A three-stage linkage hydrogen production method based on kitchen waste

Abstract

The application discloses a three-stage linkage hydrogen production method based on kitchen waste, and relates to the technical field of biological enzymes, and comprises the following steps: step I, pretreatment; step II, dark fermentation: feeding the material treated in step I into a dark fermentation tank, inoculating Clostridium beijerinckii with the preservation number of ATCC NO.35702; step III, photo fermentation: feeding the first fermentation liquor into a photo fermentation tank, inoculating Rhodopseudomonas palustris with the preservation number of CGMCC 1.2180, adding 50 mg / L-100 mg / L MoFe nanoparticles, and collecting the gas produced in the fermentation. The three-stage linkage of the synergistic pretreatment, dark fermentation and photo fermentation realizes the efficient and stable conversion of the kitchen waste into hydrogen.

Description

Technical Field

[0001] This application relates to the field of bioenzyme technology, and in particular to a three-stage hydrogen production method based on kitchen waste. Background Technology

[0002] Bio-based hydrogen production technology has garnered significant attention in the global energy transition due to its zero-carbon emission characteristics. Utilizing organic waste such as food waste through dark fermentation to produce hydrogen not only achieves waste resource recovery but also generates clean energy, making it considered one of the most promising technological pathways. However, existing food waste-based hydrogen production technologies face several challenges. Firstly, the stubborn structures like cellulose in food waste lead to low hydrolysis efficiency, resulting in low substrate utilization by microorganisms. Secondly, the end-stage metabolites of dark fermentation contain large amounts of low-molecular-weight organic acids, such as acetic acid and butyric acid. These volatile organic acids exert feedback inhibition on hydrogen-producing bacteria, limiting the rate and quantity of hydrogen production during dark fermentation. Summary of the Invention

[0003] In view of this, this application provides a three-stage hydrogen production method based on food waste, which improves the hydrogen production efficiency and hydrogen yield of food waste through synergistic pretreatment, strain selection and precise fermentation conditions.

[0004] A three-stage hydrogen production method based on food waste includes the following steps:

[0005] Step I, Pretreatment: Place the kitchen waste in an environment of 1.1MPa~1.3MPa and 160℃~180℃ for 10min~20min to obtain pretreated material. Adjust the particle size of the pretreated material to 0.5mm~2mm by grinding or sieving.

[0006] For example, the pretreatment pressure is 1.1 MPa, 1.15 MPa, 1.2 MPa, 1.25 MPa, 1.3 MPa, or any value between any two of the above. For example, the pretreatment temperature is 160°C, 165°C, 170°C, 175°C, 180°C, or any value between any two of the above. For example, the pretreatment time is 10 min, 12 min, 13 min, 15 min, 18 min, 20 min, or any value between any two of the above. For example, the particle size of the pretreated material is adjusted to 0.5 mm, 0.6 mm, 0.8 mm, 1.0 mm, 1.5 mm, 1.8 mm, 2 mm, or any value between any two of the above.

[0007] This application utilizes the synergistic regulation of high-temperature and high-pressure treatment environment and appropriate particle size selection to target and modify food waste. Furthermore, it controls the total carbon content (by dry weight) of the food waste to be ≥40%, the fat content (by dry weight) to ≤25%, and the total nitrogen content (by dry weight) to ≤6%. This means that the pretreated substrate contains ≥50% biodegradable carbohydrates, primarily small-molecule sugars (glucose, fructose) and emulsified oils. This matches the carbon source preference of Clostridium beyerridis (especially ATCC NO.35702), laying the material foundation for efficient hydrogen production through dark fermentation.

[0008] Step II, Dark Fermentation: The material pretreated in Step I is sent to a dark fermentation tank and inoculated with Clostridium beijerinckii (ATCC NO.35702) at a volume of 10% to 15% of the material. The temperature inside the dark fermentation tank is controlled at 33°C to 37°C and the pH value is 5.0 to 6.0. The fermentation is carried out for 2 to 3 days to obtain the first fermentation broth.

[0009] For example, the inoculation amount is 10%, 11%, 12%, 13%, 14%, 15% of the material volume, or any value between any two of the above. For example, the pH value in the tank is 5.0, 5.2, 5.3, 5.5, 5.6, 5.8, 5.9, 6.0, or any value between any two of the above.

[0010] In the first fermentation broth produced by dark fermentation, butyric acid accounts for 60% to 70% of the total volatile organic acids (VFAs) (compared to only 30% to 40% in traditional processes). Butyric acid is the optimal substrate for Rhodopseudomonas palustris. Through the above pretreatment process and the screening of dark fermentation strains and environment, the molar ratio of butyric acid to acetic acid in the first fermentation broth can be controlled to ≥1.5, creating ideal conditions for secondary hydrogen production in photofermentation.

