw resources. Compared with biogas, the intermediate metabolites of anaerobic digestion, short chain carboxylic acids (SCCAs), are important bio-based chemicals with wider application potential. Therefore, the development of anaerobic fermentation technology to produce SCCAs from straw has important application significance.

[0003] However, the current research and development on anaerobic fermentation to produce SCCAs mainly uses biodegradable raw materials such as sewage treatment plant sludge, restaurant kitchen waste, and food waste. The easily degradable restaurant kitchen waste / food waste with high sugar content can produce tens of grams of mixed SCCAs per liter through anaerobic fermentation. The accumulation of SCCAs using sludge fermentation is generally less than 10g / L. However, compared with straw, the resources of these waste biomass are relatively limited. Due to the difficult-to-destroy stubborn structure of straw and the difficulty of degradation of three elements, the research and development of SCCAs produced using straw as raw material is very limited. Anaerobic fermentation acid production is a metabolic process of microbial flora composed of various species with hydrolysis and fermentation functions. The flora structure, including species diversity, species uniformity, functional redundancy, etc., determines the rate, efficiency and stability of fermentation acid production. Therefore, the construction of excellent acid-producing flora is the basis for achieving efficient production of SCCAs. Summary of the invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a method for enriching anaerobic fermentation acid-producing microbial communities, and specifically relates to a method for enriching anaerobic fermentation acid-producing microbial communities, a microbial community and an application thereof.

[0005] The present invention uses anaerobic digestion sludge and fermentation acid sludge of different substrates (including kitchen waste, cellulose, sugar, fruit waste, etc.) as inoculation sources. The inoculation sources have high species richness, high diversity, and high functional redundancy. They contain a large number of species with hydrolysis and fermentation functions, providing a microbial basis for enriching excellent straw decomposition flora. Assisted by methanogen inhibition, substrate supply, and enrichment system operation parameter control strategies, a long-term continuous fermentation process is used, based on the combined effects of enrichment and domestication, to obtain a microbial flora that can efficiently produce SCCAs using straw materials. The mesophilic flora and the thermophilic flora can produce nearly 20g / L of SCCAs using straw with an initial solid content of 5%.

[0006] One of the purposes of the present invention is to provide a method for enriching anaerobic fermentation acid-producing microbial communities, comprising F1 to F3 stages:

[0007] F1 stage:

[0008] The inoculum source is added to the reactor, and nitrogen is introduced to exhaust the air before closing the reactor; the solid content of the inoculum source can be ≤5%; after starting the reactor, no material is fed in or discharged for two days, and then the substrate can be supplied under the organic load condition of 0.4-0.6g VTS / L / d; the substrate can include a mixture of straw, kitchen waste and a nitrogen source substrate; the nitrogen source substrate can specifically be residual sludge from a sewage treatment plant; the straw can specifically be steam-exploded straw.

[0009] F2 stage:

[0010] The feed solid content can be adjusted to 8-12%, and the organic load can be increased to 0.8-1.2 g VTS / L / d;

[0011] F3 stage:

[0012] In this stage, the feed solid content can be maintained at 8-12%, and the organic load can be increased to 1.8-2.2g VTS / L / d;

[0013] Collect the microbial flora in the F3 stage reactor.

[0014] Specific practices may also include the following:

[0015] In the F1 stage,

[0016] The F1 phase may run for 80 to 120 days; and / or,

[0017] The TS value of the substrate may be 4.8-5.4%; the VTS value may be 4.0-4.4%; the C / N ratio may be 18-22; and / or,

[0018] The VTS ratio of straw, kitchen waste and nitrogen source substrate in the substrate may be (0.8-1.2): (0.8-1.2): (0.8-1.2); and / or,

[0019] The material can be loaded and unloaded once every two days; and / or,

[0020] The hydraulic retention time of the reactor can be 90 to 110 days.

[0021] In the F2 stage,

[0022] The operating time of the F2 phase may be 50 to 100 days; and / or,

[0023] The substrate TS may be 9.8-10.5%, the substrate VTS may be 8.0-8.5%, and the substrate C / N ratio may be 18-22; and / or,

[0024] The VTS ratio of straw, kitchen waste and nitrogen source substrate in the substrate may be (0.8-1.2): (0.8-1.2): (0.8-1.2); and / or,

[0025] The hydraulic retention time of the reactor can be 90 to 110 days.

