CN114107040B - Portable mixing stirring enzymolysis fermentation hydrogen production method and device - Google Patents
Portable mixing stirring enzymolysis fermentation hydrogen production method and device Download PDFInfo
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- 239000001257 hydrogen Substances 0.000 title claims abstract description 111
- 229910052739 hydrogen Inorganic materials 0.000 title claims abstract description 111
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 title claims abstract description 109
- 238000003756 stirring Methods 0.000 title claims abstract description 104
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 93
- 238000000855 fermentation Methods 0.000 title claims abstract description 47
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- 230000000243 photosynthetic effect Effects 0.000 claims description 38
- 239000000243 solution Substances 0.000 claims description 28
- 239000007788 liquid Substances 0.000 claims description 25
- 241000191023 Rhodobacter capsulatus Species 0.000 claims description 16
- 230000001580 bacterial effect Effects 0.000 claims description 14
- 239000007853 buffer solution Substances 0.000 claims description 14
- 239000012295 chemical reaction liquid Substances 0.000 claims description 13
- 239000001963 growth medium Substances 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 9
- 230000000694 effects Effects 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 241000190950 Rhodopseudomonas palustris Species 0.000 claims description 7
- 238000005286 illumination Methods 0.000 claims description 7
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 241001494508 Arundo donax Species 0.000 claims description 5
- 241000191043 Rhodobacter sphaeroides Species 0.000 claims description 5
- 241000190984 Rhodospirillum rubrum Species 0.000 claims description 5
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- 239000000872 buffer Substances 0.000 claims description 4
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- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 4
- 241000316848 Rhodococcus <scale insect> Species 0.000 claims description 3
- 241000190967 Rhodospirillum Species 0.000 claims description 3
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 3
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
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- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
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- OESFSXYRSCBAQJ-UHFFFAOYSA-M sodium;3-carboxy-3,5-dihydroxy-5-oxopentanoate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.OC(=O)CC(O)(C(O)=O)CC([O-])=O OESFSXYRSCBAQJ-UHFFFAOYSA-M 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000005728 strengthening Methods 0.000 description 2
- 241000209140 Triticum Species 0.000 description 1
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- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
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- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
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- C12M21/02—Photobioreactors
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- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
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- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
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Abstract
The invention belongs to the technical field of photo-fermentation biological hydrogen production, and in particular relates to a portable mixing stirring enzymolysis fermentation hydrogen production method and device, wherein the portable mixing stirring enzymolysis fermentation hydrogen production method and device comprises a cylindrical shell with an open top end and a top cover; the top cover is detachably connected with the shell; the inner cavity of the shell is provided with a baffle plate, the baffle plate divides the inner cavity of the shell into an upper cavity and a lower cavity, and a control motor is arranged in the lower cavity; a turntable is arranged on the upper end surface of the partition plate, and an inner spiral stirring rod and an outer spiral stirring rod are arranged at the top end of the turntable; the inner spiral stirring rod and the outer spiral stirring rod are both spiral. According to the invention, by arranging the two stirring rods with reverse spirals, the stirring speed and the enzymolysis degree of the biomass raw material in the enzymolysis process can be improved, so that the contact degree between microorganisms and the biomass raw material and the metabolism degree of microorganisms are accelerated, the hydrogen production efficiency is improved to the maximum extent, and the hydrogen production efficiency in the photo-fermentation biological hydrogen production process is remarkably improved.
Description
Technical Field
The invention belongs to the technical field of photo-fermentation biological hydrogen production, and particularly relates to a portable mixing stirring enzymolysis fermentation hydrogen production method and device.
Background
As energy consumption increases dramatically, the contradiction between petroleum supply and demand becomes increasingly prominent. Meanwhile, the sustainable development of society economy is seriously affected by the problems of environmental pollution and the like caused by massive combustion of petrochemical fuel. In the age of rapid development of new energy technology, hydrogen energy is taken as a renewable new energy, and gradually takes up an important role in a modern energy stage, and the hydrogen energy has the outstanding characteristics of no secondary pollution, high efficiency, renewable property and the like in combustion, and is an ideal new energy.
The hydrogen energy content in the natural world is small, under the condition of large hydrogen energy demand, manual hydrogen production is needed, and the current hydrogen production modes mainly comprise electrolytic water hydrogen production, natural gas hydrogen production, photolytic water hydrogen production, biological hydrogen production and the like, wherein the biological hydrogen production is the most green and environment-friendly hydrogen production mode. The advantage of biological hydrogen production is that the raw materials are extensive and abundant, the hydrogen production speed is fast, and the cost is low.