[0011] Step III, Photofermentation: The first fermentation broth is transferred to a photofermenter and inoculated with Rhodopseudomonas palustris (accession number CGMCC 1.2180) at a volume of 20%–25% of the first fermentation broth. The photofermenter is made of transparent material and has a built-in LED array with a wavelength of 590±10nm. The light intensity is 5000 lux–10000 lux, the temperature is 30±1℃, and 50 mg / L–100 mg / L of MoFe nanoparticles are added. The gas produced during fermentation is collected.

[0012] For example, the inoculum amount is 20%, 21%, 22%, 23%, 24%, 25% of the first fermentation broth volume, or any value between any two of the above. For example, the amount of MoFe nanoparticles added is 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L, 100 mg / L, or any value between any two of the above.

[0013] This application uses *Rhodopseudomonas palustris* with accession number CGMCC 1.2180, whose photosynthetic pigments have the highest absorption efficiency for red light at a wavelength of 590±10 nm (30% higher than that under white light irradiation). It also incorporates an LED array at this wavelength to activate its photosynthetic system II, providing sufficient energy for hydrogen production. This further converts the organic acids (butyric acid and acetic acid) produced by dark fermentation into hydrogen, increasing the total hydrogen production by 75% compared to single dark fermentation. Therefore, *Rhodopseudomonas palustris* with accession number CGMCC 1.2180 can effectively decompose the first fermentation broth obtained after pretreatment and dark fermentation, further improving hydrogen production efficiency and hydrogen yield.

[0014] In some embodiments, the food waste meets the following requirements: total carbon content (dry weight) ≥ 40%, degradable carbohydrates accounting for ≥ 50% of the total carbon content, fat content (dry weight) ≤ 25%, total nitrogen content ≤ 6% (dry weight), salt content (as NaCl) ≤ 3%, and total heavy metal content ≤ 10 mg / kg. This makes it more suitable for matching *Clostridium beyerii* with ATCC NO. 35702.

[0015] In some embodiments, step I, the pretreatment further includes adding 0.5% to 1% of a surfactant, said surfactant including polysorbate 80.

[0016] For example, the amount of surfactant added is 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, or any value between any two of the above.

[0017] An appropriate amount of polysorbate 80 can emulsify the oils in kitchen waste (especially for substrates with a fat content of ≤25%), further reducing the solid-liquid interfacial tension, increasing the substrate dispersion by more than 40%, enhancing the contact efficiency between hydrogen-producing bacteria and carbon sources, and further improving the hydrogen production rate of dark fermentation.

[0018] In particular, when the fat content is 15%-25%, the amount of polysorbate 80 added is 0.8%~1%; when the fat content is ≤15%, the amount added is 0.5%~0.7%.

[0019] In some embodiments, in step III, the photo-fermentation tank is connected to a hydrogen permeation membrane separation system. The hydrogen permeation membrane includes a palladium-silver alloy membrane supported by porous ceramics, wherein the mass percentage of palladium in the alloy is 75% to 80%, and the residence time of the gas generated by photo-fermentation in the hydrogen permeation membrane separation system is 20 min to 30 min.

[0020] For example, the mass percentage of palladium in the alloy is 75%, 76%, 77%, 78%, 79%, 80%, or any value between any two of the above.

[0021] For example, the residence time of the gas generated by photo-fermentation in the hydrogen permeation membrane separation system is 20 min, 22 min, 24 min, 25 min, 26 min, 28 min, 30 min, or any value between any two of the above.

[0022] In some embodiments, step II further includes: adjusting the headspace oxygen content of the dark fermenter to ≤0.1% by nitrogen replacement. Precise oxygen control through nitrogen replacement creates a dedicated metabolic environment for hydrogen-producing bacteria, ensuring the yield of dark fermentation.

[0023] In some embodiments, step III further includes: adjusting the C / N ratio of the first fermentation broth to (15~20):1 by adding ammonium chloride. Adjusting the C / N ratio with ammonium chloride ensures that the carbon source is preferentially used for hydrogen production rather than cell growth, thereby achieving efficient carbon source conversion during the photofermentation stage and further increasing the total hydrogen production.