[0026] In the F3 stage,

[0027] The operating time of the F3 phase may be 120 to 200 days; and / or,

[0028] The hydraulic retention time of the reactor can be adjusted to 45 to 55 days, and / or,

[0029] The substrate C / N ratio can be increased from 18 to 22 to 38 to 42; the substrate TS can be 9.5 to 10%, and the substrate VTS can be 8.0 to 8.6%; and / or,

[0030] The VTS ratio of straw, kitchen waste and nitrogen source substrate in the substrate can be changed from (0.8-1.2):(0.8-1.2):(0.8-1.2) to (2.8-3.2):(1.8-2.2):(0.8-1.2).

[0031] A more preferred embodiment may include the following:

[0032] F1 stage:

[0033] The operation time of the F1 stage is 80 to 120 days, preferably 100 to 110 days;

[0034] The inoculum source is added to the reactor, nitrogen is introduced to exhaust the air, and then the reactor is closed; after starting the reactor, no material is fed or discharged for two days, and then the substrate is supplied under the organic load condition of 0.4-0.6g VTS / L / d; the TS value of the substrate is 4.8-5.4%; the VTS value is 4.0-4.4%; the C / N ratio is 18-22; the material can be fed and discharged once every two days, and the hydraulic retention time of the reactor is 90-110 days; the physical and chemical parameters of the reactor can be monitored once every four days;

[0035] in,

[0036] Specifically, regarding the inoculation source,

[0037] The solid content of the inoculum source may be ≤5%;

[0038] In order to inhibit the activity of methanogens, it is preferred to pre-treat the inoculum source. The pre-treatment includes:

[0039] The inoculum source is heat treated at 75-85° C. for 0.5-1.2 hours, and a methane inhibitor is added at a final concentration of 4-6 mM; the methane inhibitor can be selected from 2-bromoethanosulfophate (BES).

[0040] The inoculum source may include inoculants from different sources; the inoculants from different sources may be selected from a mixture of anaerobic digestion sludge from grain and sugar factories, anaerobic digestion sludge from restaurant waste, anaerobic digestion sludge from cellulose, anaerobic digestion sludge from glucose, and anaerobic fermentation acid production reactor sludge from fruit waste;

[0041] The cellulose anaerobic digestion sludge can be selected from cellulose high temperature anaerobic digestion sludge or cellulose medium temperature anaerobic digestion sludge; the glucose anaerobic digestion sludge can be selected from glucose high temperature anaerobic digestion sludge or glucose medium temperature anaerobic digestion sludge. Appropriate sludge types can be selected according to different reaction temperatures.

[0042] The inoculation source comprises inocula from different sources mixed according to the total solids concentration (TS), each type of sludge accounts for more than 10% of the total solids concentration (TS), and more preferably, the inoculated sludges from multiple different sources are mixed in equal proportions according to the total solids concentration (TS).

[0043] In a specific operation, the inoculation source can be crushed by a blender and then filtered (for example, filtered with 2-3 layers of gauze) to ensure that the solid content of the sludge at the start-up of the reactor is below 5%.

[0044] Specifically, regarding the substrate,

[0045] The substrate may include a mixture of straw, kitchen waste and a nitrogen source substrate; in the F1 stage, the VTS ratio of straw, kitchen waste and nitrogen source substrate in the substrate is preferably (0.8-1.2): (0.8-1.2): (0.8-1.2);

[0046] The nitrogen source substrate can be a substrate conventionally used in the field, and specifically, residual sludge from a sewage treatment plant can be used. At present, a large amount of residual sludge is generated during the sewage treatment process using the activated sludge method in urban sewage treatment plants in my country, and the sludge treatment process is still immature. Using residual sludge from a sewage treatment plant as a nitrogen source substrate can reuse the sludge, which is environmentally friendly and cost-saving.

[0047] The straw may be selected from a combination of at least one or more of corn straw, rice straw, wheat straw, reed or bean straw.

[0048] The straw is subjected to steam explosion treatment, and specifically can be steam-exploded straw. The steam-exploded straw can be conventionally steam-exploded straw in the art, and can also be prepared according to the following method:

[0049] The steam explosion treatment process mainly includes: adding straw material and water into a closed container, maintaining for a period of time (which can be several minutes), and then suddenly reducing the pressure to steam explode the straw material, so that the connecting layer of hemicellulose and lignin is destroyed, so that more active groups of cellulose are exposed, which is more conducive to degradation.

