Microbial flora for producing methane and organic acid by anaerobic degradation of biomass as well as culture method and application of microbial flora

Abstract

The invention discloses a microbial flora for producing methane and organic acid by anaerobic degradation of biomass as well as a culture method and application of the microbial flora. The microbial flora comprises the following components in percentage by weight: 5%-30% of Clostridium, 2%-10% of Paludibacter, 1%-25% of Thermovirga, 2%-20% of DMER64, 2%-20% of Mesotoga, 1%-10% of Methanosaeta and 1%-10% of Methanobacterium. The culture method of the flora comprises the following steps: S1, taking pectin, straws and fallen leaves, drying, and grinding the straws into powder by using a mortar; s2, mixing pectin degrading bacteria and residual sludge, inoculating the mixture into a culture bottle, adding a culture medium, and adding the powder obtained in the step S1 into the culture medium; s3, mixed gas of nitrogen and carbon dioxide is introduced, the culture bottle is sealed, continuous culture is performed for 30-100 days at the temperature of 30-38 DEG C, and pectin and cellulose hydrolysis flora enrichment is performed; the microbial flora can quickly hydrolyze and convert biomass such as straws and fallen leaves into methane, acetic acid, propionic acid, butyric acid or hexanoic acid products; compared with conventional pretreatment modes such as alkali treatment and high-temperature treatment, the method has the advantages of high yield, mild operation conditions, low operation cost and the like.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a microbial flora for anaerobic biodegradation to produce methane and organic acid, and a cultivation method and application thereof. Background Art

[0002] my country produces a large amount of biomass waste every year, but the utilization rate of this byproduct is low. Due to many difficulties in the utilization process, people rarely reuse it, and it is often discarded, causing environmental pollution. Although there are many directions and types of technologies for biomass utilization in my country, there are still problems in some aspects. For example, there is little secondary utilization, high processing costs, and low comprehensive benefits.

[0003] Biomass contains abundant nutrients and active substances, such as proteins, polysaccharides, amino acids, etc., among which the polysaccharide part contains a large amount of cellulose and pectin which are difficult to be degraded, resulting in difficulty in the treatment of biomass. Therefore, the present invention provides a microbial flora that is conducive to anaerobic degradation of biomass to produce methane and organic acids and a cultivation method thereof, as well as a treatment method for enriching and degrading biomass using the flora, and degrading biomass into methane and organic acid products under anaerobic conditions. Summary of the invention

[0004] In view of the above problems, the present invention aims to provide a microbial flora for anaerobic degradation of biomass to produce methane and organic acids, and a cultivation method and application thereof.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0006] The invention provides a microbial flora for anaerobic biomass degradation to produce methane and organic acid, comprising: 5%-30% Clostridium, 2%-10% Paludibacter, 1%-25% Thermovirga, 2%-20% DMER64, 2%-20% Mesotoga, 1%-10% Methanosaeta and 1%-10% Methanobacterium.

[0007] Based on the above, the present invention also provides a method for culturing a microbial flora for anaerobic biomass degradation to produce methane and organic acids, comprising the following steps:

[0008] S1: Take pectin, straw, and fallen leaves, dry them, and grind the straw into powder using a mortar;

[0009] S2: inoculating the mixed pectin-degrading bacteria and the excess sludge into a culture bottle, adding a culture medium, and adding the powder obtained in step S1 into the culture medium;

[0010] S3: introducing a mixed gas of nitrogen and carbon dioxide, sealing the culture bottle, and continuously culturing at a temperature of 30 to 38° C. for 30 to 100 days to enrich the pectin and cellulose hydrolyzing bacteria to obtain the microbial flora that anaerobically degrades biomass to produce methane and organic acids.

[0011] Furthermore, in step S1, the mass ratio of the mixture of pectin-degrading bacteria and excess sludge is 1:1-10; the excess sludge is the sludge from the secondary sedimentation tank of the sewage treatment plant.

[0012] Furthermore, the straw and fallen leaves taken in step S1 are the remaining parts of the crop plants.

