Method for producing methane through trichoderma reesei assisted artificial mixed bacteria liquid fermentation

Abstract

The invention relates to the technical field of biology, in particular to a method for producing methane through trichoderma reesei assisted artificial mixed bacteria liquid fermentation. According to the invention, biogas residue, alkali-treated corn straw and pig urine are taken as substrates for a liquid fermentation methane production test, and the contents of active carbon, Tween-80 and manganese ions in the liquid system are optimized. Meanwhile, based on an optimal liquid fermentation methane production system, trichoderma reesei RUT-C30 is added into the optimal liquid fermentation methane production system to construct a method capable of improving the methane preparation efficiency.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for producing methane by artificial mixed-bacteria liquid fermentation assisted by Trichoderma reesei. Background Art

[0002] At present, the resource utilization methods of lignocellulosic biomass mainly include fertilizer, feed, energy, base material, raw material, etc. Among them, corn straw is directly or indirectly returned to the field, burned, used as animal feed, anaerobic fermentation, edible fungus culture medium, and used as industrial production raw materials. Among the various treatment methods, anaerobic fermentation can produce biogas and biogas, with a methane content of 55%~70% in the produced biogas, and an energy of 20~25 MJ per cubic meter of biogas (Gao Zhongpo et al., Research on the Amount and Utilization of Crop Straw Resources in my country, Agricultural Mechanization Research, 2022, Vol. 44, No. 4, pp. 1-6). Therefore, the reuse of agricultural waste such as corn straw and its combination with clean energy production is a resource technology with broad application prospects (Shi Zuliang et al., Current Status of Comprehensive Utilization of Crop Straw in my country and Estimation of Carbon Emissions from Incineration, China Agricultural Resources and Zoning, 2017, Vol. 38, No. 9, pp. 32-37).

[0003] At present, good research results have been achieved in bioaugmentation and biostimulation strategies to assist microorganisms in improving the potential of methane synthesis. By rationally transforming and improving the mixed bacteria methane conversion system and improving the metabolic activity of the above system, material transfer and final product synthesis can be effectively promoted. In response to the above problems, related studies have stimulated microorganisms to rapidly synthesize methane by increasing the solid content (2%~6%) in the anaerobic fermentation process (Wang S et al., Effects of rice straw biochar onmethanogenic bacteria and metabolic function in anaerobic digestion, Sci Rep, 2022, Vol. 12, No. 1, 6971). At the same time, adjusting the mixing ratio of activated sludge and substrate can also achieve the purpose of improving the rapid conversion of substrate by mixed bacteria. At present, different proportions of activated carbon, iron oxide compounds and other substances are added exogenously to enhance the electron transfer process and the ability to adsorb small molecular acids in the mixed bacterial community system, thereby achieving the purpose of reducing the exogenous metabolic pressure of the strain (Lyu H et al., Recycling cinder in efficient methane production from wheat straw via solid-state anaerobic digestion (SS-AD), Chemical Engineering Journal, 2023, 462). In addition, by adding different types of trace heavy metal elements (Fe2+ 、Ni 2+ 、Co 2+ etc.) and surfactants to enhance intracellular energy metabolism and bacterial dispersion, which can enhance methane production capacity (Xu H et al., Enhancing direct interspecies electron transfer in syntrophic-methanogenic associations with (semi) conductive iron oxides: Effects and mechanisms, Sci Total Environ, 2019, Vol. 695, 133876).

[0004] When liquid fermentation is used to produce methane, maintaining microbial activity and improving carbon metabolic flow promotes the biological utilization and conversion of lignocellulosic biomass, so optimizing liquid fermentation conditions is crucial to improving the potential for methane synthesis. At the same time, lignocellulosic biomass has a strong "stubbornness", which hinders the microbial utilization and conversion of polysaccharides in biomass. Summary of the invention

[0005] In view of this, the present invention provides a method for producing methane by artificial mixed bacteria liquid fermentation assisted by Trichoderma reesei, by optimizing the artificial mixed bacteria-biogas residue-alkali-treated corn straw-pig urine liquid fermentation test system, and adding Trichoderma reesei to the above optimal system to increase the polysaccharide release capacity, thereby improving the methane production potential of liquid fermentation.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a methane fermentation system, comprising biogas residue, alkali-pretreated straw, and pig urine. Based on 300 mL of the fermentation system, the system further comprises 5 g of activated carbon, 50 mg / L of Tween-80, and 15 mg / L of manganese ions.