[0024] Therefore, the core invention of this application is a three-stage linkage process that combines pretreatment with dark fermentation of suitable microbial strains and environment, and further matching with suitable microbial strains and environment for photo fermentation. First, the mass ratio of degradable carbohydrates in food waste is controlled to be ≥50%, mainly small molecule sugars (glucose, fructose) and emulsified oils. Then, the food waste is targeted to adapt to the carbon source preference of ATCC NO.35702 through pretreatment. The dark fermentation environment is controlled to be suitable so that the molar ratio of butyric acid to acetic acid in the first fermentation broth is ≥1.5. Then, by selecting an appropriate amount of Rhodopseudomonas palustris with preservation number CGMCC 1.2180 as the inoculum for photo fermentation to adapt to the first fermentation broth as the substrate, the organic acids produced by dark fermentation can be further converted into hydrogen. This synergistic process achieves the targeted conversion of substrate into hydrogen, which can improve the hydrogen production efficiency and hydrogen yield of food waste. Attached Figure Description

[0025] Figure 1 This is a partial flowchart of a three-stage hydrogen production method based on kitchen waste in an embodiment of this application. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments and comparative examples. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0027] A three-stage hydrogen production method based on food waste

[0028] Food waste meets at least one of the following conditions:

[0029] Condition A: The total carbon content (on dry weight) is ≥40%, of which the mass of degradable carbohydrates accounts for ≥50% of the total carbon content of food waste;

[0030] Condition B: Its fat content (on dry weight) ≤ 25% and total nitrogen content (on dry weight) ≤ 6%;

[0031] Condition C: Its salt content (as NaCl) is ≤3%, and its total heavy metal content is ≤10mg / kg.

[0032] The three-stage hydrogen production method includes the following steps:

[0033] Step I: Preprocessing

[0034] The aforementioned food waste was treated at 1.1 MPa to 1.3 MPa and 160°C to 180°C for 10 to 20 minutes. This process disrupts the cellulose-lignin structure of the food waste, promotes carbohydrate dissolution, and increases substrate conversion rate by 20% to 30%, providing a sufficient available carbon source for dark fermentation.

[0035] Step I, the pretreatment also includes adding 0.5% to 1% of a surfactant, wherein the surfactant includes polysorbate 80. That is, 0.5% to 1% of the above-mentioned kitchen waste is added to the surfactant, and then the waste is treated in an environment of 1.1 MPa to 1.3 MPa and 160℃ to 180℃ for 10 to 20 minutes.

[0036] In this way, the oil in kitchen waste can be emulsified (especially for substrates with a fat content of ≤25%), reducing the solid-liquid interfacial tension, increasing the substrate dispersion by more than 40%, enhancing the contact efficiency between hydrogen-producing bacteria and carbon sources, and increasing the hydrogen production rate of dark fermentation.

[0037] Pretreatment also includes controlling the particle size of the pretreated material to be 0.5mm~2mm through grinding or sieving.

[0038] In this way, the specific surface area of ​​the substrate can be increased by 2 to 3 times, the contact area of ​​microorganisms can be increased, the carbon source dissolution rate can be increased by more than 50%, while avoiding the increase in viscosity of fermentation broth caused by excessively fine particle size (preventing mass transfer obstruction), ensuring the uniformity of the dark fermentation system, and improving the substrate conversion rate.

[0039] Step II: Dark Fermentation

[0040] The pretreated material from step I was fed into a dark fermenter, and the headspace oxygen content was adjusted to ≤0.1% by nitrogen replacement. Clostridium beijerinckii (ATCC NO.35702) was inoculated at 10%–15% of the material volume. The temperature inside the dark fermenter was controlled at 33℃–37℃, and the pH value was maintained at 5.0–6.0. ​​The fermentation was allowed to proceed for 2–3 days to obtain the first fermentation broth.

[0041] The ATCC NO.35702 Clostridium beyerrix depository is located in Manassas, Virginia, USA, and can be purchased through authorized ATCC distributors.

[0042] Clostridium beyerrix is ​​a strict anaerobic bacterium. By precisely controlling oxygen through nitrogen replacement, a metabolic environment is created for hydrogen-producing bacteria, further improving the yield of dark fermentation.

[0043] Step III: Photofermentation

[0044] The first fermentation broth was transferred to a photofermenter and inoculated with Rhodopseudomonas palustris (accession number CGMCC 1.2180) at a volume of 20%–25% of the first fermentation broth. The photofermenter was made of transparent material and contained an LED array with a wavelength of 590±10 nm. The light intensity was 5000 lux–10000 lux and the temperature was 30±1℃. MoFe nanoparticles at a concentration of 50 mg / L–100 mg / L were added, and the gases produced during fermentation were collected.

[0045] CGMCC 1.2180 Rhodopseudomonas palustris is deposited at the Institute of Microbiology, Chinese Academy of Sciences, and is available online.

[0046] Optionally, the C / N ratio of the first fermentation broth can be adjusted to (15~20):1 by adding ammonium chloride. The C / N range matches the optimal metabolic requirements of Rhodopseudomonas palustris, which can promote nitrogenase synthesis, increase hydrogen production by 25%~35% through photofermentation, and avoid the inhibition of hydrogenase synthesis due to insufficient nitrogen or the overgrowth of cells due to excessive nitrogen.

