A method for hydrogen production by degrading undetoxified biomass and its application

Abstract

The present invention relates to the technical field of biomass hydrogen production, and specifically discloses a method for degrading undetoxified biomass to produce hydrogen and its application. It includes the following operating steps: Step 1: Preparation of seed culture solution: Thermoanaerobacterium thermosaccharolyticum and Clostridium thermocellum are respectively inoculated into seed medium I and seed medium II, activated and expanded; Seed culture solution I and seed culture solution II are respectively obtained; Step 2: Preparation of fermentation medium: (1) Waste biomass is pretreated in a pretreatment solution, and solid-liquid separation is carried out to obtain pretreated biomass; (2) Using the pretreated biomass as a carbon source, a fermentation medium is prepared; Step 3: Under an inert gas, after sterilization, seed culture solution I and seed culture solution II are inoculated into the fermentation medium in a certain proportion, and dark fermentation is carried out at 50-60°C and a rotation speed of 100-200 rpm for 16-32 h to obtain hydrogen.

Description

Technical Field

[0001] The present invention relates to the technical field of biomass hydrogen production, and particularly to a method for producing hydrogen by degrading undetoxified biomass and its application. Background Technique

[0002] Global energy is mainly supplied by fossil fuels such as coal, oil, and natural gas. Their rapid consumption will generate a large amount of greenhouse gases and harmful particles, exacerbating the greenhouse effect and environmental pollution. In recent decades, although the use of renewable energy has increased significantly, it still cannot shake the dominant position of fossil fuels. Hydrogen is considered one of the potential alternatives to future fossil fuels. The main advantages as an energy substance are: compared with other energy substances, hydrogen has a higher energy density and net calorific value. The development of hydrogen energy will help solve the environmental and human health problems brought about by the consumption of fossil fuels.

[0003] Currently, hydrogen is mainly produced from fossil fuels and renewable resources. Among them, 50% comes from natural gas reforming, 30% from oil cracking, 18% from coal gasification, and the rest comes from water electrolysis. From the above data, most hydrogen is still derived from fossil fuels, which requires a high energy cost and the production method is not environmentally friendly. Therefore, the biological production of hydrogen has significant advantages compared with the above methods. It can operate at relatively low temperatures and pressures, theoretically requires less energy input, and will not have a significant impact on the environment.

[0004] Compared with other biological processes, dark fermentation hydrogen production has certain advantages. It does not rely on light and has a wide range of substrate sources. Lignocellulose, food waste, municipal solid waste, industrial wastewater, etc. can all be used as raw materials for dark fermentation hydrogen production. Among them, lignocellulose is an attractive raw material. It has the characteristics of being renewable, sustainable, abundantly available, and inexpensive, and has a relatively high content of carbohydrates that can be used for biological hydrogen fermentation. However, lignocellulose has a complex cell wall structure, which hinders the further utilization by microorganisms. The pretreatment process aims to destroy the natural structure of lignocellulose, remove the lignin component, and increase the bioaccessibility of the substrate. Effective chemical pretreatment methods can achieve the above purposes, but will cause partial degradation of lignocellulose components, and the degradation products have a certain inhibitory effect on microorganisms. Therefore, it is very necessary to solve the problems existing in the current dark fermentation hydrogen production process, such as the inhibition of bacteria and the incomplete degradation of substrates. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for producing hydrogen by degrading undetoxified biomass and its application to solve the problems raised in the above background technique.

[0006] A method for producing hydrogen by degrading undetoxified biomass includes the following steps:

[0007] Step 1: Preparation of seed culture solution: Anaerobiospirillum thermoglycolyticum and Clostridium thermocellum are respectively inoculated into seed medium I and seed medium II, activated and enlarged cultured; seed culture solution I and seed culture solution II are respectively obtained;

[0008] Step 2: Preparation of fermentation medium: (1) Waste biomass is pretreated in a pretreatment solution, and solid-liquid separation is carried out to obtain pretreated biomass; (2) The pretreated biomass is used as a carbon source to prepare a fermentation medium;

[0009] Step 3: Under an inert gas, after sterilization, seed culture solution I and seed culture solution II are inoculated into the fermentation medium in a certain proportion, and dark fermentation is carried out at 50-60°C and a rotation speed of 100-200 rpm for 16-32 h to obtain hydrogen.

[0010] More preferably, the Anaerobiospirillum thermoglycolyticum is a strain with the ability to ferment various soluble sugars, preferably Anaerobiospirillum thermoglycolyticum MJ2, with the preservation number of GDMCC No: 61394; it has been disclosed in the Chinese invention patent with the application number of "202110315297.0" and the name of "An Anaerobiospirillum thermoglycolyticum and its application".

[0011] The Clostridium thermocellum is a cellulose-degrading bacterium, purchased from the German Collection of Microorganisms and Cell Cultures, preferably Clostridium thermocellum DSM1313.