[0008] In some specific embodiments of the present invention, the fermentation system further comprises a bacterial strain combination;

[0009] The bacterial strain combination includes:

[0010] (a) a strain of the genus Cronobacter sp. with a deposit number of CGMCC NO. 31759; and

[0011] (b) a strain of the genus Bacteroides sp. with a deposit number of CGMCC NO. 31760; and

[0012] (c) Enterobacter sp strain with a deposit number of CGMCC NO. 31757; and

[0013] (d) Enterococcus sp strain with the deposit number of CGMCC NO. 31758.

[0014] In some specific embodiments of the present invention, the bacterial strain combination of the above fermentation system also includes:

[0015] (e) Trichoderma reesei RUT-C30 with a deposit number of CICC 13052.

[0016] In some specific embodiments of the present invention, the method for preparing the alkali-pretreated straw in the fermentation system comprises: mixing 10 g of straw and 9 mL of sodium hydroxide with a concentration of 11 g / L, and reacting at 120° C. for 30 min.

[0017] In some specific embodiments of the present invention, in the fermentation system, the inoculation ratio of the biogas residue is 2%, and the inoculation ratio of the straw is 20%, calculated by volume.

[0018] In some specific embodiments of the present invention, the straw in the above fermentation system is corn straw.

[0019] The present invention also provides a method for producing methane, which is produced based on the above fermentation system.

[0020] In some specific embodiments of the present invention, the above-mentioned production method comprises: mixing the biogas residue, the alkali-pretreated straw, the pig urine, the activated carbon, the Tween-80, the manganese ions and the bacterial species combination in the fermentation system, and fermenting to obtain methane.

[0021] In some specific embodiments of the present invention, in the above production method, OD 600 The inoculum volume of the Cronobacter sp strain with the deposit number of CGMCC NO. 31759 is 1 mL;

[0022] OD 600 The inoculation volume of the Bacteroides sp strain with the deposit number of CGMCC NO. 31760 is 1.5 is 1 mL;

[0023] OD 600The inoculation volume of the Enterobacter sp strain with the deposit number of CGMCC NO. 31757 is 1 mL;

[0024] OD 600 The inoculation volume of the Enterococcus sp strain with a deposit number of CGMCC NO. 31758 of 1.5 is 1 mL.

[0025] In some specific embodiments of the present invention, in the above production method, OD 600 The inoculation volume of Trichoderma reesei RUT-C30 with a deposit number of CICC 13052 and a concentration of 2.0 is 2 mL.

[0026] In some specific embodiments of the present invention, the fermentation time of the above production method is 17 days.

[0027] The method of the present invention has the following effects:

[0028] On the one hand, in order to address the problem of polysaccharide conversion barriers caused by the structural complexity of lignocellulosic biomass itself, which in turn leads to low product synthesis efficiency during methane fermentation, the present invention first optimizes the lignocellulosic biomass liquid fermentation methane production system, and utilizes different types of pretreatment to depolymerize the lignocellulosic biomass, thereby improving the ability of renewable resources to synthesize methane by liquid fermentation.

[0029] On the other hand, the present invention uses the response surface method to obtain a model. The test results show that the model is extremely significant (p<0.01) and the lack of fit term is not significant (p>0.05), indicating that the model has a good degree of fit and little interference from unknown factors. The model determination coefficient R 2 =0.9858, Corrected determination coefficient R 2Adj=0.9675, indicating that 96.75% of the response value changes can be explained by the model, and the methane content can be effectively analyzed and predicted. The present invention uses the response surface method to optimize the artificial mixed bacteria-assisted biogas residue-alkali pretreatment corn straw-pig urine system. The results show that when the activated carbon is 5 g, Tween-80 is 50 mg / L and the manganese ion content is 15 mg / L, the gas accumulation and methane synthesis content of the fermentation system are increased to 550 mL and 5.37%, respectively. Furthermore, the original strain Trichoderma reesei RUT-C30 (Trichoderma reesei RUT-C30) with a preservation number of CICC 13052 and T. reesei TRB1 constructed by T-DNA mutagenesis technology were used to assist the artificial mixed bacteria-biogas residue-alkali pretreatment corn straw-pig urine system to carry out liquid fermentation methane production experiments. The results successfully further improved the gas accumulation and methane synthesis efficiency, where the gas accumulation and methane content were 900 mL and 6.01%, respectively.