[0047] Step IV: Separate hydrogen gas

[0048] The photofermentation tank is connected to a hydrogen permeation membrane separation system. The residence time of the gas produced by photofermentation in the hydrogen permeation membrane separation system is 20-30 minutes. The hydrogen permeation membrane consists of a palladium-silver alloy membrane supported by porous ceramics, wherein palladium accounts for 75%-80% of the alloy by mass. The palladium-silver alloy membrane (Pd 75%-80%) supported by porous ceramics has a selective permeability coefficient of 10 for hydrogen. -8 ~10 -7 mol·m -1 ·s -1 ·Pa -1 It can separate hydrogen from fermentation gas in real time, avoiding hydrogen dissolution and loss, and at the same time improve the hydrogen purity to over 99%.

[0049] The following describes a three-stage hydrogen production method based on food waste, using specific embodiments. Those skilled in the art will understand that the preparation method described in this application is merely an example, and any other suitable preparation method is within the scope of this application.

[0050] Preparation of experimental materials

[0051] Food waste: Collect mixed waste of rice, vegetable leaves and noodles from the canteen (dry weight total carbon 42%, biodegradable carbohydrates account for 58% of total carbon, fat 18%, total nitrogen 4.5%, salt 2.2%, heavy metals 7.3mg / kg).

[0052] Bacterial strain: Clostridium beyerridis (ATCC NO.35702), activated on enhanced Clostridium medium (containing 20 g / L glucose and 5 g / L yeast extract, anaerobic culture at 35°C for 48 h).

[0053] Rhodopseudomonas palustris (CGMCC 1.2180) was activated on RCVBN medium (containing 1 g / L sodium acetate, irradiated with 590 nm red light, and incubated at 30°C for 72 h).

[0054] Reagents: MoFe nanoparticles (average particle size 50 nm, purity 99.9%), polysorbate 80 (analytical grade), ammonium chloride (analytical grade), phosphate buffer (0.1 mol / L, pH 5.5).

[0055] Equipment: High-pressure steam treatment vessel (5L capacity), colloid mill (3000r / min speed), anaerobic fermenter (2L, with online pH monitoring), photo-fermentation vessel (1L, borosilicate glass), 590±10nm LED array (adjustable light intensity), gas chromatograph (Agilent 7890A, DB-FFAP column).

[0056] Detection indicators and methods

[0057] Hydrogen purity: Gas chromatography TCD detector (5A molecular sieve column, column temperature 80℃, argon carrier gas);

[0058] Total hydrogen production: Conversion of cumulative water displacement (under standard conditions);

[0059] Fermentation broth composition: HPLC (Aminex HPX-87H column, 0.05 mol / L sulfuric acid mobile phase, column temperature 45℃). Example 1

[0060] Step I: Preprocessing

[0061] Steam explosion treatment: Put 500g of kitchen waste (wet weight) into an autoclave, set the pressure to 1.2MPa and the temperature to 170℃, maintain for 15min and then quickly depressurize and collect the treated material;

[0062] Particle size control: Transfer the steam-explosion treated material to a colloid mill, grind for 5 minutes, pass through a 1 mm standard sieve, collect the sieve material (particle size 0.5 mm ~ 1 mm), and determine the moisture content (controlled at 80 ± 2%).

[0063] Step II: Dark Fermentation

[0064] Add 1L of pretreated material to a 2L anaerobic fermenter, add 120mL of Clostridium beyerii seed culture (12% inoculum), and adjust the initial pH to 5.5 with phosphate buffer.

[0065] Environmental parameter control: High-purity nitrogen (99.99%) was introduced for 10 minutes to replace the headspace (oxygen content ≤0.1%), the temperature was set at 35℃, the stirring rate was 120r / min, and the fermentation was carried out in a sealed environment;

[0066] Process monitoring: 5 mL of sample was taken every 12 h, centrifuged at 12000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. The concentrations of butyric acid and acetic acid were then detected by gas chromatography (chromatographic conditions: column temperature 120℃, FID detector 200℃, nitrogen carrier gas 1 mL / min).

[0067] Endpoint determination: After 60 hours of fermentation, the concentration of butyric acid was measured to be 8.2 g / L, the concentration of acetic acid was 4.9 g / L, and the molar ratio was 1.67 (≥1.5). Fermentation was stopped and the first fermentation broth was collected.