[0012] More preferably, the pH values of the seed medium I and the seed medium II are both 6.8-7.2, and the compositions of the two are the same except for the carbon source, including the following components: carbon source 3-15 g / L, ammonium sulfate 1-1.5 g / L, magnesium chloride hexahydrate 2.5-3 g / L, potassium dihydrogen phosphate 1.4-1.5 g / L, dipotassium hydrogen phosphate trihydrate 5-6 g / L, calcium chloride dihydrate 0.1-0.15 g / L, β-glycerophosphate sodium pentahydrate 5-7 g / L, reduced glutathione 0.2-0.3 g / L, yeast powder 4-5 g / L, ferrous sulfate heptahydrate aqueous solution 1-1.2 mL / L; the concentration of the ferrous sulfate heptahydrate solution is 0.1 w / v%;

[0013] The carbon source of the seed medium I is xylan; the carbon source of the seed medium II is microcrystalline cellulose.

[0014] More preferably: The seed medium I includes the following components: xylan 10 g / L, ammonium sulfate 1.3 g / L, magnesium chloride hexahydrate 2.6 g / L, potassium dihydrogen phosphate 1.43 g / L, dipotassium hydrogen phosphate trihydrate 5.5 g / L, calcium chloride dihydrate 0.13 g / L, β-glycerophosphate sodium pentahydrate 6 g / L, reduced glutathione 0.25 g / L, yeast powder 4.5 g / L, ferrous sulfate heptahydrate aqueous solution 1.1 mL / L;

[0015] More preferably, the seed culture medium II comprises the following components: microcrystalline cellulose 10 g / L, ammonium sulfate 1.3 g / L, magnesium chloride hexahydrate 2.6 g / L, potassium dihydrogen phosphate 1.43 g / L, dipotassium hydrogen phosphate trihydrate 5.5 g / L, calcium chloride dihydrate 0.13 g / L, β-glycerophosphate disodium pentahydrate 6 g / L, reduced glutathione 0.25 g / L, yeast powder 4.5 g / L, ferrous sulfate heptahydrate aqueous solution 1.1 mL / L;

[0016] More preferably, the pH values of the seed culture medium I and the seed culture medium II are 7.0.

[0017] More optimally, in the preparation process of the seed culture solution I, the activation conditions of Anaerobacillus thermoglycolyticus are as follows: adding the seed culture medium I into a serum bottle, evacuating and filling with inert gas, inoculating the above-mentioned Anaerobacillus thermoglycolyticus after sterilization, and shaking for 16 - 20 h under the culture conditions of 50 - 60 °C and 120 - 180 rpm; more preferably: adding the seed culture medium I into a serum bottle, evacuating and filling with inert gas, inoculating the above-mentioned Anaerobacillus thermoglycolyticus after sterilization, and shaking for 18 h under the culture conditions of 55 °C and 150 rpm.

[0018] In the preparation process of the seed culture solution II, the activation conditions of Clostridium thermocellum are as follows: adding the seed culture medium II into a serum bottle, evacuating and filling with inert gas, inoculating the above-mentioned Clostridium thermocellum after sterilization, and shaking for 20 - 28 h under the culture conditions of 50 - 60 °C and 120 - 180 rpm; more preferably: adding the seed culture medium I into a serum bottle, evacuating and filling with inert gas, inoculating the above-mentioned Clostridium thermocellum after sterilization, and shaking for 24 h under the culture conditions of 55 °C and 150 rpm.

[0019] In the preparation process of the seed culture solution I, the subculture conditions of Anaerobacillus thermoglycolyticus are as follows: adding the seed culture medium I into a serum bottle, evacuating and filling with inert gas, inoculating the above-mentioned activated culture solution of Anaerobacillus thermoglycolyticus after sterilization, and shaking for 10 - 14 h under the culture conditions of 50 - 60 °C and 120 - 180 rpm; more preferably: adding the seed culture medium I into a serum bottle, evacuating and filling with inert gas, inoculating the above-mentioned activated culture solution of Anaerobacillus thermoglycolyticus after sterilization, and shaking for 12 h under the culture conditions of 55 °C and 150 rpm.

[0020] During the preparation of the seed culture solution II, the enlarged culture conditions of Clostridium thermocellum are as follows: Add seed medium II into a serum bottle, evacuate and fill with inert gas, inoculate the above-activated culture solution of Clostridium thermocellum after sterilization, and shake for 14 - 18 h under the culture conditions of 50 - 60 °C and 120 - 180 rpm; More preferably: Add seed medium II into a serum bottle, evacuate and fill with inert gas, inoculate the above-activated culture solution of Clostridium thermocellum after sterilization, and shake for 16 h under the culture conditions of 55 °C and 150 rpm.

[0021] The inoculation amount of the seed culture solution I of Anaerobacillus thermoglycolyticus is 2.5 - 10% (v / v);

[0022] The inoculation amount of the seed culture solution II of Clostridium thermocellum is 2.5 - 10% (v / v).

[0023] More preferably, the pretreatment solution comprises the following components: by weight / volume percentage, hydrogen peroxide 0.25 - 4%, sodium hydroxide 0.25 - 4%; More preferably: the pretreatment solution comprises 2% hydrogen peroxide and 1% sodium hydroxide.

[0024] The ratio of the waste biomass to the pretreatment solution is 1 g: 20 - 30 mL; More preferably: the ratio is 1 g: 25 mL.

[0025] The conditions of the pretreatment are: shake at 30 - 80 °C and 120 - 180 rpm for 4 - 8 h; More preferably: shake at 55 °C and 150 rpm for 6 h.