[0050] The food waste can be common food waste, or homemade, such as mixing wet weight of crushed fruits, vegetables, and grains;

[0051] The substrate can be stored at low temperature (eg 4° C.) and diluted to a target solid content when used.

[0052] Specifically, regarding the reactor,

[0053] The reactor used in the method of the present application can be a high temperature reactor or a medium temperature reactor. The reactor can be a conventional reactor in the art, such as a conventional mechanical stirring tank. The stirring speed of the reactor can be 50 to 150 ppm to ensure that the materials are mixed evenly.

[0054] The pH value of the reactor may be 5.5 to 6.0;

[0055] The operating temperature of the high temperature reactor may specifically be 50-55°C; and / or the operating temperature of the medium temperature reactor may specifically be 35-40°C.

[0056] When the reactor is a high-temperature reactor, the cellulose anaerobic digestion sludge can be selected from cellulose high-temperature anaerobic digestion sludge, and the glucose anaerobic digestion sludge can be selected from glucose high-temperature anaerobic digestion sludge;

[0057] When the reactor is a mesophilic reactor, the cellulose anaerobic digestion sludge can be selected from cellulose mesophilic anaerobic digestion sludge, and the glucose anaerobic digestion sludge can be selected from glucose mesophilic anaerobic digestion sludge.

[0058] In specific practice, a semi-continuous fully mixed flow anaerobic fermentation acid production reactor system can be constructed, and a high-temperature reactor or a medium-temperature reactor can be used to enrich the high-temperature bacterial community and the medium-temperature bacterial community respectively.

[0059] F2 stage:

[0060] The operation time of the F2 stage is 50 to 100 days, preferably 75 to 90 days; the pH value of the reactor is 5.5 to 6.0; the feed solid content is adjusted to 8 to 12%, the organic load is increased to 0.8 to 1.2 g VTS / L / d, the substrate is supplied, the substrate TS is 9.8 to 10.5%, the substrate VTS is 8.0 to 8.5%, and the VTS ratio of straw, kitchen waste and sludge in the substrate is (0.8 to 1.2): (0.8 to 1.2): (0.8 to 1.2); the substrate C / N ratio is 18 to 22; the material can be loaded and unloaded once every two days; the hydraulic retention time of the reactor can be 90 to 110 days; and the operation is continued until it is stable.

[0061] F3 stage:

[0062] The operation time of the F3 stage is 120 to 200 days, preferably 165 to 185 days; the pH value of the reactor is 5.5 to 6.0; the feed solid content is kept unchanged in this stage (the feed solid content is kept at 8 to 12% in this stage), and the organic load is increased to 1.8 to 2.2 g The substrate is supplied at VTS / L / d, the hydraulic retention time is adjusted to 45-55 days, and the VTS ratio of straw, food waste and sludge in the feed is changed from (0.8-1.2):(0.8-1.2):(0.8-1.2) to (2.8-3.2):(1.8-2.2):(0.8-1.2), and the substrate C / N ratio is increased from 18-22 to 38-42; the substrate TS is 9.5-10%, and the substrate VTS is 8.0-8.6%; the material can be loaded and discharged once every two days; the operation is continued until it is stable; and the microbial flora in the reactor during the stable operation of the F3 stage is collected.

[0063] The entire operation process of the reactor of the present invention can be divided into F1, F2 and F3 stages, and the organic load of the straw is gradually increased in the three stages until stable operation in the F3 stage. Among them, the F1 stage is the start-up stage of the reactor, and the solid content and load are low. The solid content and load are increased to enter the F2 stage. After the acid production in F2 is stable, the proportion of straw in the material is increased, the C / N ratio is increased, and the F3 stage is entered to adapt the bacterial community to the high C / N ratio straw material.

[0064] The second object of the present invention is to provide a microbial community obtained by the method for enriching anaerobic fermentation acid-producing microbial communities as described in one of the objects of the present invention.

