A method for co-producing hydrogen and butanol from waste

Abstract

This invention discloses a method for co-producing hydrogen and butanol from waste, relating to the field of biochemical technology. The method includes the following steps: adding NADH and NADPH precursors, as well as amylase-dependent metal ions, to a fermentation system for anaerobic saccharification and fermentation to achieve co-production of hydrogen and butanol from waste; the fermentation system includes a fermentation strain, a culture medium, and waste substrate; the fermentation strain is Clostridium perfringens. This invention achieves efficient, green co-production of hydrogen and butanol from organic solid waste, providing technical support for the industrial conversion of low-value biomass into hydrogen and butanol bioenergy.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, and in particular to a method for co-producing hydrogen and butanol from waste. Background Technology

[0002] In recent years, with the depletion of global oil resources and the continuous rise in world oil prices, the global climate has shown a deteriorating trend. Energy issues have become an urgent problem to be solved for the sustainable development of the world economy and industry. Developing new renewable and environmentally friendly energy sources has become one of the energy development strategies of countries around the world. Hydrogen is a high-quality, efficient, and sustainable new energy source, considered one of the most promising clean energy sources of the 21st century that can replace coal and oil. Biobutanol is not only a basic biological chemical, but also an excellent organic solvent and an important chemical raw material. Furthermore, it is a highly promising second-generation biofuel, with a higher calorific value and better fuel economy compared to bioethanol. With the implementation of energy substitution strategies, hydrogen and butanol have become a focus of attention for countries seeking to replace fossil fuels. Today, biomass energy has become the world's fourth largest energy source after oil, natural gas, and coal. Due to its renewable and environmentally friendly advantages, it is of great significance to social and economic development and has broad application prospects.

[0003] Bioenergy has a vast consumer market, with demand in the global chemical and energy sectors maintaining continuous growth and possessing significant potential. Currently, the raw materials for bioenergy production via biomass fermentation suffer from problems such as complex composition, unsuitable nutrient components and ratios, and numerous inhibitors. Furthermore, mismatched fermentation processes lead to issues like abnormal strain growth, excessive byproducts, and low yields during hydrogen and butanol fermentation. Therefore, research on bioenergy production has become a hot topic in the development and utilization of renewable green energy. To date, various efforts have been made to improve the concentration, yield, and production efficiency of hydrogen and butanol, such as classical chemical mutagenesis, a series of enrichment programs, and overexpression of targeted functional genes in engineered hosts to overcome the butanol toxicity barrier in microorganisms. However, despite advancements in the technology of genetically engineered strains or mutants, the resulting instability in hydrogen and butanol production indicates that transferring relevant pathways to modified strains or host strains remains difficult and complex due to the inherent instability or inactive expression of wild-type strains.

[0004] A paper from Nanjing Agricultural University, titled "Research on Bio-hydrogen Production from Anaerobic Digestion of Food Waste," disclosed a technical scheme for hydrogen production using Clostridium difficile fermentation of waste; however, its hydrogen yield was low. Although significant progress has been made in understanding the physiological metabolism and regulatory mechanisms of typical wild-type fermentation strains, research progress on improving the utilization efficiency and production intensity of hydrogen and butanol fermentation substrates through strain modification has been extremely slow, mainly due to the lack of effective target proteins for targeted metabolic engineering. While existing technologies disclose some methods for producing hydrogen or butanol from food waste, in-depth research on the co-production of hydrogen and butanol has not yet been conducted. Summary of the Invention

[0005] Based on the above, this invention provides a method for co-producing hydrogen and butanol from waste. This method is simple, produces few byproducts, and yields high levels of hydrogen and butanol energy. It can efficiently utilize organic solid waste, ensure a virtuous cycle in the environmental ecological chain and diversify energy supply, and provide a scientific basis for the green and sustainable industrialization of bioenergy from organic waste biomass.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] One of the technical solutions of this invention is a method for co-producing hydrogen and butanol from waste, comprising the following steps:

[0008] Adding NADH and NADPH precursors and amylase to the fermentation system to catalyze metal ions enables anaerobic biological fermentation, achieving co-production of hydrogen and butanol from waste.

[0009] The fermentation system includes fermentation strains, inoculum culture medium, and waste substrate;

[0010] The fermentation strain is Clostridium perfringens.

[0011] Furthermore, the concentration of the waste substrate in the fermentation system is 160-250 g / L; preferably, it is 180 g / L.