[0026] The conditions of the solid-liquid separation are: centrifuge at 2000 - 4000 rpm for 5 - 15 min; More preferably: centrifuge at 3000 rpm for 10 min.

[0027] More preferably, the inoculation ratio of the seed culture solution I and the seed culture solution II is 1:1; The inoculation amount of the total seed culture solution is 5 - 15 v / v%. More preferably 10 v / v%.

[0028] More preferably, the pH value of the fermentation medium is 6.8 - 7.2, and it comprises the following components: pretreated biomass 5 - 20 g / L, ammonium sulfate 1 - 1.5 g / L, magnesium chloride hexahydrate 2.5 - 3 g / L, potassium dihydrogen phosphate 1.4 - 1.5 g / L, dipotassium hydrogen phosphate trihydrate 5 - 6 g / L, calcium chloride dihydrate 0.1 - 0.15 g / L, β-glycerophosphate sodium pentahydrate 5 - 7 g / L, reduced glutathione 0.2 - 0.3 g / L, yeast powder 4 - 5 g / L, ferrous sulfate heptahydrate aqueous solution 1 - 1.2 mL / L; The concentration of the ferrous sulfate heptahydrate solution is 0.1 w / v%.

[0029] Among them, the pre-treated biomass is non-detoxified pre-treated biomass;

[0030] More preferably, the pH value of the culture medium of the fermentation medium is preferably 7.0; the culture medium of the fermentation medium comprises the following components: 15 g / L of non-detoxified pre-treated biomass, 1.3 g / L of ammonium sulfate, 2.6 g / L of magnesium chloride hexahydrate, 1.43 g / L of potassium dihydrogen phosphate, 5.5 g / L of dipotassium hydrogen phosphate trihydrate, 0.13 g / L of calcium chloride dihydrate, 6 g / L of β-glycerophosphate disodium pentahydrate, 0.25 g / L of reduced glutathione, 4.5 g / L of yeast powder, 1.1 mL / L of aqueous solution of ferrous sulfate heptahydrate.

[0031] Among them, in the dark fermentation process of step 3, a fermentation medium is added to a sealable fermentation vessel, evacuated and filled with an inert gas, and after sterilization, a certain proportion of seed culture solution I and seed culture solution II are inoculated to carry out dark fermentation to produce hydrogen; more preferably, a fermentation medium is added to a sealable fermentation vessel, evacuated and filled with an inert gas, and after sterilization, seed culture solution I and seed culture solution II with a volume ratio of 1:1 are inoculated to carry out dark fermentation to produce hydrogen.

[0032] The sealable fermentation vessel is preferably a serum bottle.

[0033] The degree of vacuum in the evacuation condition is preferably -0.085 to -0.095 MPa; more preferably -0.09 MPa.

[0034] The inert gas is preferably nitrogen.

[0035] The filling of the inert gas is preferably to fill the inert gas at 0.03 to 0.05 MPa; more preferably to fill the inert gas at 0.04 MPa.

[0036] The sterilization condition is preferably sterilization at 115 to 121 °C for 20 to 30 min; more preferably sterilization at 115 °C for 20 min.

[0037] The dark fermentation condition is more preferably fermentation at 55 °C at a rotation speed of 150 rpm for 24 h.

[0038] More optimally, in (2) of step 2, the specific process is to mix the pre-treated biomass with a solid bacteria carrier as a carbon source to prepare a fermentation medium; the proportion of the solid bacteria carrier in the fermentation medium is 1 to 3 g / L.

[0039] Preferably, the method for preparing the solid bacterium carrier is as follows: (1) Mix, crush, grind, and sieve dry anaerobic sludge and orange peel with a mass ratio of (3-5):1 in sequence to obtain a mixture; (2) Add iron oxide nanoparticles to cobalt chloride solution and mix evenly, then add the mixture, stir for 10-12 h and then dry; then, under a nitrogen atmosphere, heat-treat at a temperature of 500-650 °C for 1-2 h, cool, wash, and dry to obtain the solid bacterium carrier;

[0040] The raw materials in the solid bacterium carrier include the following components: by mass, 2-3 parts of iron oxide nanoparticles, 5-6 parts of cobalt chloride solution, and 1-2.5 parts of the mixture; the concentration of the cobalt chloride solution is 30-50 g / mL.

[0041] Application of the above method for promoting the degradation and hydrogen production of unpretreated biomass without detoxification in the microbial energy conversion of lignocellulose.

[0042] The waste biomass involved in the process is waste biomass containing cellulose, hemicellulose, and lignin, including but not limited to one or more of garden waste, bagasse, crop straw, and agricultural and forestry residues.

[0043] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0044] (1) The present invention can use a large amount of waste biomass as raw materials, and its stubborn structure can be destroyed through simple mild pretreatment, having good sustainability.

[0045] (2) The present invention directly uses unpretreated biomass without detoxification as a substrate. Compared with traditional pretreatment methods, it does not require washing or detoxification treatment, can simplify the operation steps and reduce production costs, and avoids the environmental pollution caused by the detoxification process, having good application prospects.