[0068] Step III: Photofermentation

[0069] Take 500 mL of the first fermentation broth, inoculate it with 125 mL of Rhodopseudomonas palustris seed culture (inoculation amount 25%), add 75 mg of MoFe nanoparticles (final concentration 75 mg / L), and transfer it to a photofermenter;

[0070] Illumination and temperature control: Turn on the 590nm LED array, set the light intensity to 8000 lux, maintain the temperature at 30℃, and set the stirring rate to 80 r / min;

[0071] Gas collection: The generated gas was collected by water displacement, and the gas production was recorded every 24 hours. Fermentation continued for 96 hours.

[0072] Expected results:

[0073] The hydrogen production rate during the dark fermentation stage is 120~150 mL / g volatile solids, and the hydrogen production rate during the light fermentation stage is an additional 80~100 mL / g volatile solids. The total hydrogen production rate can be increased by 75% compared with the single dark fermentation, and the hydrogen purity can reach more than 99.2% after membrane separation. Example 2

[0074] Step I: Preprocessing

[0075] Steam explosion treatment: Put 500g of kitchen waste (wet weight) into an autoclave, add 0.8% of polysorbate 80, set the pressure to 1.2MPa and the temperature to 170℃, maintain for 15min and then quickly depressurize and collect the treated material;

[0076] Particle size control: Transfer the steam-treated material to a colloid mill, grind for 5 minutes, pass through a 1 mm standard sieve, collect the sieve material (particle size 0.5~1 mm), and determine the moisture content (controlled at 80±2%).

[0077] Step II: Dark Fermentation

[0078] Add 1L of pretreated material to a 2L anaerobic fermenter, add 120mL of Clostridium beyerii seed culture (12% inoculum), and adjust the initial pH to 5.5 with phosphate buffer.

[0079] Environmental parameter control: High-purity nitrogen (99.99%) was introduced for 10 minutes to replace the headspace (oxygen content ≤0.1%), the temperature was set at 35℃, the stirring rate was 120r / min, and the fermentation was carried out in a sealed environment;

[0080] Process monitoring: 5 mL of sample was taken every 12 h, centrifuged at 12000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. The concentrations of butyric acid and acetic acid were then detected by gas chromatography (chromatographic conditions: column temperature 120℃, FID detector 200℃, nitrogen carrier gas 1 mL / min).

[0081] Endpoint determination: After 60 hours of fermentation, the concentration of butyric acid was measured to be 8.2 g / L, the concentration of acetic acid was 4.9 g / L, and the molar ratio was 1.67 (≥1.5). Fermentation was stopped and the first fermentation broth was collected.

[0082] Step III: Photofermentation

[0083] Take 500 mL of the first fermentation broth, inoculate it with 125 mL of Rhodopseudomonas palustris seed culture (inoculation amount 25%), add 75 mg of MoFe nanoparticles (final concentration 75 mg / L), and transfer it to a photofermenter;

[0084] Illumination and temperature control: Turn on the 590nm LED array, set the light intensity to 8000 lux, maintain the temperature at 30℃, and set the stirring rate to 80 r / min;

[0085] Gas collection: The generated gas was collected by water displacement, and the gas production was recorded every 24 hours. Fermentation continued for 96 hours.

[0086] Test results:

[0087] During the dark fermentation stage, hydrogen production increased to 135-155 mL / g volatile solids.

[0088] Compared to Example 1, Example 2 maintains the same core steam explosion parameters (pressure 1.2 MPa, temperature 170°C, and holding time 15 min) during the pretreatment stage, but adds an additional 0.6% (mass fraction) of polysorbate 80. As a nonionic surfactant, polysorbate 80 can reduce the solid-liquid interfacial tension of food waste, thereby promoting the efficiency of steam explosion in destroying the material structure. This allows for more complete degradation of large organic molecules such as cellulose and hemicellulose into small soluble carbon sources (such as glucose and fructose), providing more readily available substrates for subsequent microbial metabolism and effectively improving substrate accessibility.

[0089] During the dark fermentation stage, the operating parameters of Example 2 were completely consistent with those of Example 1: 1L of pretreated material was added to a 2L anaerobic fermenter, and 120mL of Clostridium beyerii (ATCC NO.35702) seed culture (inoculation amount 12%) was added. The initial pH was adjusted to 5.5 using phosphate buffer. 99.99% high-purity nitrogen was introduced for 10min to replace the headspace (ensuring the headspace oxygen content ≤0.1%). The fermentation temperature was set to 35℃ and the stirring rate to 120r / min. Anaerobic fermentation was carried out in a sealed container. 5mL samples were taken every 12h, centrifuged at 12000r / min for 10min, filtered through a 0.22μm filter membrane, and the concentrations of butyric acid and acetic acid were detected by gas chromatography (column temperature 120℃, FID detector temperature 200℃, nitrogen carrier gas flow rate 1mL / min). After 60 hours of fermentation, the butyric acid concentration in Example 3 was measured to be 8.2 g / L and the acetic acid concentration to be 4.9 g / L, with a butyric acid to acetic acid molar ratio of 1.67 (meeting the process requirement of ≥1.5). This result was completely consistent with that in Example 1, indicating that the addition of polysorbate 80 in the pretreatment stage did not have a negative impact on the metabolic pathway and product concentration ratio of Clostridium beyerrix under the condition that the dark fermentation parameters remained unchanged, thus ensuring the metabolic stability of the dark fermentation stage.