[0013] Furthermore, the formula of the culture medium is 450-550 mg / L NH4Cl; 130-180 mg / L KH2PO4; 45-55 mg / L Na2SO4; 14-18 g / L Na2HPO4·12H2O; 45-55 mg / L KCl; 8-12 mg / L CaCl2; 65-75 mg / L MgCl2·6H2O; 0.6-1 mg / L MnCl2·4H2O; 1.0-1.4 mg / L CoCl2·2H2O; 3.0-3.4 mg / L FeSO4·7H2O; 0.4-0.6 mg / L AlCl3; 0.05-0.15 mg / L NaMO4·2H2O; 0.1-0.25 mg / L H3BO3; 0.4~0.6mg / LNiCl2·6H2O; 1.0~1.2mg / L CuCl2·2H2O; 3.0~3.5mg / L ZnSO4·7H2O; 2.8~3.2mg / L EDTA-2Na.

[0014] Furthermore, the pH of the culture medium is adjusted to 7.0-7.4 by acid or alkali.

[0015] Furthermore, in step S2, the concentration of pectin and cellulose in the culture medium is 5-10 g / L.

[0016] Furthermore, in step S3, the volume ratio of nitrogen to carbon dioxide in the mixed gas of nitrogen and carbon dioxide is 70% to 85%: 15% to 25%.

[0017] The microbial flora can be applied to anaerobic degradation of biomass to produce methane and organic acids. The method for anaerobic degradation of biomass by the microbial flora to produce methane and organic acids comprises the following steps:

[0018] The above-mentioned microbial flora is inoculated into an anaerobic reactor, with straw as substrate, and the inoculation amount is 5-10 g / L;

[0019] A mixed gas of excess nitrogen and carbon dioxide is introduced, the anaerobic reactor is sealed, and cultured at a temperature of 34 to 36° C. for 3 to 17 days to obtain metabolites of methane and organic acids.

[0020] Furthermore, the volume ratio of nitrogen to carbon dioxide in the mixed gas of nitrogen and carbon dioxide is 70% to 85%:15% to 25%; the organic acid is one or more of acetic acid, propionic acid, butyric acid or caproic acid.

[0021] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0022] The microbial flora cultivation method and core flora for anaerobic degradation of biomass such as straw and fallen leaves to produce methane and organic acids provided by the present invention have the following advantages: first, they can quickly hydrolyze and transform biomass such as straw and fallen leaves; second, they can transform biomass into methane, acetic acid, propionic acid, butyric acid or caproic acid products; third, compared with conventional pretreatment methods such as alkali treatment and high temperature treatment, they have the advantages of high yield, mild operating conditions and low operating costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the amount of methane produced by the microbial flora provided by the present invention in the degradation of biomass at different concentrations; in the figure, 0 g / L straw powder is a blank control group; 5 g / L straw powder represents an experimental group with 0.3 g straw powder added; and 10 g / L straw powder represents an experimental group with 0.6 g straw powder added. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0025] It should also be noted that, in order to avoid obscuring the technical solution of the present invention due to unnecessary details, only the technical solutions and / or processing steps closely related to the solution of the present invention are shown in the embodiments, while other details that are not very relevant are omitted.

[0026] The present embodiment provides a microbial flora for anaerobic biomass degradation to produce methane and organic acids, including: 5%-30% Clostridium, 2%-10% Paludibacter, 1%-25% Thermovirga, 2%-20% DMER64, 2%-20% Mesotoga, which have the functions of secreting pectinase, cellulase, xylanase, etc., and can degrade biomass such as straw and fallen leaves to produce acetic acid, propionic acid, butyric acid, lactic acid and caproic acid, etc.; 1%-10% Methanosaeta and 1%-10% Methanobacterium, which have the function of producing methane.

[0027] Based on the above, this embodiment also provides a method for culturing a microbial flora that anaerobically degrades biomass to produce methane and organic acids, comprising the following steps:

[0028] S1: Take pectin, straw, and fallen leaves (the straw and fallen leaves are the remaining parts of crop plants), dry them, and grind the straw into powder using a mortar.