[0012] Furthermore, the waste substrate is any one or more of the following: kitchen waste, low-value cassava residue, molasses wastewater, vinegar residue, and soy sauce residue.

[0013] Furthermore, the anaerobic biological fermentation time is 72-120 hours; preferably, it is 72 hours.

[0014] Furthermore, the precursor is one or more of nicotinic acid, aspartic acid, and tryptophan; the amylase catalyzes the metal ion Ca2+. 2+ and / or Zn 2+ .

[0015] Furthermore, the concentration of nicotinic acid in the fermentation system is 0-20 mg / L, the concentration of aspartic acid is 0-2.5 g / L, the concentration of tryptophan is 0-2 g / L, and the concentration of Ca... 2+ and / or Zn 2+ The concentration is 0-100 mM. Preferably, the concentration of nicotinic acid is 10 mg / L, the concentration of tryptophan is 1.0 g / L, the concentration of aspartic acid is 2.0 g / L, and the concentration of Ca... 2+ The concentration was 8 mM.

[0016] Furthermore, in the initial stage of fermentation, the pH of the fermentation system is adjusted to 5.5-6.0 and fermented for 6-8 hours. Afterward, the pH of the fermentation system is adjusted to 5.0-5.5 until fermentation is complete. Preferably, in the initial stage of fermentation, the pH of the fermentation system is adjusted to 6.0 and fermented for 6-8 hours. Afterward, the pH of the fermentation system is adjusted to 5.0 until fermentation is complete.

[0017] Furthermore, the culture medium is a carbon source culture medium suitable for Clostridium difficile.

[0018] Furthermore, the inoculation process of the fermentation strain specifically involves: inoculating the fermentation system with seed liquid; the inoculation amount of the seed liquid is 6% v / v.

[0019] Furthermore, the method for preparing the seed liquid includes the following steps:

[0020] After adding inorganic salt solution and trace element solution to the culture medium, seal and sterilize. Then add sterile glucose stock solution and sterile yeast extract stock solution, inoculate with live cells of the strain, and culture with shaking for 24-30 hours to obtain the seed culture.

[0021] The second technical solution of this invention is a method to improve the efficiency of hydrogen production from waste by Clostridium difficile fermentation, which involves adding NADH and NADPH precursors and amylase-catalyzed metal ions to the fermentation system for anaerobic biological fermentation.

[0022] Furthermore, the precursor is one or more of nicotinic acid, aspartic acid, and tryptophan; the amylase catalyzes the metal ion Ca2+. 2+ and / or Zn 2+ .

[0023] The third technical solution of the present invention is a strain used in the above-mentioned method for co-producing hydrogen and butanol from waste, characterized in that the strain is Clostridium sp. Strain M6, deposited at the China General Microbiological Culture Collection Center on August 29, 2022, with accession number CGMCC No. 40305.

[0024] Invention concept:

[0025] Coenzyme factors have become key targets for inducing metabolic changes, playing a crucial role in numerous biochemical reactions and the production of various fermentation products. Cofactors may become a significant factor in cofactor-dependent production systems, particularly in hemolytic Clostridium difficile involving the reducing coenzymes NADH and NADPH-dependent butanol dehydrogenases, potentially proving to be a powerful tool for enhancing overall process yield and productivity.

[0026] This invention utilizes niacin (NA), a precursor of NADH and NADPH, tryptophan, aspartic acid (AA), and amylase to catalyze the conversion of metal ions into Ca2+. 2+ and / or Zn 2+ Added to the fermentation medium of Clostridium difficile. Among them, Ca... 2+ or Zn 2+ The addition of these substances can enhance the activity of metalloenzymes such as starch-degrading enzymes in the reaction system, increase the yield of glucose conversion from waste starch, and provide sufficient substrates for the conversion to hydrogen and butanol products. Nicotinic acid, tryptophan, and aspartic acid, through their synergistic effects, can improve the availability and intracellular levels of the reducing coenzymes NADH and NADPH of butanol dehydrogenase, thereby enhancing the NADH... - and NADPH - The activity of butanol dehydrogenase was enhanced, thereby increasing the production of hydrogen and butanol. Results showed that under optimal fermentation conditions, the highest yields of both hydrogen and butanol were achieved simultaneously, at 313.5 mmol of hydrogen and 15.2 g / L of butanol, respectively. This invention reveals that by shifting the metabolic focus more towards reducing metabolic byproducts, improving the availability and levels of coenzymes, and altering the pH of the fermentation broth, metabolic flux can be redirected towards hydrogen and butanol, achieving metabolic engineering goals and increasing the co-production of hydrogen and butanol.