[0090] The final test results showed that the hydrogen production during the dark fermentation stage of Example 2 was increased to 135~155 mL / g volatile solids (VS), which was a significant improvement over the 120~150 mL / g VS of Example 1. Example 3

[0091] Step I: Preprocessing

[0092] Steam explosion treatment: Put 500g of kitchen waste (wet weight) into an autoclave, set the pressure to 1.2MPa and the temperature to 170℃, maintain for 15min and then quickly depressurize and collect the treated material;

[0093] Particle size control: Transfer the steam-exploded material to a colloid mill, grind for 5 minutes, pass through a 1 mm standard sieve, collect the sieve material (particle size 0.5 mm to 1 mm), and measure the moisture content (controlled at 80 ± 2%).

[0094] Step II: Dark Fermentation

[0095] Add 1L of pretreated material to a 2L anaerobic fermenter, add 120mL of Clostridium beyerii seed culture (12% inoculum), and adjust the initial pH to 5.5 with phosphate buffer.

[0096] Environmental parameter control: High-purity nitrogen (99.99%) was introduced for 10 minutes to replace the headspace (oxygen content ≤0.1%), the temperature was set at 35℃, the stirring rate was 120r / min, and the fermentation was carried out in a sealed environment;

[0097] Process monitoring: 5 mL of sample was taken every 12 h, centrifuged at 12000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. The concentrations of butyric acid and acetic acid were then detected by gas chromatography (chromatographic conditions: column temperature 120℃, FID detector 200℃, nitrogen carrier gas 1 mL / min).

[0098] Endpoint determination: After 60 hours of fermentation, the concentration of butyric acid was measured to be 8.2 g / L, the concentration of acetic acid was 4.9 g / L, and the molar ratio was 1.67 (≥1.5). Fermentation was stopped and the first fermentation broth was collected.

[0099] Step III: Photofermentation

[0100] Substrate conditioning: Take 500 mL of the first fermentation broth, add ammonium chloride to adjust the C / N ratio to 18:1, measure the pH and adjust it to 7.0;

[0101] Inoculation and parameter settings: Inoculate 125 mL of Rhodopseudomonas palustris seed culture (inoculation amount 25%), add 75 mg MoFe nanoparticles (final concentration 75 mg / L), and transfer to a photofermenter;

[0102] Illumination and temperature control: Turn on the 590nm LED array, set the light intensity to 8000 lux, maintain the temperature at 30℃, and set the stirring rate to 80 r / min;

[0103] Gas collection: The generated gas was collected by water displacement, and the gas production was recorded every 24 hours. Fermentation continued for 96 hours.

[0104] Test results:

[0105] The photofermentation stage produces an additional 90-110 mL / g of hydrogen in volatile solids.

[0106] As can be seen, in the photofermentation stage, compared with Example 1, the C / N ratio of the first fermentation broth in Example 3 was adjusted to 18:1 by adding ammonium chloride, and the pH value was adjusted to 7.0. The C / N ratio is a core parameter affecting the growth and hydrogen production activity of the photofermentation strain (Rhodopseudomonas palustris, CGMCC1.2180). A suitable C / N ratio is beneficial to promoting the growth, reproduction, and hydrogen production metabolism of the strain. The subsequent photofermentation operation parameters were kept consistent with those in Example 1: 125 mL of Rhodopseudomonas palustris seed culture (inoculum 25%) was inoculated, 75 mg of MoFe nanoparticles (final concentration 75 mg / L) were added, and the mixture was transferred to a photofermenter; a 590 nm LED array (light intensity 8000 lux) was turned on, the fermentation temperature was maintained at 30 °C, the stirring rate was 80 r / min, the fermentation gas was collected by water displacement, and the fermentation was carried out continuously for 96 h.

[0107] Comparative Example 1

[0108] Step I: Preprocessing

[0109] Steam explosion treatment: Put 500g of kitchen waste (wet weight) into an autoclave, set the pressure to 1.2MPa and the temperature to 130℃, maintain for 15min and then quickly depressurize and collect the treated material;

[0110] Particle size control: Transfer the steam-treated material to a colloid mill, grind for 5 minutes, pass through a 1 mm standard sieve, collect the sieve material (particle size 0.5~1 mm), and determine the moisture content (controlled at 80±2%).