[0046] (3) The thermophilic saccharolytic anaerobe used in the present invention has good tolerance to pretreatment inhibitors, significantly improving the tolerance of co-cultured microorganisms to insoluble substrates; the present invention adds a solid bacterium carrier to the fermentation medium to provide more microbial attachment points and a stable microbial attachment environment, enabling anaerobic microorganisms to attach and grow, thereby being beneficial to increasing the biomass and activity of microorganisms, and thus enhancing the hydrogen production. The iron oxide and cobalt catalysts loaded in the solid bacterium carrier can promote the generation of hydrogen during the dark fermentation process; the iron oxide provides magnetism, which helps to separate and recover the catalyst, while cobalt has good catalytic activity and can accelerate the reaction; the high surface area of the solid bacterium carrier improves the physical contact of the substrate, enabling the substrate to be more effectively degraded, thereby enhancing hydrogen production.

[0047] (4) The present invention adopts an artificially constructed co - culture system of thermophilic bacteria, which can significantly improve the utilization rate of substrates, thereby enhancing the biological hydrogen production efficiency. The hydrogen yield can reach 22.48 mmol / g. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention, but do not constitute a limitation to the present invention. In the drawings:

[0049] Figure 1 is a graph showing the hydrogen production results of the co - cultured thermophilic bacteria provided by the present invention using untreated pretreated biomass at different substrate loadings;

[0050] Figure 2 is a graph showing the cell protein amount results of the co - cultured thermophilic bacteria provided by the present invention using untreated pretreated biomass at different substrate loadings;

[0051] Figure 3 is a graph showing the hydrogen production results of different co - culture ratios of thermophilic bacteria provided by the present invention using untreated pretreated biomass;

[0052] Figure 4 is a graph showing the cell protein amount results of different co - culture ratios of thermophilic bacteria provided by the present invention using untreated pretreated biomass;

[0053] Figure 5 is a graph showing the hydrogen production results of pure - cultured Clostridium thermocellum using untreated pretreated biomass at different substrate loadings;

[0054] Figure 6 is a graph showing the cell protein amount results of pure - cultured Clostridium thermocellum using untreated pretreated biomass at different substrate loadings;

[0055] Figure 7 is a graph showing the hydrogen production results of using untreated pretreated biomass at different substrate loadings under different culture modes provided by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0056] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0057] The preparation method of the solid bacteria carrier is as follows: (1) Mix, crush, grind, and sieve dry anaerobic sludge and orange peel with a mass ratio of 3:1 in sequence to obtain a mixture; (2) Add iron oxide nanoparticles to cobalt chloride solution and mix evenly, then add the mixture, stir for 10 h and then dry; then, under a nitrogen atmosphere, heat-treat at a temperature of 500 °C for 1 h, cool, wash, and dry to obtain the solid bacteria carrier;

[0058] Among them, the raw materials in the solid bacteria carrier include the following components: by mass, 2 parts of iron oxide nanoparticles, 5 parts of cobalt chloride solution, and 1.8 parts of the mixture; the concentration of the cobalt chloride solution is 30 g / mL.

[0059] The specific implementation plan of the present invention is as follows:

[0060] Example 1: Hydrogen production capacity and influence on cell protein content under different substrate concentrations in the co-culture mode of thermophilic bacteria;

[0061] Step 1: The preparation method of the seed culture solution I is as follows: First, activate Thermoanaerobacterium thermosaccharolyticum MJ2 preserved at -80 °C by shaking culture in a 120 mL serum bottle (containing 45 mL of seed medium I) at 55 °C and 150 rpm for 16 h, and then expand by shaking culture in a 120 mL serum bottle (containing 45 mL of seed medium I) at 55 °C and 150 rpm for 12 h to obtain the seed culture solution I;

[0062] Among them, the seed medium I is a liquid medium containing 10 g / L xylan, and its main components are: xylan 10 g / L (carbon source), ammonium sulfate 1.3 g / L, magnesium chloride hexahydrate 2.6 g / L, potassium dihydrogen phosphate 1.43 g / L, dipotassium hydrogen phosphate trihydrate 5.5 g / L, calcium chloride dihydrate 0.13 g / L, β-glycerophosphate sodium pentahydrate 6 g / L, reduced glutathione 0.25 g / L, yeast powder 4.5 g / L, and aqueous solution of ferrous sulfate heptahydrate 1.1 mL / L;

[0063] (2) The preparation method of the seed culture solution II is as follows: First, activate Clostridium thermocellum DSM1313 preserved at -80 °C by shaking culture in a 120 mL serum bottle (containing 45 mL of seed medium II) at 55 °C and 150 rpm for 24 h, and then expand by shaking culture in a 120 mL serum bottle (containing 45 mL of seed medium II) at 55 °C and 150 rpm for 16 h to obtain the seed culture solution II;

[0064] Among them, the seed medium II is a liquid medium containing 10 g / L microcrystalline cellulose, and its main components are: microcrystalline cellulose 10 g / L (carbon source), ammonium sulfate 1.3 g / L, magnesium chloride hexahydrate 2.6 g / L, potassium dihydrogen phosphate 1.43 g / L, dipotassium hydrogen phosphate trihydrate 5.5 g / L, calcium chloride dihydrate 0.13 g / L, β-glycerophosphate sodium pentahydrate 6 g / L, reduced glutathione 0.25 g / L, yeast powder 4.5 g / L, ferrous sulfate heptahydrate aqueous solution 1.1 mL / L;