[0027] The present invention discloses the following technical effects:

[0028] The raw materials for fermentation in this invention come from solid waste such as kitchen waste, low-value cassava residue, molasses wastewater, vinegar residue, soy sauce, and low-value cassava. These materials are inexpensive and readily available, which can greatly reduce the production cost of bio-fermentation.

[0029] The fermentation method provided by this invention has the advantages of low cost and high yield, and does not require pretreatment such as saccharification. It is a green and sustainable bioenergy manufacturing technology.

[0030] By adding niacin, tryptophan, aspartic acid, and amylase—precursors to NADH and NADPH—and catalyzing the metal ion Ca2+, 2+ and / or Zn 2+By leveraging their synergistic effects to enhance the availability and regeneration of reducing coenzymes, and through phased pH-regulated metabolism, a single microorganism can complete the synthesis of hydrolytic / saccharifying enzymes, polysaccharide fermentation, and hydrogen / butanol co-production in a one-step process, thereby enhancing the integrated transformation of biological processes.

[0031] This invention addresses the limiting problem between the lack of reducing coenzymes and the dependence on reducing coenzymes in bioenergy production systems. It increases the availability of reducing coenzymes through multiple pathways, clarifies the master-controlling regulatory factors of NAD(P)H synthesis rate and the acid-alcohol phase partitioning mechanism, and achieves a one-step bioprocess transformation of hydrolysis / saccharification enzyme synthesis, polysaccharide fermentation, and hydrogen / butanol co-production using a single microorganism. This breakthrough significantly improves the efficiency of synthesizing biohydrogen and butanol from low-value waste biomass. Therefore, this technology enables efficient, green, and sustainable directed bioconversion of hydrogen and butanol from organic solid waste, providing technical support for the industrial-scale conversion of hydrogen and butanol from low-value biomass into bioenergy. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 This invention illustrates the effects of nicotinic acid concentration on hydrogen and butanol production, enzyme activity, and intracellular NADP and NADPH levels.

[0034] Figure 2 This invention illustrates the effects of tryptophan concentration on hydrogen and butanol production, enzyme activity, and intracellular NADP and NADPH levels.

[0035] Figure 3 This invention illustrates the effects of aspartic acid concentration on hydrogen and butanol production, enzyme activity, and intracellular NADP and NADPH levels.

[0036] Figure 4 For the embodiments of the present invention, Ca 2+ Effects on enzyme activity;

[0037] Figure 5 This invention illustrates the relationship between pH and the maximum rates of butanol and hydrogen formation during fermentation in this embodiment.

[0038] Figure 6 This invention illustrates the effect of kitchen waste concentration on the concentration of hydrogen, butanol, and intermediate products produced by strain M6 during fermentation.

[0039] Figure 7 This invention relates to the effect of fermentation time on the concentration of hydrogen, butanol, and intermediate products produced by strain M6 from fermented kitchen waste. Detailed Implementation

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0043] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0044] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0045] Unless otherwise specified, all raw materials used in the embodiments of this invention were obtained through purchase.

[0046] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; the culture media used should be understood as any conventional culture media applicable to Clostridium butanol-producing bacteria in the prior art.

[0047] Unless otherwise specified, all reagents used in the embodiments of this invention are of analytical grade or higher.

[0048] The strain M6 used in this embodiment of the invention is a wild-type Clostridium sp. strain isolated from rice, deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCCNo.40305, and classified as Clostridium sp. Strain M6.

[0049] Other Clostridium species can utilize waste to produce butanol. Under the same fermentation conditions as M6, Clostridium acetobutylicum ATCC824 (type strain, US Culture Collection ATCC824) produced 7.1 g / L of butanol, only 46.1% of that of M6; for example, Clostridium beijerinckii NCIMB8052 (National Industrial, Marine and Food Culture Collection (NCIMB)) produced 10.5 g / L of butanol, only 69% of that of M6; and Clostridium acetobutylicum CICC8008 (China Industrial Microbial Culture Collection Center) produced 4.2 g / L of butanol, only 27.6% of that of M6.