[0111] Step II: Dark Fermentation

[0112] Add 1L of pretreated material to a 2L anaerobic fermenter, add 120mL of Clostridium beyerii seed liquid (inoculation amount 12%), adjust the initial pH to 6.0 with phosphate buffer, set the temperature to 35℃, the stirring rate to 120r / min, and seal for fermentation;

[0113] Process monitoring: 5 mL of sample was taken every 12 h, centrifuged at 12000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. The concentrations of butyric acid and acetic acid were then detected by gas chromatography (chromatographic conditions: column temperature 120℃, FID detector 200℃, nitrogen carrier gas 1 mL / min).

[0114] Endpoint determination: After 60 hours of fermentation, the concentration of butyric acid was measured to be 7.14 g / L and the concentration of acetic acid was 5.1 g / L, with a molar ratio of 1.4 (<1.5). Fermentation was stopped and the first fermentation broth was collected.

[0115] Step III: Photofermentation

[0116] Take 500 mL of the first fermentation broth, inoculate it with 125 mL of Rhodopseudomonas palustris seed culture (inoculation amount 25%), add 75 mg of MoFe nanoparticles (final concentration 75 mg / L), and transfer it to a photofermenter;

[0117] Illumination and temperature control: Turn on the 590nm LED array, set the light intensity to 8000 lux, maintain the temperature at 30℃, and set the stirring rate to 80 r / min;

[0118] Gas collection: The generated gas was collected by water displacement, and the gas production was recorded every 24 hours. Fermentation continued for 96 hours.

[0119] Test results:

[0120] The hydrogen production during the dark fermentation stage is 90~120 mL / g of volatile solids, while the hydrogen production during the light fermentation stage is an additional 30~50 mL / g of volatile solids.

[0121] In Comparative Example 1, the temperature was set to 130℃ during the pretreatment steam explosion stage, significantly lower than the 170℃ in Example 1. Following the same pace as Example 1, butyric acid and acetic acid concentrations were measured every 12 hours. After 60 hours of fermentation, the butyric acid concentration was 7.14 g / L and the acetic acid concentration was 5.1 g / L, with a molar ratio of 1.4. Compared to Example 1, the butyric acid concentration was lower, the acetic acid concentration was slightly higher, and the molar ratio did not meet the standard of Example 1. During the photofermentation stage, the operation of Comparative Example 1 was basically the same as that of Example 1. 125 mL of *Rhodopseudomonas palustris* seed culture was inoculated, 75 mg of MoFe nanoparticles (final concentration 75 mg / L) were added, a 590 nm LED array was turned on, the light intensity was set to 8000 lux, the temperature was maintained at 30℃, the stirring rate was 80 r / min, and the gas was collected by water displacement. Fermentation continued for 96 hours. However, due to the differences in the dark fermentation stage, the final hydrogen production was also affected. Compared with Comparative Example 1 and Example 1, the hydrogen production in the dark fermentation stage decreased by about 60 mL / g volatile solids, and the additional hydrogen production in the light fermentation stage decreased by about 50 mL / g volatile solids.

[0122] Comparative Example 2

[0123] Step 1: Dark fermentation

[0124] Add 1L of material to a 2L anaerobic fermenter, add 120mL of Clostridium beyerii seed liquid (inoculation amount 12%), adjust the initial pH to 6.0 with phosphate buffer, set the temperature to 35℃, the stirring rate to 120r / min, and seal for fermentation;

[0125] Process monitoring: 5 mL of sample was taken every 12 h, centrifuged at 12000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. The concentrations of butyric acid and acetic acid were then detected by gas chromatography (chromatographic conditions: column temperature 120℃, FID detector 200℃, nitrogen carrier gas 1 mL / min).

[0126] After 60 hours of fermentation, the concentrations of butyric acid and acetic acid were measured, fermentation was stopped, and the first fermentation broth was collected.

[0127] Step II, Photofermentation

[0128] Take 500 mL of the first fermentation broth, inoculate it with 125 mL of Rhodopseudomonas palustris seed culture (inoculation amount 25%), add 75 mg of MoFe nanoparticles (final concentration 75 mg / L), and transfer it to a photofermenter;

[0129] Illumination and temperature control: Turn on the 590nm LED array, set the light intensity to 8000 lux, maintain the temperature at 30℃, and set the stirring rate to 80 r / min;

[0130] Gas collection: The generated gas was collected by water displacement, and the gas production was recorded every 24 hours. Fermentation continued for 96 hours.