[0065] The third object of the present invention is to provide the method for enriching anaerobic fermentation acid-producing microbial communities described in the first object of the present invention and the application of the microbial flora obtained by the method for enriching anaerobic fermentation acid-producing microbial communities described in the second object of the present invention, preferably in the production of short-chain carboxylic acids from cellulosic materials. DETAILED DESCRIPTION

[0066] The present invention is described in detail below in conjunction with specific embodiments. It is necessary to point out that the following embodiments are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made to the present invention by those skilled in the art based on the contents of the present invention still fall within the scope of protection of the present invention.

[0067] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0068] Source of raw materials

[0069] Anaerobic digestion sludge from grain and sugar factories: COFCO Biochemical (Chengdu) Co., Ltd.;

[0070] Anaerobic digestion sludge of restaurant kitchen waste: The anaerobic methanogenetic reactor stably operated by the Environmental Biotechnology Research Center of the School of Architecture and Environment of Sichuan University. The restaurant kitchen waste was taken from the student cafeteria of Sichuan University;

[0071] Cellulose high temperature anaerobic digestion sludge: The high temperature anaerobic methanogenic reactor stably operated by the Environmental Biotechnology Research Center of the School of Architecture and Environment of Sichuan University uses commercially available carboxymethyl cellulose as the carbon source;

[0072] Cellulose mesophilic anaerobic digestion sludge: The mesophilic anaerobic methanogenic reactor stably operated by the Environmental Biotechnology Research Center of the School of Architecture and Environment of Sichuan University uses commercially available carboxymethyl cellulose as the carbon source;

[0073] Glucose thermophilic anaerobic digestion sludge: The thermophilic anaerobic methanogenic reactor stably operated by the Environmental Biotechnology Research Center of the School of Architecture and Environment of Sichuan University uses commercially available glucose as the carbon source;

[0074] Glucose mesophilic anaerobic digestion sludge: The mesophilic anaerobic methanogenetic reactor stably operated by the Environmental Biotechnology Research Center of the School of Architecture and Environment of Sichuan University uses commercially available glucose as the carbon source;

[0075] Anaerobic fermentation acid production reactor sludge from fruit waste: The anaerobic fermentation acid production reactor stably operated by the Environmental Biotechnology Research Center of the School of Architecture and Environment of Sichuan University uses commercially available fruits (banana, watermelon, and orange mixed in a weight ratio of 1:1:1) as raw materials;

[0076] The wastewater plant residual sludge is the dewatered sludge from a municipal wastewater treatment plant in Chengdu;

[0077] The artificially prepared food waste is a mixture of bananas, oranges, cabbages and rice at equal wet weight after crushing;

[0078] Steam-exploded straw refers to steam-exploded straw, wherein the steam explosion treatment comprises: adding corn straw and water into a closed container, and maintaining the pressure at 1.7 MPa for 2 minutes, and then suddenly reducing the pressure to steam-explode the straw material.

[0079] The raw materials used in the examples and comparative examples, unless otherwise specified, are disclosed in the prior art, for example, they can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0080] Table 1 TS and VTS of inoculum and substrate

[0081]

[0082] In the embodiment, one high-temperature reactor (working volume 4 L) and one medium-temperature reactor (working volume 2.4 L) were prepared to enrich the bacterial flora adapted to high temperature and the bacterial flora adapted to medium temperature, respectively.

[0083] Example 1

[0084] 1. Reactor Construction

[0085] Preparation of inoculum source: In order to enrich the anaerobic fermentation and acid-producing microbial community, the anaerobic digestion sludge of grain sugar factory, the anaerobic digestion sludge of restaurant waste, the high-temperature anaerobic digestion sludge of cellulose, the high-temperature anaerobic digestion sludge of glucose and the anaerobic fermentation and acid-producing reactor sludge of fruit waste were mixed and used as the inoculum source; inocula from different sources were mixed in equal proportions (i.e., 1:1:1:1:1) according to the total solids (TS) concentration (TS and VTS of inocula from different sources are shown in Table 1).

[0086] Preparation of substrate: Steam-exploded straw, artificially prepared food waste (banana, orange, cabbage and rice were crushed and mixed with equal wet weight) and wastewater plant residual sludge were used as substrates. Different substrates were mixed in equal proportions according to the volatile total solids (VTS) concentration (TS and VTS of various substrates are shown in Table 1). The mixed substrates were stored at 4°C and diluted to the target solid content when used.