[0029] S2: Pectin-degrading bacteria and excess sludge (the mass ratio of pectin-degrading bacteria to excess sludge is 1:1-10; in this embodiment, the excess sludge is the sludge from the secondary sedimentation tank of Fuzhou Jinshan Sewage Treatment Plant) are mixed and inoculated in a culture bottle, and a culture medium is added. The concentration of pectin and cellulose in the culture medium is 5-10 g / L, and the powder obtained in step S1 is added to the culture medium. The formula of the culture medium in this embodiment is: 450-550 mg / L NH4Cl; 130-180 mg / L KH2PO4; 45-55 mg / LNa2SO4; 14-18 g / LNa2HPO4·12H2O; 45-55 mg / L KCl; 8-12 mg / L CaCl2; 65-75 mg / LMgCl2·6H2O; 0.6-1 mg / L MnCl2·4H2O; 1.0-1.4 mg / L CoCl2·2H2O; 3.0~3.4mg / LFeSO4·7H2O; 0.4~0.6mg / L AlCl3; 0.05~0.15mg / L NaMO4·2H2O; 0.1~0.25mg / LH3BO3; 0.4~0.6mg / LNiCl2·6H2O; 1.0~1.2mg / L CuCl2·2H2O; 3.0~3.5mg / LZnSO4·7H2O; 2.8~3.2mg / L EDTA-2Na; the pH of the culture medium is adjusted to 7.0~7.4 by acid or alkali.

[0030] S3: A mixed gas of nitrogen and carbon dioxide is introduced (the volume ratio of nitrogen and carbon dioxide in the mixed gas is 70% to 85%: 15% to 25%), the culture bottle is sealed, and continuous culture is carried out at a temperature of 30 to 38° C. for 30 to 100 days to enrich the pectin and cellulose hydrolyzing bacteria to obtain a microbial flora that anaerobically degrades biomass to produce methane and organic acids.

[0031] This embodiment also provides a method for producing methane and organic acids by anaerobic degradation of biomass using the above-mentioned microbial flora, comprising the following steps:

[0032] The microbial flora is inoculated into an anaerobic reactor, with straw as a substrate and an inoculation amount of 5 to 10 g / L; an excessive amount of a mixed gas of nitrogen and carbon dioxide (the volume ratio of nitrogen to carbon dioxide in the mixed gas is 70% to 85%: 15% to 25%) is introduced, the anaerobic reactor is sealed, and cultured at a temperature of 34 to 36° C. for 3 to 17 days to obtain metabolites of methane and organic acids; wherein the organic acid is one or more of acetic acid, propionic acid, butyric acid or caproic acid.

[0033] Example 1

[0034] The microbial flora obtained by the above culture was inoculated into a 1L culture bottle, and a culture medium (the culture medium formula is the same as above) was added. 2.5g of straw powder was added to the culture medium, the liquid phase was 500mL, and an excess of a mixed gas of nitrogen and carbon dioxide (the volume ratio of nitrogen and carbon dioxide in the mixed gas was 70% to 85%: 15% to 25%) was introduced, the original air in the culture bottle was removed, the culture bottle was sealed, and continuous culture was carried out at a temperature of 34 to 36°C.

[0035] The culture flask was sealed with a butyl rubber stopper and an aluminum cap and maintained at 35° C. The main metabolites were analyzed every 2 days. The main metabolite was methane, with a maximum methane yield of 465 mL, and pectin and cellulose hydrolyzing bacteria were obtained.

[0036] Example 2

[0037] In this example, the methane production by straw degradation at different concentrations was compared. 20 mL of the microbial flora obtained by the above culture was inoculated into three 120 mL culture bottles, and culture medium (the culture medium formula was the same as above) was added. No straw powder was added to the blank control group. 0.3 g and 0.6 g of straw powder were added to the culture medium in the experimental group, respectively. The liquid phase was 60 mL. An excess of nitrogen and carbon dioxide mixed gas (the volume ratio of nitrogen and carbon dioxide in the mixed gas was 70% to 85%: 15% to 25%) was introduced, the original air in the culture bottle was removed, the culture bottle was sealed, and continuous culture was carried out at a temperature of 34 to 36°C.

[0038] The above culture bottles were sealed with butyl rubber stoppers and aluminum caps, and the temperature was maintained at 35°C. The main metabolites were analyzed every 2 days. The main metabolite was methane. The final methane production in the experimental group with added straw powder was as high as 85mL (i.e., the experimental group with added 0.6g straw powder), which was 96% higher than the methane production of 3.3mL in the blank control group and 32% higher than the methane production of 58mL with added 0.3g straw powder (as shown in the attached figure). Figure 1 shown).