[0065] Step 2: Preparation and sterilization of the fermentation medium: The components and dosages of the fermentation medium are as follows: 5 - 20 g / L of non-detoxified pretreated biomass (different fermentation media are set, and the difference between different fermentation media lies in the content of non-detoxified pretreated biomass), ammonium sulfate 1.3 g / L, magnesium chloride hexahydrate 2.6 g / L, potassium dihydrogen phosphate 1.43 g / L, dipotassium hydrogen phosphate trihydrate 5.5 g / L, calcium chloride dihydrate 0.13 g / L, β-glycerophosphate sodium pentahydrate 6 g / L, reduced glutathione 0.25 g / L, yeast powder 4.5 g / L, ferrous sulfate heptahydrate aqueous solution 1.1 mL / L; Among them, the non-detoxified pretreated biomass is mixed with the solid bacteria carrier as the carbon source, and the proportion of the solid bacteria carrier in the fermentation medium is 2.2 g / L;

[0066] Seal the serum bottle containing the fermentation medium with a rubber stopper and an aluminum cap, and repeat the process of pumping -0.09 MPa vacuum and filling with 0.04 MPa nitrogen 4 times, and finally sterilize at 115 °C for 20 min;

[0067] Step 3: Co-culture of thermophilic bacteria to utilize the hydrogen production of non-detoxified pretreated biomass at different substrate loadings: Inject the prepared total seed culture solution (the volume ratio of seed culture solution I to seed culture solution II is 1:1) into the above fermentation medium at an inoculation amount of 10% (v / v), the fermentation temperature is 55 °C, the shaker speed is 150 rpm, and the fermentation is terminated after 24 h; Then measure the effects of different substrate loadings on hydrogen production and cell protein content;

[0068] The results are as Figure 1As shown: The measured hydrogen production increased with the increase of substrate loading. The hydrogen production was 78.78 mmol / L (5 g / L substrate), 143.88 mmol / L (10 g / L substrate), 204.37 mmol / L (15 g / L substrate), and 210.26 mmol / L (20 g / L substrate) respectively. The hydrogen yields were 19.74 mmol / g substrate, 20.20 mmol / g substrate, 22.48 mmol / g substrate, and 20.96 mmol / g substrate respectively. Higher concentrations of substrate did not significantly affect the hydrogen production yield of the co-culture system, indicating that the dark fermentation system of the co-culture of Thermoanaerobacterium thermosaccharolyticum MJ2 and Clostridium thermocellum DSM1313 could well utilize the unpretreated biomass without detoxification for hydrogen production, and the hydrogen production efficiency was at a relatively high level.

[0069] The results are as Figure 2 shown: The measured cell protein content first increased and then decreased with the increase of substrate loading. The cell protein contents were 0.22 g / L (5 g / L substrate), 0.44 g / L (10 g / L substrate), 0.59 g / L (15 g / L substrate), and 0.53 g / L (20 g / L substrate) respectively. The cell yields were 60.72 mg / g substrate, 70.02 mg / g substrate, 74.91 mg / g substrate, and 61.52 mg / g substrate respectively. The highest cell yield was obtained when the substrate loading was 15 g / L, indicating that the dark fermentation system of the co-culture of Thermoanaerobacterium thermosaccharolyticum MJ2 and Clostridium thermocellum DSM1313 had good substrate tolerance.

[0070] Example 2: Hydrogen production capacity and influence on cell protein content under different inoculation ratios in the co-culture mode of thermophilic bacteria;

[0071] Step 1: The preparation method of seed culture solution I was as follows: Thermoanaerobacterium thermosaccharolyticum MJ2 preserved at -80 °C was first activated by shaking culture at 55 °C and 150 rpm for 16 h in a 120 mL serum bottle (containing 45 mL of seed medium I), and then expanded by shaking culture at 55 °C and 150 rpm for 12 h in a 120 mL serum bottle (containing 45 mL of seed medium I) to obtain seed culture solution I;

[0072] Among them, seed medium I was a liquid medium containing 10 g / L xylan, and its main components were: xylan 10 g / L (carbon source), ammonium sulfate 1.3 g / L, magnesium chloride hexahydrate 2.6 g / L, potassium dihydrogen phosphate 1.43 g / L, dipotassium hydrogen phosphate trihydrate 5.5 g / L, calcium chloride dihydrate 0.13 g / L, β-glycerophosphate sodium pentahydrate 6 g / L, reduced glutathione 0.25 g / L, yeast powder 4.5 g / L, and aqueous solution of ferrous sulfate heptahydrate 1.1 mL / L;

[0073] (2) The preparation method of the seed culture solution II is as follows: First, the Clostridium thermocellum DSM1313 preserved at -80°C is activated by shaking culture in a 120 mL serum bottle (containing 45 mL of seed medium II) at 55°C and 150 rpm for 24 h, and then expanded by shaking culture in a 120 mL serum bottle (containing 45 mL of seed medium II) at 55°C and 150 rpm for 16 h to obtain the seed culture solution II;