[0087] A high temperature reactor (working volume 4 L, named H) was prepared.

[0088] 2. Reactor startup and operation

[0089] The present invention starts up a high temperature reactor (H).

[0090] The whole operation process of the reactor is divided into F1, F2 and F3 stages. The organic load of straw is gradually increased in the three stages until it operates stably in the F3 stage. The operating conditions of the reactor in the three stages are shown in Table 2.

[0091] Days 0 to 105 are the F1 stage.

[0092] The startup and operation temperature of the high-temperature reactor is 53°C, the stirring speed is 100rpm, and the pH value is controlled at 5.5-6.0. According to TS, the inoculation source obtained by mixing inoculum sludge from various sources in equal proportions is crushed with a blender and filtered with 2-3 layers of gauze to ensure that the solid content of the reactor startup sludge is less than 5%. In order to inhibit the activity of methanogens, the startup sludge needs to be pretreated. The startup sludge is heat-treated at 80°C for 1 hour, and a methane inhibitor 2-bromoethane sulfonic acid (2-bromoethanosulfophate, BES) is added at a final concentration of 5mM. The pretreated mixed inoculum is injected into the working volume of the reactor, and the reactor is closed after nitrogen is introduced to exhaust the air to reach an anaerobic state. After starting the reactor, no material was added or removed for two days. After that, a substrate containing straw, food waste and sludge (TS: 5.2%; VTS: 4.1%; C / N ratio: 20) was supplied under an organic loading condition of 0.5 g VTS / L / d. The VTS ratio of straw, food waste and sludge in the substrate was 1:1:1. The material was added and removed every two days. The hydraulic retention time of the reactor was 100 days. The physical and chemical parameters of the reactor were monitored every four days.

[0093] Days 105 to 189 are the F2 stage, during which the hydraulic retention time is maintained at 100 days, the feed solid content is adjusted to 10%, the substrate TS is 10.3%, the substrate VTS is 8.2%, the VTS ratio of straw, food waste and sludge in the substrate is 1:1:1; the substrate C / N ratio is 20; the organic load is increased to 1g VTS / L / d; and the operation is continued until stability.

[0094] Days 189 to 368 were the F3 stage, during which the feed solid content was maintained at 10%, the organic load was increased to 2 gVTS / L / d, the hydraulic retention time was adjusted to 50 days, the substrate TS was 9.7%, the substrate VTS was 8.3%, and the VTS ratio of straw, food waste and sludge in the feed was changed from 1:1:1 to 3:2:1, which increased the proportion of straw, reduced the proportion of sludge, and increased the feed C / N ratio from 20 to 40.

[0095] Acid production capacity of continuous fermentation process in F3 stage:

[0096] High temperature reactor: The production of short-chain carboxylic acids reached about 22 g / L, and the main short-chain carboxylic acids were acetic acid (about 55%), butyric acid (about 30%), propionic acid (about 10%) and valeric acid (about 5%).

[0097] The microbial flora of the reactor during the stable operation of the F3 stage was collected. The specific method was to take the fermentation liquid, centrifuge it at 4°C for 10 minutes, remove the supernatant, and the precipitate was the flora, which was used for flora structure detection and batch fermentation. The results are as follows:

[0098] Description of enriched bacterial flora structure:

[0099] The species diversity of the microbial flora gradually decreases with the operation time, and the flora structure tends to be stable in the F3 stage:

[0100] Firmicutes species are absolutely dominant in the thermophilic bacterial community, and the dominant species include Caproiciproducens, unclassified Hungateiclostridiacea, norankEthanoligenenaceae, Thermoanaerobacterium, unclassified Bacillaceae, Lactobacillus, Symbiobacterium, Herbinix, Thermomonas, etc.

[0101] Evaluation of the acid production capacity of enriched bacterial flora based on batch fermentation:

[0102] Steam exploded straw was used as the sole substrate and wastewater sludge as the nitrogen source. Batch fermentation was carried out under the conditions of initial TS of straw substrate of 5% and 10% to evaluate the acid production capacity of the bacterial community.

[0103] Thermostatic bacteria ferment under high temperature 53℃:

[0104] When the substrate TS was 5%, the production of short-chain carboxylic acids reached about 17.3 g / L after 8 days of fermentation, and the main short-chain carboxylic acids were lactic acid (about 71%), acetic acid (about 14%) and propionic acid (about 11%).