[0030] Biological Deposit Description

[0031] Biological material: HK-EPT-7, classification name: Enterobacter sp., deposited on August 26, 2024 at the General Microbiology Center of China Culture Collection Administration. The address of the collection center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the collection number is CGMCC No. 31757.

[0032] Biological material: HK-EPT-8, classification name: Enterococcus sp., deposited on August 26, 2024 in the General Microbiology Center of China Culture Collection Administration. The address of the collection center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the collection number is CGMCC No. 31758.

[0033] Biological material: HK-EPT-9, classification name: Cronobacter sp., deposited on August 26, 2024 in the General Microbiology Center of China Culture Collection Administration. The address of the collection center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the collection number is CGMCC No. 31759.

[0034] Biological material: HK-EPT-10, classification name: Bacteroides sp., deposited on August 26, 2024 in the General Microbiology Center of China Culture Collection Administration. The address of the collection center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the collection number is CGMCC No. 31760. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0036] Figure 1 The effects of different exogenous additives on anaerobic fermentation of methane are shown, where (a) and (b) represent the response surfaces and contour lines of Tween-80 and activated carbon on methane content, respectively; (c) and (d) represent the response surfaces and contour lines of manganese ions and activated carbon on methane content, respectively; (e) and (f) represent the response surfaces and contour lines of manganese ions and Tween-80 on methane content, respectively;

[0037] Figure 2 Shown are (a) biogas volume and (b) methane content under different treatments. DETAILED DESCRIPTION

[0038] The present invention discloses a method for producing methane by artificial mixed-bacteria liquid fermentation assisted by Trichoderma reesei. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the method. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0039] The present invention optimizes the artificial mixed bacteria-biogas residue-alkali treated corn straw-pig urine liquid fermentation test system, and adds Trichoderma reesei to the above optimal system to increase the polysaccharide release capacity, so as to achieve the improvement of the methane production potential of liquid fermentation.

[0040] The present invention conducts a liquid fermentation methane production experiment based on biogas residue, alkali-treated corn straw and pig urine as substrates. The liquid fermentation system is 300 mL, the fermentation cycle is 17 d, the inoculum volume ratio is 2%, the solid inoculum ratio is 20% (volume ratio), and pig urine is used to make up the remaining liquid volume.

[0041] In a specific embodiment, the response surface analysis method was used to optimize the amount of activated carbon, surfactant (Tween-80) and trace heavy metal (manganese) added to the artificial mixed bacteria system: Cronobacter. sp strains, Bacteroides. sp strains, Enterobacter. sp strains, and Enterococcus. sp strains assisted digestate-alkali pretreatment corn straw-pig urine system. In addition, the inoculation amount of the above strains was 1 mL (OD 600 is 1.5).

[0042] In some specific embodiments, the Cronobacter sp strain is a strain with a deposit number of CGMCC NO. 31759.

[0043] In some specific embodiments, the Bacteroides sp strain is a strain with a deposit number of CGMCC NO. 31760.

[0044] In some specific embodiments, the Enterobacter sp strain is a strain with a deposit number of CGMCC NO. 31757.

[0045] In some specific embodiments, the Enterococcus sp strain is a strain with a deposit number of CGMCC NO. 31758.

[0046] In the specific embodiment, the original strain Trichoderma reesei RUT-C30 with a preservation number of CICC 13052 and T. reesei TRB1 constructed by T-DNA mutagenesis technology were used to assist the artificial mixed bacteria-biogas residue-alkali pretreated corn straw-pig urine system to carry out liquid fermentation methane production experiments. In addition, the inoculation amount of the above strains was 2 mL (OD 600 is 2.0).

[0047] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0048] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0049] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0050] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present application.

[0051] In addition, the numerical ranges and parameters used to define the present application are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0052] The biogas residue involved in this application comes from the biogas digesters of farmers' homes and is made through long-term fermentation of livestock manure and straw.

[0053] The Shewanella oneidensis MR-1△nagR involved in the present invention is a gift bacterium, which has been disclosed in an existing patent (Construction method for co-utilization metabolism of xylose and glucose in Shewanella, 2023-11-07, China, CN201911096689.1).

[0054] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0055] The present invention will be further described below in conjunction with the embodiments.

[0056] Example 1

[0057] First, the amount of biomass added in the process of preparing methane by feces-corn straw-liquid fermentation was optimized. Among them, the amount of corn straw added was set to 4%, 8%, 15%, 20% and 30% (volume ratio) respectively. At the same time, the liquid fermentation system of the present invention is 300mL, the liquid fermentation is 17 days, the volume of the inoculum (pig feces) is 2% (volume ratio), and the rest of the reaction system is supplemented by pig urine. The results are shown in Table 1.