The light fermentation biological hydrogen production process involves an enzymolysis stage, and enzymolysis reaction needs to be carried out in specific equipment, for example, chinese patent publication No. CN107488578B discloses a synchronous saccharification fermentation hydrogen production reactor with enzyme recycling. However, it can be seen that most of the photo-fermentation biological hydrogen production devices at the present stage have the problems of large device volume, inconvenient carrying and difficult transportation, and can not realize hydrogen production anytime and anywhere.
Therefore, the inventor designs a portable mixing and stirring enzymolysis device based on the photo-fermentation biological hydrogen production process and the existing hydrogen production equipment, and simultaneously provides a corresponding hydrogen production process.
Disclosure of Invention
The invention provides a portable mixing stirring enzymolysis fermentation hydrogen production method and device, which can improve the stirring speed and the enzymolysis degree of biomass raw materials in the enzymolysis process by arranging two reverse spiral stirring rods, thereby accelerating the contact degree between microorganisms and the biomass raw materials and the metabolism degree of microorganisms, furthest improving the hydrogen production efficiency and remarkably improving the hydrogen production efficiency in the photo-fermentation biological hydrogen production process.
Based on the above purpose, the invention adopts the following technical scheme:
a portable light fermentation hydrogen production mixing stirring enzymolysis device comprises a cylindrical shell with an open top end and a top cover; the top cover is detachably connected with the shell;
the inner cavity of the shell is provided with a baffle plate, the baffle plate divides the inner cavity of the shell into an upper cavity and a lower cavity, and a control motor is arranged in the lower cavity;
a turntable is arranged on the upper end surface of the partition plate, and an inner spiral stirring rod and an outer spiral stirring rod are arranged at the top end of the turntable; the inner spiral stirring rod and the outer spiral stirring rod are both spiral;
one end of the external spiral stirring rod is fixedly arranged on the upper end surface of the turntable, and the other end of the external spiral stirring rod extends upwards in a spiral manner along the vertical direction;
the motor shaft of the control motor extends upwards to penetrate through the partition plate and is fixedly connected with the center of the rotary table, the motor shaft of the control motor rotates and can drive the rotary table to rotate, and the rotary table drives the inner spiral stirring rod and the outer spiral stirring rod to rotate by taking the axial center line of the rotary table as the center.
Further, the partition plate is horizontally arranged.
Further, the upper chamber is an enzymolysis reaction chamber, and reaction liquid is arranged in the upper chamber.
Further, in order to prevent the reaction liquid in the upper chamber from leaking into the lower chamber, waterproof layers are arranged on the upper surface and the lower surface of the partition plate.
Further, the inner spiral stirring rod extends spirally along the horizontal direction, and the side walls of the inner spiral stirring rod are fixed on the upper end face of the rotary table, namely, the inner spiral stirring rod forms a spiral disc shape.
Further, both end parts of the inner spiral stirring rod are provided with vortex stirring blocks, and the vortex stirring blocks are in a pointed tower shape extending vertically upwards.
Further, the diameter of the disk-shaped structure formed by the inner spiral stirring rod is smaller than that of the outer spiral stirring rod.
Further, the top cap includes arched roof and cylindric barrel, roof and the top fixed connection of barrel are equipped with the internal thread in the barrel, and casing top outer wall is equipped with the external screw thread with barrel internal thread looks adaptation, through the cooperation connection of internal thread and external screw thread, can realize that top cap and casing top can dismantle the connection.
Further, in order to ensure the stability of the reaction temperature, a heat-insulating layer is arranged outside the shell.
Further, in order to increase the enzymolysis efficiency, the inner spiral stirring rod and the outer spiral stirring rod are arranged in opposite extending directions, so that the opposite rotating directions are realized, and the stirring and enzymolysis efficiency in the enzymolysis reaction chamber (upper chamber) is improved.
The method for preparing hydrogen by utilizing the portable light fermentation hydrogen preparation mixing stirring enzymolysis device comprises the following steps:
1) Firstly, sterilizing the enzymolysis device, and checking the air tightness of the device; adding biomass raw materials, buffer solution and enzyme into the enzymolysis device to obtain initial reaction solution, and adjusting the initial reaction temperature in a shell of the enzymolysis device to be 45-50 ℃;
2) Then, a top cover is covered for sealing enzymolysis reaction, and different enzyme loads are set by adding different amounts of enzymes, so that the substrate enzymolysis rate is changed, the rotation speed of a motor is set and controlled in the reaction process, the stirring speeds of an inner spiral stirring rod and an outer spiral stirring rod are set, and the temperature of a reaction solution is ensured by a heat preservation layer in the reaction process, so that an enzymolysis reaction solution is obtained;
3) After the enzymolysis reaction is finished, opening a top cover, taking out enzymolysis reaction liquid, centrifuging to obtain enzymolysis supernatant, adding photosynthetic bacteria HAU-M1 initial culture liquid, adding hydrogen production culture medium, and placing in a photo-fermentation hydrogen production reactor to perform photo-fermentation biological hydrogen production reaction.