[0105] When the substrate TS was 10%, the production of short-chain carboxylic acids reached about 26.3 g / L after 8 days of fermentation, and the main short-chain carboxylic acids were acetic acid (about 48%), lactic acid (about 40%) and propionic acid (about 8%).

[0106] Example 2

[0107] 1. Reactor Construction

[0108] Preparation of inoculum source: In order to enrich the anaerobic fermentation acid-producing microbial community, the anaerobic digestion sludge of grain sugar factory, the anaerobic digestion sludge of restaurant waste, the mesophilic anaerobic digestion sludge of cellulose, the mesophilic anaerobic digestion sludge of glucose and the anaerobic fermentation acid-producing reactor sludge of fruit waste were mixed and used as the inoculum source; inocula from different sources were mixed in equal proportions according to the total solids (TS) concentration (TS and VTS of inocula from different sources are shown in Table 1).

[0109] Preparation of substrate: Steam-exploded straw, artificially prepared food waste (banana, orange, cabbage and rice were crushed and mixed with equal wet weight) and wastewater plant residual sludge were used as substrates. Different substrates were mixed in equal proportions according to the volatile total solids (VTS) concentration. The TS and VTS of various substrates are shown in Table 1. The mixed substrates were stored at 4 °C and diluted to the target solid content when used.

[0110] Prepare 1 mesophilic reactor (working volume 2.4 L).

[0111] 2. Reactor startup and operation

[0112] The present invention starts up a medium temperature reactor.

[0113] The whole operation process of the reactor is divided into F1, F2 and F3 stages. The organic load of straw is gradually increased in the three stages until it operates stably in the F3 stage. The operating conditions of the reactor in the three stages are shown in Table 2.

[0114] Days 0 to 105 are the F1 stage.

[0115] The startup and operation temperature of the mesophilic reactor is 37°C, the stirring speed is 100rpm, and the pH value is controlled at 5.5-6.0. According to TS, the inoculation source obtained by mixing inoculum sludge from various sources in equal proportions is crushed with a blender and filtered with 2-3 layers of gauze to ensure that the solid content of the reactor startup sludge is less than 5%. In order to inhibit the activity of methanogens, the startup sludge needs to be pretreated. The startup sludge is heat treated at 80°C for 1 hour, and a methane inhibitor 2-bromoethane sulfonic acid (2-bromoethanosulfophate, BES) is added at a final concentration of 5mM. The pretreated mixed inoculum is injected into the working volume of the reactor, and the reactor is closed after nitrogen is introduced to exhaust the air to reach an anaerobic state. After starting the reactor, no material was added or removed for two days. After that, a substrate containing straw, food waste and sludge (TS: 5.2%; VTS: 4.1%; C / N ratio: 20) was supplied under an organic loading condition of 0.5 g VTS / L / d. The VTS ratio of straw, food waste and sludge in the substrate was 1:1:1. The material was added and removed every two days. The hydraulic retention time of the reactor was 100 days. The physical and chemical parameters of the reactor were monitored every four days.

[0116] Days 105 to 189 are the F2 stage, during which the hydraulic retention time is maintained at 100 days, the feed solid content is adjusted to 10%, the substrate TS is 10.3%, the substrate VTS is 8.2%, the VTS ratio of straw, food waste and sludge in the substrate is 1:1:1; the substrate C / N ratio is 20; the organic load is increased to 1g VTS / L / d, and the operation is continued until stability.

[0117] Days 189 to 368 were the F3 stage, during which the feed solid content was maintained at 10%, the organic load was increased to 2 gVTS / L / d, the hydraulic retention time was adjusted to 50 days, the substrate TS was 9.7%, the substrate VTS was 8.3%, and the VTS ratio of straw, kitchen waste and sludge in the feed was changed from 1:1:1 to 3:2:1, increasing the proportion of straw, reducing the proportion of sludge, and increasing the feed C / N ratio from 20 to 40. Continue to operate until stable.

[0118] Acid production capacity of continuous fermentation process in F3 stage:

[0119] Medium temperature reactor: The production of short-chain carboxylic acids is about 23 g / L, and the main short-chain carboxylic acids are acetic acid (about 60%), butyric acid (about 20%), propionic acid (about 10%) and valeric acid (about 10%).