[0058] Table 1: Liquid fermentation capacity at different corn straw addition ratios

[0059]

[0060] The results showed that when liquid fermentation was carried out using different corn straw addition ratios, the gas accumulation showed a trend of first increasing and then decreasing with the increase of corn straw addition ratio (Table 1). Among them, when the corn straw addition ratio was 15%, the gas volume was higher than that of other experimental groups. As for the methane synthesis efficiency, when the corn straw addition ratio was 15%, its methane content was the highest and reached 1.57% (Table 1).

[0061] Then, corn straw was depolymerized by different types of pretreatments - alkali pretreatment and ethylenediamine pretreatment. The alkali pretreatment conditions were: 10 g corn straw, 9 mL sodium hydroxide (11 g / L), reaction temperature 120°C, and reaction time 30 min; the ethylenediamine pretreatment conditions were: 10 g corn straw, 9 mL ethylenediamine, reaction temperature 130°C, and reaction time 60 min. The corn straw after the above pretreatment was dried, and the methane production test was carried out with different types of pretreated corn straw inoculation ratios (4%, 8%, 15%, 20%, and 30%). The results are shown in Table 2.

[0062] Table 2: Liquid fermentation capacity of corn straw with different types of pretreatment

[0063]

[0064] From the methane production test of manure-ethylenediamine pretreated corn straw-liquid fermentation in Table 2, it can be seen that with the increase in the proportion of pretreated biomass added in the above system, the gas volume gradually increases and the methane content is low. Compared with the above pretreated corn straw, when 20% pretreated biomass is added to the manure-sodium hydroxide pretreated corn straw-liquid fermentation system, the gas accumulation volume reaches 325 mL. At the same time, the methane content in the above system has increased to 2.35%.

[0065] Example 2

[0066] Liquid fermentation and methane production experiments were carried out with artificial mixed bacteria, biogas residue, alkali-treated corn stalks and pig urine as substrates. The liquid fermentation system was 300 mL, the fermentation cycle was 17 d, the inoculum (biogas residue) inoculation ratio was 2%, the solid inoculation ratio was 20% (i.e., the biomass inoculation ratio, in this embodiment, alkali-pretreated corn stalks), and pig urine was used to make up the remaining liquid volume. The artificial mixed bacteria were Cronobacter. sp strains with a preservation number of CGMCC NO. 31759, Bacteroides. sp strains with a preservation number of CGMCC NO. 31760, Enterobacter. sp strains with a preservation number of CGMCC NO. 31757, and Enterococcus. sp strains with a preservation number of CGMCC NO. 31758, and the inoculation amount was 1 mL (OD 600 is 1.5).

[0067] The conditions of the alkaline pretreatment are: 10 g corn stalks and 9 mL of sodium hydroxide with a concentration of 11 g / L are mixed, the reaction temperature is 120°C, and the reaction time is 30 min. The specific steps are: 10 g corn stalks (dry weight) and 9 mL of sodium hydroxide solution are added to the reactor and stirred evenly. After tightening the reactor, put the reactor into a 120°C constant temperature oil bath. After reacting for 30 min, take out the reactor and cool it to room temperature with tap water. The reacted corn stalks are washed 3 times with 100 mL of distilled water each time and filtered with gauze. The washed stalks are dried in a 60°C oven overnight to a moisture content of less than 10%, and stored in a ziplock bag at room temperature.

[0068] The response surface analysis software Design-Expert8.0 was used for experimental design. The amount of activated carbon added, Tween-80 content, and trace heavy metal (manganese) content were used as response variables, and the methane content was used as the response value. The experiment was carried out according to the response surface design scheme and the response values ​​were filled in for data analysis (Table 3). The quadratic polynomial regression equation obtained was:

[0069] Y=5.19 - 0.021 A + 0.016 B + 0.032 C - 0.070 AB +0.096 AC - 0.13 BC -0.75 A 2 – 0.52 B 2 -1.59 C 2 .