[0131] Test results:

[0132] The butyric acid to acetic acid molar ratio is 0.76. The hydrogen production during the dark fermentation stage is 80-100 mL / g volatile solids (about 40% lower than in Example 1), and the additional hydrogen production during the light fermentation stage is 30-50 mL / g volatile solids.

[0133] Comparative Example 3

[0134] Food waste: Collect mixed waste from canteen rice, vegetable leaves, fatty meat, and fried food (42% total carbon by dry weight, 46% biodegradable carbohydrates, 33% fat, and 7% total nitrogen).

[0135] The experimental procedure was completely consistent with that of Example 1.

[0136] The test results showed that with a butyric acid / acetic acid molar ratio of 0.52, the hydrogen production during the dark fermentation stage was 70-90 L / g volatile solids (approximately 40% lower than in Example 1), and the additional hydrogen production during the photofermentation stage was 20-35 mL / g volatile solids (approximately 65% ​​lower than in Example 1). In Comparative Example 3, the insufficient biodegradable carbohydrates in the kitchen waste led to insufficient available sugar sources during the dark fermentation stage, resulting in a decrease in butyric acid production. Furthermore, the excessively high fat content inhibited the activity of ATCC NO.35702 and reduced cell activity during the photofermentation stage; CGMCC 1.2180 is sensitive to high-oil environments. Therefore, even with appropriate pretreatment, dark fermentation, and photofermentation, efficient hydrogen production cannot be achieved using kitchen waste with unsuitable parameter ranges.

[0137] The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A three-stage hydrogen production method based on food waste, characterized in that, The food waste meets the following requirements: On a dry weight basis, its total carbon content is ≥40%, of which the mass of degradable carbohydrates accounts for ≥50% of the total carbon content of food waste; and its fat content is ≤25% and its total nitrogen content is ≤6%. The salt content of the kitchen waste is ≤3% (based on NaCl), and the total heavy metal content is ≤10mg / kg. And it includes the following steps: Step 1: Preprocessing; The food waste is treated in an environment of 1.1MPa~1.3MPa and 160℃~180℃. The pretreatment process also includes: adjusting the particle size of the pretreated material to 0.5 mm to 2 mm by grinding or sieving; Step II: Dark fermentation; The material pretreated in step I is fed into a dark fermentation tank and inoculated with Clostridium beijerinckii, which has the preservation number ATCC NO.35702. The temperature inside the dark fermentation tank is controlled at 33℃~37℃ and the pH value inside the tank is 5.0~6.

0. The fermentation is carried out for 2~3 days to obtain the first fermentation broth. This also includes: controlling the headspace oxygen content of the dark fermentation tank to ≤0.1% through nitrogen replacement; Step III: Photofermentation; The first fermentation broth was transferred to a photofermenter and inoculated with Rhodopseudomonas palustris, preservation number CGMCC 1.2180. The photofermenter was made of transparent material and contained an LED array with a wavelength of 590±10nm. The light intensity was 5000lux~10000lux and the temperature was 30±1℃. 50mg / L~100mg / L of MoFe nanoparticles were added, and the gas produced during fermentation was collected.

2. The three-stage hydrogen production method based on kitchen waste according to claim 1, characterized in that, The kitchen waste was treated in an environment of 1.1MPa~1.3MPa and 160℃~180℃ for 10min~20min.

3. The three-stage hydrogen production method based on kitchen waste according to claim 1 or 2, characterized in that, In step I, the preprocessing further includes: Add 0.5% to 1% of a surfactant, said surfactant including polysorbate 80.

4. The three-stage hydrogen production method based on kitchen waste according to claim 1, characterized in that, In step II, the inoculation amount is 10% to 15% of the volume of the material; and / or, In the first fermentation broth, the molar ratio of butyric acid to acetic acid is ≥1.

5.

5. The three-stage hydrogen production method based on kitchen waste according to claim 1, characterized in that, In step III, the inoculum size is 20% to 25% of the volume of the first fermentation broth; and / or, The photo-fermentation tank is connected to a hydrogen permeation membrane separation system. The hydrogen permeation membrane includes a palladium-silver alloy membrane supported by porous ceramics, wherein the mass percentage of palladium in the alloy is 75% to 80%.

6. The three-stage hydrogen production method based on kitchen waste according to claim 5, characterized in that, The residence time of the gas generated by photo-fermentation in the hydrogen permeation membrane separation system is 20 min to 30 min.

7. The three-stage hydrogen production method based on kitchen waste according to claim 1 or 2, characterized in that, Step III also includes: The C / N ratio of the first fermentation broth was adjusted to (15~20):1 by adding ammonium chloride, and the pH value was adjusted to 6.0~7.0.

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