[0074] Among them, the seed medium II is a liquid medium containing 10 g / L of microcrystalline cellulose, and its main components are: 10 g / L of microcrystalline cellulose (carbon source), 1.3 g / L of ammonium sulfate, 2.6 g / L of magnesium chloride hexahydrate, 1.43 g / L of potassium dihydrogen phosphate, 5.5 g / L of dipotassium hydrogen phosphate trihydrate, 0.13 g / L of calcium chloride dihydrate, 6 g / L of β-glycerophosphate sodium pentahydrate, 0.25 g / L of reduced glutathione, 4.5 g / L of yeast powder, and 1.1 mL / L of aqueous solution of ferrous sulfate heptahydrate;

[0075] Step 2: Preparation and sterilization of the fermentation medium: The components and dosages of the fermentation medium are: 15 g / L of undetoxified pretreated biomass (different fermentation media are set, and the difference between different fermentation media lies in the different contents of undetoxified pretreated biomass), 1.3 g / L of ammonium sulfate, 2.6 g / L of magnesium chloride hexahydrate, 1.43 g / L of potassium dihydrogen phosphate, 5.5 g / L of dipotassium hydrogen phosphate trihydrate, 0.13 g / L of calcium chloride dihydrate, 6 g / L of β-glycerophosphate sodium pentahydrate, 0.25 g / L of reduced glutathione, 4.5 g / L of yeast powder, and 1.1 mL / L of aqueous solution of ferrous sulfate heptahydrate; Among them, the undetoxified pretreated biomass and the solid bacteria carrier are mixed as the carbon source, and the proportion of the solid bacteria carrier in the fermentation medium is 2.2 g / L;

[0076] The serum bottle containing the fermentation medium is sealed with a rubber stopper and an aluminum cap, and the vacuum of -0.09 MPa and the nitrogen of 0.04 MPa are repeatedly pumped and filled 4 times, and finally sterilized at 115°C for 20 min;

[0077] Step 3: Hydrogen production at different inoculation ratios in the co-culture mode of thermophilic bacteria: Inject the prepared total seed culture solution into the above fermentation medium at an inoculation amount of 10% (v / v). The volume ratio of the seed culture solution I to the seed culture solution II is 3:1, 1:1, 1:3 (different inoculation ratios are set, and the difference between different inoculation ratios lies in the relative contents of the seed culture solution I and the seed culture solution II). The fermentation temperature is 55°C, the shaking speed of the shaker is 150 rpm, and the fermentation is terminated after 24 h; Measure the hydrogen production at different inoculation ratios in the co-culture mode of thermophilic bacteria and the influence on the amount of cell protein;

[0078] The results are as Figure 3As shown in the figure: The measured hydrogen production and yield both reached their maximum values when the inoculation ratio of Thermoanaerobacterium thermosaccharolyticum to Clostridium thermocellum was 1:1. The maximum hydrogen production was 183.49 mM, and the maximum hydrogen yield was 22.99 mmol / g of substrate. When the inoculation ratio was 3:1, due to the decrease in the relative abundance of Clostridium thermocellum, the degradation performance of the substrate decreased, and the hydrogen production dropped to 87.66 mM, and the hydrogen yield dropped to 13.71 mmol / g of substrate. When the inoculation ratio was 1:3, due to the decrease in the relative abundance of Thermoanaerobacterium thermosaccharolyticum, its tolerance to the undetoxified substrate decreased, resulting in a decline in hydrogen production performance. The hydrogen production dropped to 110.76 mM, and the hydrogen yield dropped to 16.71 mmol / g of substrate;

[0079] The results are as Figure 4 shown: The measured cell protein content and yield both reached their maximum values when the inoculation ratio of Thermoanaerobacterium thermosaccharolyticum to Clostridium thermocellum was 1:1. The maximum cell protein content was 0.60 g / L, and the maximum cell yield was 74.55 mg / g of substrate. When the inoculation ratio was 3:1, due to the decrease in the relative abundance of Clostridium thermocellum, the degradation performance of the substrate decreased, and there was not enough fermentable carbon source to supply the growth of the bacteria. The cell protein content dropped to 0.44 g / L, and the cell yield dropped to 68.20 mg / g of substrate. When the inoculation ratio was 1:3, due to the decrease in the relative abundance of Thermoanaerobacterium thermosaccharolyticum, its tolerance to the undetoxified substrate decreased, resulting in the inhibition of the bacteria; the cell protein content dropped to 0.48 g / L, and the cell yield dropped to 72.53 mmol / g of substrate.

[0080] Comparative Example 1: Hydrogen production capacity and the effect on cell protein content under different substrate concentrations in the pure culture mode of Clostridium thermocellum;

[0081] Step 1: The preparation method of the seed expansion culture solution II is as follows: First, activate Clostridium thermocellum DSM1313 stored at -80°C by shaking culture in a 120 mL serum bottle (containing 45 mL of seed medium II) at 55°C and 150 rpm for 24 h, and then expand it by shaking culture in a 120 mL serum bottle (containing 45 mL of seed medium II) at 55°C and 150 rpm for 16 h to obtain the seed expansion culture solution II;