[0050] The isolation method of strain M6 used in the embodiments of the present invention is as follows:

[0051] (1) Anaerobic enrichment: In an anaerobic glove box, open a 60 mL serum bottle containing 30 mL of culture medium (with 0.6 g starch added), place the sample into the serum bottle, and then seal the serum bottle with a butyl rubber stopper and aluminum cap. Incubate at 37°C and 180 rpm for 3 days, and repeat the enrichment culture process twice. Take the supernatant, centrifuge at 14000 rpm for 10 minutes at 4°C, and detect the concentration of butanol product by gas chromatography; at the same time, collect biogas using a gas bag and detect hydrogen production by gas chromatography.

[0052] (2) Selection and screening: Open the anaerobic enrichment medium described above in an anaerobic glove box, add 10 mL of enrichment culture to 100 mL of screening medium (with 10 g of organic waste added to the medium), and continue to incubate for 7 days at 37°C and 180 rpm. After 7 days of incubation, bottles with significant consumption of organic waste and obvious generation of hydrogen and butanol were selected for subsequent strain screening.

[0053] (3) Plate separation: The final fermentation broth was serially diluted using anaerobic sterile ultrapure water. 0.1 mL of each plate was then plated. -1 10 -2 10 -3 10 -4 10 -510 -6 10 -7 10 -8 10 -9 The diluted solution was spread onto 9 cm agar plates. After incubation at 37°C for 48 h, colonies with different morphologies and colors growing on the surface of the medium were picked and isolated and purified by streaking multiple times to obtain single strains.

[0054] (4) Selection of optimal strains: The selected strains were inoculated into the fermentation medium and cultured at 37℃ and 180 rpm for 120 h. The organic waste conversion rate, hydrogen and butanol production were measured to explore the strains' capabilities, and the strains with the best conversion effect and activity were selected for identification. Through 16S rRNA gene sequencing and identification, the strain was identified as Clostridium sp. Strain M6.

[0055] In this embodiment of the invention, the detection of bioenergy substances such as hydrogen and butanol was performed using gas chromatography. Specifically, the temperature program was as follows: column temperature, 60℃ (held for 2 minutes) to 240℃ (held for 2 minutes), with the temperature increasing at 15℃ / min; detector temperature, 250℃; helium carrier gas flow rate, 2 mL / min; injected sample volume, 1 μL; split ratio, 20:1; hydrogen flow rate, 30 mL / min; air flow rate, 40 mL / min.

[0056] Example 1

[0057] Step 1, Preparation of culture medium: Add 10 mL of inorganic salt culture medium and 1 mL of trace elements to 1 L of deionized water; the composition of the inorganic salt culture medium is: CaCO3, 3 g / L; KH2PO4, 0.75 g / L; K2HPO4, 0.75 g / L; CH3COONH4, 2 g / L.

[0058] Step 2, Preparation of Seed Culture: Boil the 1L of culture medium mentioned above and cool to room temperature under nitrogen flow. Dispense 42mL into 160mL serum bottles, seal with butyl rubber stoppers and aluminum caps, autoclave for 30 minutes, cool to room temperature, and then add 5mL of sterile glucose stock solution (300g / L) and 1mL of sterile yeast extract stock solution (150g / L). Subsequently, inoculate the viable cells of the strain (4%, vol / vol) into the serum bottles and culture on a shaker at 37℃ for 26 hours to obtain the seed culture.

[0059] Step 3: Add 50 mL of the culture medium prepared in Step 1, 180 g / L food residue, and different concentrations of nicotinic acid (0 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L), tryptophan (0 g / L, 0.5 g / L, 1 g / L, 1.5 g / L, 2.0 g / L), aspartic acid (0 g / L, 0.5 g / L, 1 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L), and calcium to a 160 mL serum bottle. 2+ (0mM, 8mM) Inoculate the prepared seed solution (inoculation amount 6% (v / v)) into the solution containing nicotinic acid, tryptophan, aspartic acid, and Ca. 2+ Anaerobic fermentation was carried out in the culture medium at 37℃ in a shaking incubator for 120 h. The yields of hydrogen and butanol, the activities of NADH-dependent butyraldehyde dehydrogenase, NADPH-dependent butanol dehydrogenase, and NADH-dependent butanol dehydrogenase, as well as the levels of intracellular and extracellular NADP and NADPH were measured. Results are as follows: Figure 1 , 2 As shown in Figures 3 and 4; Figure 1 The effects of nicotinic acid concentration on hydrogen and butanol production, enzyme activity, and intracellular NADP and NADPH levels; Figure 2 The effects of tryptophan concentration on hydrogen and butanol production, enzyme activity, and intracellular NADP and NADPH levels; Figure 3 The effects of aspartate concentration on hydrogen and butanol production, enzyme activity, and intracellular NADP and NADPH levels; Figure 4 For Ca 2+ Effects on enzyme activity.