[0120] The microbial flora of the reactor during the stable operation of the F3 stage was collected. The specific collection method can be as follows: take the fermentation liquid, centrifuge it at 4°C for 10 minutes, remove the supernatant, and the precipitate is the flora, which is used for flora structure detection and batch fermentation. The results are as follows:

[0121] Description of enriched bacterial flora structure:

[0122] The species diversity of the microbial flora gradually decreases with the operation time, and the flora structure tends to be stable in the F3 stage:

[0123] Species of Firmicutes, Proteobacteria, Thermococcus, and Bacteroidetes are dominant in the mesophilic bacterial community, and the dominant species include Defluviitoga, Rikenellaceae_RC9_gut_group, Lachnospiraceae_NK3A20_group, Leuconostoc, norank Run.SP154, Dechloromonas, norank Saccharimonadales, Defluviimonas, Lactobacillus, etc.

[0124] Evaluation of the acid production capacity of enriched bacterial flora based on batch fermentation:

[0125] Steam exploded straw was used as the sole substrate and wastewater sludge as the nitrogen source. Batch fermentation was carried out under the conditions of initial TS of straw substrate of 5% and 10% to evaluate the acid production capacity of the bacterial community.

[0126] Mesophilic bacteria ferment under medium temperature 37℃ condition:

[0127] When the substrate TS was 5%, the production of short-chain carboxylic acids reached about 19.2 g / L after 8 days of fermentation. The main short-chain carboxylic acids were acetic acid (about 49%), lactic acid (about 23%), butyric acid (about 14%) and propionic acid (about 12%).

[0128] When the substrate TS was 10%, the production of short-chain carboxylic acids reached about 40 g / L after 8 days of fermentation, and the main short-chain carboxylic acids were lactic acid (about 53%), acetic acid (about 34%) and propionic acid (about 10%).

[0129] As can be seen from the above, the methods of Examples 1 and 2 of the present application obtain microbial flora that can efficiently produce SCCAs using straw materials. The mesophilic flora and thermophilic flora can produce nearly 20 g / L of SCCAs using straw, the only substrate with an initial solid content of 5%, which has a significant effect.

[0130] At present, there are very limited research reports on the production of short-chain carboxylic acids using acid-producing bacteria with straw as the only substrate. Under the same straw solid content conditions, the accumulated acid concentration is low. In order to increase the acid concentration, straw and other easily degradable wastes such as kitchen waste and livestock and poultry manure are basically used for fermentation. The high-temperature bacterial flora and the medium-temperature bacterial flora enriched by the specific method described in this application can use straw as the only substrate for fermentation, and the acid concentration of the short-chain carboxylic acid obtained is high, which has a good application prospect.

[0131] Table 2 Operating conditions of the reactors at different stages of Example 1 and Example 2

[0132] F1 F2 F3 Substrate TS (%) 5.2 10.3 9.7 Substrate VTS (%) 4.1 8.2 8.3 VTS ratio of straw, kitchen waste and sludge in substrate 1:1:1 1:1:1 3:2:1 Substrate C / N ratio 20 20 40 Organic load (gVTS / L / d) 0.5 1 2 Residence time (d) 100 100 50

Claims

1. A method for enriching anaerobic fermentation acid-producing microbial communities, characterized in that Including F1 stage ~ F3 stage: F1 stage: The inoculum source is added to the reactor, nitrogen is introduced to exhaust the air, and the reactor is closed; the solid content of the inoculum source is ≤5%; no material is fed or discharged for two days after the reactor is started, and then a substrate is supplied under an organic load condition of 0.4-0.6 g VTS / L / d; the substrate comprises a mixture of straw, food waste and a nitrogen source substrate; F2 stage: Adjust the feed solid content to 8-12% and increase the organic load to 0.8-1.2 g VTS / L / d; F3 stage: In this stage, the feed solid content is maintained at 8-12%, and the organic load is increased to 1.8-2.2 g VTS / L / d; Collect the microbial flora in the F3 stage reactor.