Specifically, in the step 1), the biomass raw material is arundo donax, corn stalk or wheat stalk.
Specifically, in step 1), the Total Solids (TS) content of the biomass feedstock is between 5 and 8g.
Specifically, in the step 1), the buffer solution is a citric acid-sodium citrate buffer solution with the pH of 4.8 and the volume of 80-100mL.
Specifically, the total volume of the initial reaction liquid in the step 1) is 100-120mL.
Specifically, the total solid-to-liquid ratio of the solid material and the initial reaction liquid in the enzymolysis reaction process in the step 2) is 1g (20-30) mL.
Specifically, in the step 2), the stirring speed of the inner spiral stirring rod and the outer spiral stirring rod is 120-150rpm, the enzymolysis temperature is 30-32 ℃, the enzymolysis time is 0-72h, and different enzyme loads of 10-50FPU are set, so that the substrate enzymolysis rate is changed.
Preferably, the enzyme load in step 2) is set to 10FPU, 20FPU, 30FPU, 40FPU, 50FPU.
Specifically, the photosynthetic bacteria HAU-M1 used in the hydrogen production in the step 3) mainly comprise rhodospirillum rubrum (R.rhodospirillum rubrum), rhodopseudomonas capsulata (R.capsuloata), rhodopseudomonas palustris (R.pulstrasis), rhodobacter sphaeroides (R.rhodobacter sphaeroides) and rhodobacter capsulatus (Rhodobacter capsulatus).
Specifically, the volume of the hydrogen-producing medium added in the step 3) is 5-10mL.
Specifically, the volume of the initial culture solution of the photosynthetic bacteria HAU-M1 added in the step 3) is 20-30% (v/v) of the volume of the enzymolysis supernatant, and the working volume of the fermentation solution in the process of the photo-fermentation biological hydrogen production reaction is 120-150mL.
Specifically, the reaction conditions of the photo-fermentation biological hydrogen production in the step 3) are as follows: the temperature is 30-32 ℃, and the illumination intensity is 1000-3000Lux.
Specifically, in the step 3), the method for culturing the initial culture solution of the photosynthetic bacteria HAU-M1 comprises the following steps: placing photosynthetic bacteria HAU-M1 into a growth medium, fermenting and culturing for 48-72h at the temperature of 30-32 ℃ and the illumination intensity of 2000-3000Lux to obtain initial culture solution of the photosynthetic bacteria HAU-M1 for later use.
Specifically, the culture medium and the reagent used in the hydrogen production process are as follows:
growth medium: NH (NH) 4 Cl 0.5g/L；NaHCO 3 1g/L; yeast extract 0.5g/L; k (K) 2 HPO 4 0.1g/L；CH 3 COONa 2g/L；MgSO 4 0.1g/L；NaCl 1g/L。
Hydrogen-producing medium: NH (NH) 4 Cl 0.4g/L；MgCl 2 0.2g/L; yeast extract 0.1g/L; k (K) 2 HPO 4 0.5g/L; naCl 2g/L; sodium glutamate 3.56g/L.
Cellulase: enzyme activity is more than or equal to 1800U/mg, and the enzyme is purchased from Shanghai source leaf biotechnology Co., ltd, plays a biological catalysis role in decomposing cellulose, and can decompose the cellulose into monosaccharide.
The buffer solution is as follows: pH4.8 citrate-sodium citrate buffer.
Compared with the prior art, the application has the advantages that:
1. by utilizing the portable mixing stirring enzymolysis fermentation hydrogen production method and device, the reaction time can be shorter, and the material flow is smoother.
2. By utilizing the portable mixing stirring enzymolysis fermentation hydrogen production method and device, the enzymolysis degree of biomass raw materials is improved by changing the enzyme load in the hydrogen production process, so that the contact degree between microorganisms and the biomass raw materials is accelerated, the hydrogen production rate can be improved, and the utilization rate of the biomass raw materials can be improved. The application provides a new strengthening way for improving the hydrogen production efficiency of microorganisms.
Drawings
FIG. 1 is a schematic diagram of a portable mixing and stirring enzymolysis device according to embodiment 1;
fig. 2 is a top view of the internal structure of the portable mixing and stirring enzymolysis device in example 1;
FIG. 3 is a graph showing the effect of enzyme loading on the cumulative hydrogen production and hydrogen production rate of a photo-fermentative biological hydrogen production;
FIG. 4 is a graph showing the effect of enzyme loading on the concentration of byproducts in a photo-fermented biological hydrogen production fermentation broth.
Detailed Description
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit or scope of the invention, which is therefore not limited to the specific embodiments disclosed below.