[0060] Depend on Figure 1 , 2 Figure 3 shows that the yields of butanol and hydrogen initially increase and then decrease with increasing concentrations of nicotinic acid, tryptophan, and aspartic acid. The yields of both butanol and hydrogen reach their maximum values ​​when the concentrations of nicotinic acid are 10 mg / L, tryptophan is 1.0 g / L, and aspartic acid is 2.0 g / L. Further analysis of the NAD(P)H / NAD(P) ratio in the fermentation system... + The same conclusion was reached regarding enzyme activity and intracellular and extracellular NADP and NADPH levels, indicating that a certain amount of nicotinic acid can enhance the NAD(P)H and NAD(P) levels in the fermentation system. + The ratio of NADH-dependent butyraldehyde dehydrogenase, the activity of NADPH-dependent butanol dehydrogenase and NADH-dependent butanol dehydrogenase, as well as the levels of intracellular and extracellular NADP and NADPH, promote the metabolic level of M6 and enhance the ability of food waste to ferment and produce butanol and hydrogen.

[0061] Depend on Figure 4 It can be seen that when 8mMCa is added 2+At the specified concentration, the activities of amylase, NADH-dependent butyraldehyde dehydrogenase (NADH-BADH), NADPH-dependent butanol dehydrogenase (NADPH-BDH), and NADH-dependent butanol dehydrogenase (NADH-BDH) in the fermentation system were all higher than those in the control group. Therefore, calcium ions can also promote the metabolic level of M6 and enhance the ability of food waste to produce butanol and hydrogen through fermentation.

[0062] Example 2

[0063] In Example 1, the technical solution (nicotinic acid concentration was 10 mg / L, tryptophan concentration was 1.0 g / L, aspartic acid concentration was 2.0 g / L, and Ca...) 2+ Based on a concentration of 8 mM, the pH, butanol yield, and hydrogen production of the fermentation broth were measured at different fermentation times. Then, the maximum specific butanol formation rate and the maximum specific hydrogen formation rate were calculated. The results are as follows: Figure 5 As shown.

[0064] Depend on Figure 5 It can be seen that no hydrogen and butanol were produced when the pH was less than 4.5 or greater than 8.0, indicating that the suitable pH for hydrogen and butanol production is 4.5–8.0. Within the pH range of 5.0–5.5, the formation rates of both hydrogen and butanol reach their maximum values. From pH 5.5 to 6.0, the formation rates of both gradually decrease, but still remain relatively high. Therefore, in the early stages of fermentation, to control the forward reaction, it is suitable to maintain a relatively high pH value (e.g., pH 5.5–6.0). In the middle and later stages of fermentation, to maximize the production of butanol and hydrogen, a lower pH value (e.g., pH 5.0–5.5) should be chosen.

[0065] Example 3

[0066] (1) By increasing the fermentation volume in Example 1 and changing the concentration of the substrate food waste, the optimal Ca under the following conditions was studied. 2+ The effects of food waste concentration on the production of hydrogen and butanol by strain M6 were investigated at concentrations of 8 mM, nicotinic acid 10 mg / L, tryptophan 1.0 g / L, and aspartic acid 2.0 g / L. The fermenter had a volume of 2 L and an effective volume of 1 L. The fermentation time was 120 h. The pH of the fermentation system was adjusted, maintaining pH 6.0 for 6 hours, and then decreasing to 5.0 until fermentation ended. The specific amounts of food waste added were 30 g / L, 60 g / L, 90 g / L, 120 g / L, 150 g / L, 180 g / L, 210 g / L, and 240 g / L.

[0067] (2) Samples were prepared using a batch extraction method, and the solutions and gases were analyzed. Gas samples collected using gas bags were analyzed by gas chromatography. The culture broth extracted from the fermenter was centrifuged at 10,000 rpm and 4°C for 10 minutes. The supernatant was stored at -20°C, and the contents of the products (hydrogen and butanol) and intermediates (acetone and ethanol) were determined by gas chromatography. The results are as follows: Figure 6 As shown.