[0039] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microbial flora for anaerobic biomass degradation to produce methane and organic acids, characterized in that: include: 5%-30% Clostridium spp., 2%-10% Paludibacter spp., 1%-25% Thermovirga spp., 2%-20% DMER64 spp., 2%-20% Mesotoga spp., 1%-10% Methanosaeta spp. and 1%-10% Methanobacterium spp.

2. A method for culturing a microbial flora for anaerobic biomass degradation to produce methane and organic acids as claimed in claim 1, characterized in that: The steps include: S1: Take pectin, straw, and fallen leaves, dry them, and grind the straw into powder using a mortar; S2: inoculating the mixed pectin-degrading bacteria and the excess sludge into a culture bottle, adding a culture medium, and adding the powder obtained in step S1 into the culture medium; S3: introducing a mixed gas of nitrogen and carbon dioxide, sealing the culture bottle, and continuously culturing at a temperature of 30 to 38° C. for 30 to 100 days to enrich the pectin and cellulose hydrolyzing bacteria to obtain the microbial flora that anaerobically degrades biomass to produce methane and organic acids.

3. The method for culturing a microbial flora for anaerobic biomass degradation to produce methane and organic acids according to claim 2, characterized in that: In step S1, the mass ratio of the mixture of pectin-degrading bacteria and excess sludge is 1:1-10; the excess sludge is the sludge from the secondary sedimentation tank of the sewage treatment plant.

4. The method for culturing a microbial flora for anaerobic biomass degradation to produce methane and organic acids according to claim 2, characterized in that: The straw and fallen leaves taken in step S1 are the remaining parts of the crop plants.

5. The method for culturing a microbial flora for anaerobic biomass degradation to produce methane and organic acids according to claim 2, characterized in that: The formula of the culture medium is 450-550 mg / L NH4Cl; 130-180 mg / L KH2PO4; 45-55 mg / L Na2SO4; 14-18 g / L Na2HPO4·12H2O; 45-55 mg / L KCl; 8-12 mg / L CaCl2; 65-75 mg / L MgCl2·6H2O; 0.6-1 mg / L MnCl2·4H2O; 1.0-1.4 mg / L CoCl2·2H2O; 3.0-3.4 mg / L FeSO4·7H2O; 0.4-0.6 mg / L AlCl3; 0.05-0.15 mg / L NaMO4·2H2O; 0.1-0.25 mg / L H3BO3; 0.4-0.6 mg / L NiCl2·6H2O; 1.0-1.2 mg / L CuCl2·2H2O; 3.0~3.5mg / LZnSO4·7H2O; 2.8~3.2mg / L EDTA-2Na.

6. The method for culturing a microbial flora for anaerobic biomass degradation to produce methane and organic acids according to claim 5, characterized in that: The pH of the culture medium is adjusted to 7.0-7.4 by acid or alkali.

7. The method for culturing a microbial flora for anaerobic biomass degradation to produce methane and organic acids according to claim 2, characterized in that: In step S2, the concentration of pectin and cellulose in the culture medium is 5-10 g / L.

8. The method for culturing a microbial flora for anaerobic biomass degradation to produce methane and organic acids according to claim 2, characterized in that: The volume ratio of nitrogen to carbon dioxide in the mixed gas of nitrogen and carbon dioxide in step S3 is 70% to 85%:15% to 25%.

9. Use of the microbial flora as claimed in claim 1 in anaerobic degradation of biomass to produce methane and organic acids, characterized in that: The method for producing methane and organic acid by anaerobic degradation of biomass by microbial flora comprises the following steps: Inoculate the microbial flora as claimed in claim 1 into an anaerobic reactor, using straw as a substrate, and the inoculation amount is 5 to 10 g / L; A mixed gas of excessive nitrogen and carbon dioxide is introduced, the anaerobic reactor is sealed, and cultured at a temperature of 34 to 36° C. for 3 to 17 days to obtain metabolites of methane and organic acids.

10. The use according to claim 9, characterized in that: The volume ratio of nitrogen to carbon dioxide in the mixed gas of nitrogen and carbon dioxide is 70% to 85%:15% to 25%; the organic acid is one or more of acetic acid, propionic acid, butyric acid or caproic acid.

Images