[0082] Among them, the seed medium II is a liquid medium containing 10 g / L of microcrystalline cellulose, and its main components are: 10 g / L of microcrystalline cellulose (carbon source), 1.3 g / L of ammonium sulfate, 2.6 g / L of magnesium chloride hexahydrate, 1.43 g / L of potassium dihydrogen phosphate, 5.5 g / L of dipotassium hydrogen phosphate trihydrate, 0.13 g / L of calcium chloride dihydrate, 6 g / L of β-glycerophosphate sodium pentahydrate, 0.25 g / L of reduced glutathione, 4.5 g / L of yeast powder, and 1.1 mL / L of aqueous solution of ferrous sulfate heptahydrate;

[0083] Step 2: Preparation and sterilization of the fermentation medium: The components and dosages of the fermentation medium are as follows: 5 - 20 g / L of unpretreated biomass without detoxification (different fermentation media are set, and the difference between different fermentation media lies in the content of unpretreated biomass without detoxification), 1.3 g / L of ammonium sulfate, 2.6 g / L of magnesium chloride hexahydrate, 1.43 g / L of potassium dihydrogen phosphate, 5.5 g / L of dipotassium hydrogen phosphate trihydrate, 0.13 g / L of calcium chloride dihydrate, 6 g / L of β-glycerophosphate sodium pentahydrate, 0.25 g / L of reduced glutathione, 4.5 g / L of yeast powder, 1.1 mL / L of aqueous solution of ferrous sulfate heptahydrate; among them, the unpretreated biomass without detoxification is mixed with the solid bacteria carrier as the carbon source, and the proportion of the solid bacteria carrier in the fermentation medium is 2.2 g / L;

[0084] Seal the serum bottle containing the fermentation medium with a rubber stopper and an aluminum cap, and repeat the process of pumping -0.09 MPa vacuum and filling with 0.04 MPa nitrogen 4 times. Finally, sterilize at 115 °C for 20 min;

[0085] Hydrogen production of Clostridium thermocellum in pure culture mode using unpretreated and undetoxified garden waste at different substrate loadings: Inject the prepared total seed culture solution II into the above fermentation medium at an inoculation amount of 10% (v / v). The fermentation temperature is 55 °C, the shaker speed is 150 rpm, and the fermentation is terminated after 24 h; Measure the hydrogen production capacity at different substrate concentrations and the effect on the amount of cell protein in the pure culture system of Clostridium thermocellum;

[0086] The results are as Figure 5 shown: Both the measured hydrogen production and yield first increase and then decrease with the increase of substrate loading. The hydrogen productions are 33.84 mmol / L (5 g / L garden waste), 54.08 mmol / L (10 g / L garden waste), 50.24 mmol / L (15 g / L garden waste), and 42.98 mmol / L (20 g / L garden waste) respectively. The hydrogen yields are 9.30 mmol / g substrate, 9.58 mmol / g substrate, 7.95 mmol / g substrate, and 7.48 mmol / g substrate respectively. Higher concentrations of substrate significantly reduce the hydrogen production and yield in the pure culture system of Clostridium thermocellum, indicating that high-concentration substrate has a significant inhibitory effect on Clostridium thermocellum.

[0087] The results are as Figure 6As shown in the figure, both the measured cell protein content and the yield first increased and then decreased with the increase of the substrate loading. The cell protein contents were 0.21 g / L (5 g / L of garden waste), 0.39 g / L (10 g / L of garden waste), 0.40 g / L (15 g / L of garden waste), and 0.31 g / L (20 g / L of garden waste), respectively. The cell yields were 58.99 mg / g of substrate, 68.60 mg / g of substrate, 63.46 mg / g of substrate, and 54.39 mg / g of substrate, respectively. A higher concentration of substrate significantly reduced the cell protein content and the yield in the pure culture system of Clostridium thermocellum, indicating that a high concentration of substrate has a significant substrate inhibition effect on Clostridium thermocellum.

[0088] It can be found from the examples and comparative examples that at different substrate loadings, the hydrogen production of the co-culture system using Anaerobacterium thermoglycolicum MJ2 was increased by 132.80 - 389.20% compared with that of the pure culture system of Clostridium thermocellum, and the hydrogen yield was increased by 110.86 - 182.77%. The highest hydrogen yield could reach 22.48 mmol / g of substrate. It demonstrated the excellent synergistic degradation performance of the co-cultured thermophiles on the substrate, which could effectively improve the inhibitor tolerance of the bacteria to the unpretreated garden waste without detoxification and enhance the dark fermentation efficiency of hydrogen.