[0068] Depend on Figure 6 It can be seen that the yields of hydrogen, butanol, and the intermediate products ethanol and acetone all increase with increasing food waste concentration. When the substrate concentration is 180 g / L, the yields of hydrogen and butanol reach their maximum values ​​of 313.5 mmol and 15.2 g / L, respectively, with yields of 4.98 mmol / g reducing sugar and 0.241 g / g reducing sugar, respectively. As the substrate concentration continues to increase, the yields of both substances reach equilibrium and no longer increase. Therefore, a substrate concentration of 180 g / L is most favorable for the fermentation production of hydrogen and butanol.

[0069] Example 4

[0070] Based on the experimental results in Example 3, a food waste concentration of 180 g / L was selected to investigate the effect of fermentation time on the production of hydrogen, butanol, and other intermediate products by strain M6 from fermented food waste. Gas and liquid samples were collected and analyzed at fermentation times of 0 h, 12 h, 24 h, 30 h, 36 h, 48 h, 60 h, 72 h, 96 h, and 120 h. The results are as follows: Figure 7 As shown.

[0071] Depend on Figure 7 It can be seen that, except for acetic acid and butyric acid which showed a trend of first increasing and then decreasing, the yields of hydrogen, butanol and other intermediate products all showed an increasing trend with fermentation time. This indicates that the acetic acid and butyric acid produced in the early stage of fermentation were gradually utilized by microorganisms to produce hydrogen and butanol, and reached equilibrium at 72 hours of fermentation.

[0072] Based on Examples 1-4, the optimal fermentation conditions for this invention were determined to be: adding amylase to catalyze the metal ion Ca2+. 2+ The concentration was 8 mM, and the concentrations of the precursors nicotinic acid, tryptophan, and aspartic acid were 10 mg / L, 1.0 g / L, and 2.0 g / L, respectively. Fermentation was maintained at pH 6.0 for 6 hours, and then gradually decreased to pH 5.0 until fermentation ended. The fermentation time was 72 hours, and the substrate concentration was 180 g / L. Under optimal fermentation conditions, the hydrogen yield was 313.5 mmol, and the butanol yield was 15.2 g / L.

[0073] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for co-producing hydrogen and butanol from waste, characterized in that, Includes the following steps: Adding niacin, aspartic acid, and tryptophan, precursors of NADH and NADPH, to the fermentation system, along with amylase-catalyzed metal ion Ca2+, 2+ Anaerobic biological fermentation is carried out to achieve co-production of hydrogen and butanol from waste; The fermentation system contains nicotinic acid at a concentration of 5-20 mg / L, aspartic acid at a concentration of 0.5-2.5 g / L, tryptophan at a concentration of 0.5-2.0 g / L, and Ca... 2+ The concentration was 8 mM; The anaerobic biological fermentation includes: adjusting the pH of the fermentation system to 5.5-6.0 in the early stage of fermentation and fermenting for 6-8 hours, and then adjusting the pH of the fermentation system to 5.0-5.5 until the fermentation is completed; The fermentation system includes fermentation strains, inoculum culture medium, and waste substrate; The concentration of the waste substrate in the fermentation system is 160-250 g / L; The fermentation strain is Clostridium sp. Strain M6, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 29, 2022, with accession number CGMCC No. 40305.

2. The method for co-producing hydrogen and butanol from waste according to claim 1, characterized in that, The waste substrate is any one or more of the following: kitchen waste, low-value cassava residue, molasses wastewater, vinegar residue, and soy sauce residue.

3. The method for co-producing hydrogen and butanol from waste according to claim 1, characterized in that, The anaerobic biological fermentation time is 72-120 hours.

4. A method for improving the efficiency of hydrogen production from waste by Clostridium difficile fermentation, characterized in that, Includes the following steps: Adding niacin, aspartic acid, and tryptophan, precursors of NADH and NADPH, to the fermentation system, along with amylase-catalyzed metal ion Ca2+, 2+ Anaerobic biological fermentation is carried out; The fermentation system contains nicotinic acid at a concentration of 5-20 mg / L, aspartic acid at a concentration of 0.5-2.5 g / L, tryptophan at a concentration of 0.5-2.0 g / L, and Ca... 2+ The concentration was 8 mM; The anaerobic biological fermentation includes: adjusting the pH of the fermentation system to 5.5-6.0 in the early stage of fermentation and fermenting for 6-8 hours, and then adjusting the pH of the fermentation system to 5.0-5.5 until the fermentation is completed; The fermentation system includes fermentation strains, inoculum culture medium, and waste substrate; The concentration of the waste substrate in the fermentation system is 160-250 g / L; The fermentation strain is Clostridium sp. Strain M6, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 29, 2022, with accession number CGMCC No. 40305.

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