[0070] Table 3: Box-Behnken test plan and results

[0071]

[0072] Table 4: Regression model and ANOVA

[0073]

[0074] like Figure 1 As shown in Table 3, the model is extremely significant (p < 0.01) and the lack of fit term is not significant (p > 0.05), indicating that the model has a good fit and little interference from unknown factors. 2 =0.9858, Corrected determination coefficient R 2 Adj=0.9675. The above results show that 96.75% of the response value changes can be explained by this model, and it can effectively analyze and predict the methane content. 2 , B 2 and C 2 The influence on methane content was significant (p<0.01). According to the p value of each factor, the order of influence of each parameter on methane content can be obtained: trace heavy metal (manganese) content > activated carbon addition > Tween-80 content.

[0075] The response surface methodology was used to optimize the artificial mixed bacteria-assisted biogas residue-alkali pretreatment corn straw-pig urine system. The results showed that when the activated carbon was 5 g, Tween-80 was 50 mg / L and the manganese ion content was 15 mg / L, the gas accumulation and methane synthesis content increased to 550 mL and 5.37%, respectively (Table 5).

[0076] Furthermore, the original strain Trichoderma reesei RUT-C30 (CICC 13052) and T. reesei TRB1 constructed by T-DNA mutagenesis technology were used to assist the artificial mixed bacteria-biogas residue-alkali pretreated corn straw-pig urine system to carry out liquid fermentation methane production experiments in order to further improve the methane production. The inoculation amount was 2 mL (OD 600 is 2.0).

[0077] Based on the optimized artificial mixed bacteria-biogas residue-alkali pretreated corn straw-pig urine liquid fermentation system, the gas accumulation and methane synthesis efficiency can be further improved by adding T. reesei RUT-C30 to the above system ( Figure 2 , Table 5). Among them, the gas accumulation and methane content were 900 mL and 6.01%, respectively.

[0078] Table 5: Gas production capacity of different types of liquid fermentation

[0079]

[0080] In summary, the present invention provides a strategy with reference significance for preparing CH4 by biomass liquid fermentation.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A methane fermentation system comprising biogas residue, alkali-pretreated straw, and pig urine, characterized in that: Based on 300 mL of the fermentation system, 5 g of activated carbon, 50 mg / L of Tween-80, and 15 mg / L of manganese ions are also included.

2. The fermentation system according to claim 1, characterized in that It also includes bacterial strain combinations; The bacterial strain combination includes: (a) a strain of the genus Cronobacter sp. with a deposit number of CGMCC NO. 31759; and (b) a strain of the genus Bacteroides sp. with a deposit number of CGMCC NO. 31760; and (c) Enterobacter sp strain with a deposit number of CGMCC NO. 31757; and (d) Enterococcus sp strain with the deposit number of CGMCC NO. 31758.

3. The fermentation system according to claim 2, characterized in that The bacterial strain combination also includes: (e) Trichoderma reesei RUT-C30 with a deposit number of CICC 13052.

4. The fermentation system according to any one of claims 1 to 3, characterized in that: The preparation method of the alkali-pretreated straw comprises: mixing 10 g of straw and 9 mL of sodium hydroxide with a concentration of 11 g / L, and reacting at 120° C. for 30 minutes.

5. The fermentation system according to any one of claims 1 to 4, characterized in that: Calculated by volume ratio, the inoculation ratio of the biogas residue is 2%, and the inoculation ratio of the straw is 20%.

6. The fermentation system according to any one of claims 1 to 5, characterized in that: The straw is corn straw.

7. A method for producing methane, characterized in that: Produced based on the fermentation system according to any one of claims 1 to 6.

8. The production method according to claim 7, characterized in that include: The biogas residue, the alkali-pretreated straw, the pig urine, the activated carbon, the Tween-80, the manganese ions and the bacterial species combination in the fermentation system according to claim 2 or 3 are mixed and fermented to obtain methane.

9. The production method according to claim 8, characterized in that OD 600 The inoculation volume of the Cronobacter sp strain with the deposit number of CGMCC NO.31759 is 1.5 and is 1 mL; OD 600 The inoculation volume of the Bacteroides sp strain with the deposit number of CGMCC NO. 31760 is 1.5 is 1 mL; OD 600 The inoculation volume of the Enterobacter sp strain with the deposit number of CGMCC NO. 31757 is 1 mL; OD 600 The inoculation volume of the Enterococcus sp strain with a deposit number of CGMCC NO. 31758 of 1.5 is 1 mL.

10. The production method according to claim 8 or 9, characterized in that: OD 600 The inoculation volume of Trichoderma reesei RUT-C30 with a deposit number of CICC13052 of 2.0 is 2 mL.

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