2. The method for enriching anaerobic fermentation acid-producing microbial communities according to claim 1, characterized in that: In the F1 stage, The operating period of the F1 phase is 80 to 120 days; and / or, The TS value of the substrate is 4.8-5.4%; the VTS value is 4.0-4.4%; the C / N ratio is 18-22; and / or, The VTS ratio of straw, kitchen waste and nitrogen source substrate in the substrate is (0.8-1.2): (0.8-1.2): (0.8-1.2); and / or, Load and unload once every two days; and / or, The hydraulic retention time of the reactor is 90 to 110 days.

3. The method for enriching anaerobic fermentation acid-producing microbial communities according to claim 1, characterized in that: In the F2 stage, The operating time of the F2 phase is 50 to 100 days; and / or, The substrate TS is 9.8-10.5%, the substrate VTS is 8.0-8.5%, and the substrate C / N ratio is 18-22; and / or, The VTS ratio of straw, kitchen waste and nitrogen source substrate in the substrate is (0.8-1.2): (0.8-1.2): (0.8-1.2); and / or, The hydraulic retention time of the reactor is 90 to 110 days.

4. The method for enriching anaerobic fermentation acid-producing microbial communities according to claim 1, characterized in that: In the F3 stage, The operating time of the F3 phase is 120 to 200 days; and / or, The hydraulic retention time of the reactor is adjusted to 45 to 55 days, and / or, The substrate C / N ratio is increased from 18-22 to 38-42; the substrate TS is 9.5-10%, and the substrate VTS is 8.0-8.6%; and / or, The VTS ratio of straw, kitchen waste and nitrogen source substrate in the substrate was changed from (0.8~1.2):(0.8~1.2):(0.8~1.2) to (2.8~3.2):(1.8~2.2):(0.8~1.2).

5. The method for enriching anaerobic fermentation acid-producing microbial communities according to claim 1, characterized in that: The nitrogen source substrate is excess sludge from a sewage treatment plant; and / or, The straw is steam-exploded straw.

6. The method for enriching anaerobic fermentation acid-producing microbial communities according to claim 1, characterized in that: The inoculum source comprises inocula from different sources; The inoculum from different sources is selected from a mixture of anaerobic digestion sludge from grain sugar factories, anaerobic digestion sludge from kitchen waste, anaerobic digestion sludge from cellulose, anaerobic digestion sludge from glucose, and anaerobic fermentation acid production reactor sludge from fruit waste.

7. The method for enriching anaerobic fermentation acid-producing microbial communities according to claim 6, characterized in that: The cellulose anaerobic digestion sludge is selected from cellulose high-temperature anaerobic digestion sludge or cellulose medium-temperature anaerobic digestion sludge; and / or, The glucose anaerobic digestion sludge is selected from glucose high-temperature anaerobic digestion sludge or glucose medium-temperature anaerobic digestion sludge.

8. The method for enriching anaerobic fermentation acid-producing microbial communities according to claim 6, characterized in that: The inoculum source comprises inocula from different sources mixed according to the total solids (TS), each type of sludge accounts for more than 10% of the total solids (TS), and more preferably, they are mixed in equal proportions according to the total solids (TS).

9. The method for enriching anaerobic fermentation acid-producing microbial communities according to any one of claims 1 to 8, characterized in that: The reactor is a high temperature reactor or a medium temperature reactor; The pH value of the reactor is preferably 5.5 to 6.0; Preferably, The operating temperature of the high temperature reactor is 50-55°C; and / or, The working temperature of the medium temperature reactor is 35-40°C.

10. The method for enriching anaerobic fermentation acid-producing microbial communities according to claim 9, characterized in that: When the reactor is a high-temperature reactor, the cellulose anaerobic digestion sludge is selected from cellulose high-temperature anaerobic digestion sludge, and the glucose anaerobic digestion sludge is selected from glucose high-temperature anaerobic digestion sludge; When the reactor is a mesophilic reactor, the cellulose anaerobic digestion sludge is selected from cellulose mesophilic anaerobic digestion sludge, and the glucose anaerobic digestion sludge is selected from glucose mesophilic anaerobic digestion sludge.

11. A bacterial community obtained by the method for enriching anaerobic fermentation acid-producing microbial communities according to any one of claims 1 to 10.

12. The method for enriching anaerobic fermentation acid-producing microbial communities according to any one of claims 1 to 10 or the use of the bacterial community according to claim 11, preferably in the production of short-chain carboxylic acids from cellulosic materials.

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