[0089] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variation thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0090] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for producing hydrogen by degrading undetoxified biomass, characterized in that: The following steps are involved: Step 1: Preparation of seed expansion culture medium: inoculate Anaerobic Bacillus saccharolyticus and Clostridium thermocellum into seed culture medium I and seed culture medium II respectively, activate and expand the culture; The seed expansion liquid I and seed expansion liquid II were obtained respectively; Step 2: Preparation of fermentation medium: (1) Mixing, crushing, grinding and sieving dried anaerobic sludge and orange peel in a mass ratio of (3-5):1 in sequence to obtain a mixture; (2) Adding ferroferric oxide nanoparticles to a cobalt chloride solution and mixing them evenly, adding the mixture, stirring for 10-12 h and then drying; then heat treating at a temperature of 500-650°C for 1-2 h under a nitrogen atmosphere, cooling, washing and drying to obtain a solid bacterial carrier; (3) placing the waste biomass in a pretreatment liquid, pretreating it at 30-80°C and 120-180 rpm for 4-8 h, and centrifuging it at 2000-4000 rpm for 5-15 min to separate the solid and liquid, thereby obtaining the pretreated biomass; (4) uniformly mixing the pretreated biomass with the solid bacteria carrier to prepare a fermentation medium with a pH value of 6.8-7.2; Step 3: Under inert gas, after sterilization, inoculate the seed expansion solution I and the seed expansion solution II into the fermentation medium at a mass ratio of 1:1, and perform dark fermentation at 50-60° C. and a rotation speed of 100-200 rpm for 16-32 h to obtain hydrogen; The pretreatment liquid includes the following components: 0.25-4% hydrogen peroxide and 0.25-4% sodium hydroxide by weight / volume percentage; the ratio of the waste biomass to the pretreatment liquid is 1g:20-30mL; the inoculation amount of the total seed expansion liquid is 5-15v / v; The raw materials in the solid bacteria carrier include the following components: 2-3 parts of ferroferric oxide nanoparticles, 5-6 parts of cobalt chloride solution, and 1-2.5 parts of a mixture, by mass; the concentration of the cobalt chloride solution is 30-50 g / mL.

2. The method for producing hydrogen by degrading non-detoxified biomass according to claim 1, characterized in that: The thermosaccharolytic anaerobic bacteria are strains with multiple soluble sugar fermentation capabilities, including thermosaccharolytic anaerobic bacteria MJ2, with a preservation number of GDMCC No: 61394; The Clostridium thermocellum is a cellulose degrading bacterium, including Clostridium thermocellum DSM1313.

3. The method for producing hydrogen by degrading non-detoxified biomass according to claim 1, characterized in that: The pH values ​​of the seed culture medium I and the seed culture medium II are both 6.8-7.2, and the components of the two are the same except for the carbon source, including the following components: 3-15 g / L carbon source, 1-1.5 g / L ammonium sulfate, 2.5-3 g / L magnesium chloride hexahydrate, 1.4-1.5 g / L potassium dihydrogen phosphate, 5-6 g / L dipotassium hydrogen phosphate trihydrate, 0.1-0.15 g / L calcium chloride dihydrate, 5-7 g / L sodium β-glycerophosphate pentahydrate, 0.2-0.3 g / L reduced glutathione, 4-5 g / L yeast powder, 1-1.2 mL / L ferrous sulfate heptahydrate aqueous solution; the concentration of the ferrous sulfate heptahydrate solution is 0.1w / v; The carbon source of the seed culture medium I is xylan; the carbon source of the seed culture medium II is microcrystalline cellulose.

4. The method for producing hydrogen by degrading non-detoxified biomass according to claim 1, characterized in that: In the process of preparing the seed expansion culture solution I, the activation conditions of the thermosaccharolytic anaerobic bacillus are as follows: adding seed culture medium I to a serum bottle, evacuating and filling with inert gas, inoculating the thermosaccharolytic anaerobic bacillus after sterilization, and shaking at 50-60° C. and 120-180 rpm for 16-20 h; In the process of preparing the seed expansion culture solution II, the activation conditions of the thermocellum Clostridium are as follows: adding the seed culture medium II into the serum bottle, evacuating and filling with inert gas, inoculating the above-mentioned thermocellum Clostridium after sterilization, and shaking at 50-60° C. and 120-180 rpm for 20-28 h; In the process of preparing the seed expansion culture solution I, the expansion culture conditions of the thermosaccharolytic anaerobic bacillus are as follows: adding seed culture medium I to a serum bottle, evacuating and filling with inert gas, inoculating the above thermosaccharolytic anaerobic bacillus activation culture solution after sterilization, and shaking at 50-60° C. and 120-180 rpm for 10-14 h; During the preparation of the seed expansion culture solution II, the expansion culture conditions of Clostridium thermocellum are as follows: adding seed culture medium II into a serum bottle, evacuating and filling with inert gas, inoculating the above-mentioned Clostridium thermocellum activation culture solution after sterilization, and shaking at 50-60° C. and 120-180 rpm for 14-18 hours.

5. The method for producing hydrogen by degrading undetoxified biomass according to claim 1, characterized in that: The fermentation medium comprises the following components: 5-20 g / L of pretreated biomass, 1-3 g / L of solid bacteria carrier, 1-1.5 g / L of ammonium sulfate, 2.5-3 g / L of magnesium chloride hexahydrate, 1.4-1.5 g / L of potassium dihydrogen phosphate, 5-6 g / L of dipotassium hydrogen phosphate trihydrate, 0.1-0.15 g / L of calcium chloride dihydrate, 5-7 g / L of sodium β-glycerophosphate pentahydrate, 0.2-0.3 g / L of reduced glutathione, 4-5 g / L of yeast powder, and 1-1.2 mL / L of ferrous sulfate heptahydrate aqueous solution; the concentration of the ferrous sulfate heptahydrate solution is 0.1w / v; The waste biomass is waste biomass containing cellulose, hemicellulose and lignin, including one or more of garden waste, bagasse, crop straw, and agricultural and forestry residues.

6. Use of the method for producing hydrogen by degrading non-detoxified biomass according to any one of claims 1 to 5 in microbial energy conversion of waste biomass.

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