Example 1
1-2, the portable light fermentation hydrogen production mixing stirring enzymolysis device comprises a cylindrical shell 1 with an open top end and a top cover 2; the top cover 2 is detachably connected with the shell 1;
the inner cavity of the shell 1 is provided with a baffle plate 3, the baffle plate 3 is horizontally arranged, the inner cavity of the shell is divided into an upper cavity and a lower cavity by the baffle plate 3, the upper cavity is an enzymolysis reaction chamber, the upper cavity is internally provided with a reaction liquid, and the lower cavity is internally provided with a control motor 4; in order to prevent the reaction liquid in the upper chamber from leaking into the lower chamber, the upper surface and the lower surface of the partition plate 3 are provided with waterproof layers.
A rotary table 31 is arranged on the upper end surface of the partition plate 3, and an inner spiral stirring rod 5 and an outer spiral stirring rod 6 are arranged at the top end of the rotary table 31; the inner spiral stirring rod 5 and the outer spiral stirring rod 6 are both spiral;
specifically, the inner spiral stirring rod 5 extends spirally along the horizontal direction, and the side walls of the inner spiral stirring rod 5 are all fixed on the upper end surface of the turntable 31, namely, the inner spiral stirring rod 5 forms a spiral disk shape; the two tail end parts of the inner spiral stirring rod 5 are respectively provided with a vortex stirring block, and the vortex stirring blocks are respectively in a pointed tower shape extending vertically upwards;
one end of the external spiral stirring rod 6 is fixedly arranged on the upper end surface of the rotary disc 31, and the other end of the external spiral stirring rod extends upwards in a spiral manner along the vertical direction;
the diameter of the disk-shaped structure formed by the inner spiral stirring rod 5 is smaller than the spiral diameter of the outer spiral stirring rod 6.
The motor shaft 41 of the control motor 4 extends upwards to pass through the partition plate 3 and is fixedly connected with the center of the turntable 31, when the control motor 4 is started, the motor shaft 41 of the control motor 4 rotates to drive the turntable 31 to rotate, and the turntable 31 drives the inner spiral stirring rod 5 and the outer spiral stirring rod 6 to rotate by taking the axial center line of the turntable 31 as the center.
The top cap 2 includes arched roof and cylindric barrel, roof and the top fixed connection of barrel are equipped with the internal thread in the barrel, and casing 1 top outer wall is equipped with the external screw thread with barrel internal thread looks adaptation, through the cooperation connection of internal thread and external screw thread, can realize that top cap 2 can dismantle with casing 1 top and be connected.
In order to ensure the stability of the reaction temperature, a heat preservation layer 11 is arranged outside the shell 1.
When the device is used, the top cover 2 is opened, biomass raw materials, buffer solution, enzyme and bacterial solution are poured from the top end opening of the shell 1, the top cover 2 is covered for sealing enzymolysis reaction, the inner spiral stirring rod 5 and the outer spiral stirring rod 6 arranged in the enzymolysis reaction chamber (upper cavity) are driven to rotate by the control motor 4, so that the inner spiral stirring rod 5 and the outer spiral stirring rod 6 are driven to rotate by taking the axial center line of the rotary table 31 as the center, and the biomass raw materials, the buffer solution and the enzyme are fully stirred, so that enzymolysis is realized.
In order to increase the enzymolysis efficiency, the inner spiral stirring rod 5 spirally extends anticlockwise along the horizontal direction, one end of the outer spiral stirring rod 6 is fixedly arranged on the upper end face of the rotary table 31, the other end of the outer spiral stirring rod extends clockwise upwards along the vertical direction, and in the rotating process of the rotary table 31, the inner spiral stirring rod 5 and the outer spiral stirring rod 6 are arranged in opposite extending directions, so that the opposite rotating directions are realized, and the stirring and enzymolysis efficiency in the enzymolysis reaction chamber (upper chamber) is improved.
Example 2
The method for preparing hydrogen by utilizing the portable light fermentation hydrogen preparation mixing stirring enzymolysis device in the embodiment 1 comprises the following specific steps:
1) Firstly, placing the enzymolysis device in a sterilizing pot for sterilization, taking out the enzymolysis device after sterilization, and checking the air tightness of the device; adding biomass raw materials, buffer solution and enzyme into the enzymolysis device to obtain initial reaction liquid, and adjusting the initial reaction temperature in a shell of the enzymolysis device to be 50 ℃;
2) Then, a top cover 2 is covered for sealing enzymolysis reaction, and different enzyme loads are set by adding different amounts of enzymes, so that the substrate enzymolysis rate is changed, the rotation speed of a motor 4 is set and controlled in the reaction process, the stirring speeds of an inner spiral stirring rod 5 and an outer spiral stirring rod 6 are set, and the temperature of a reaction solution is ensured by a heat preservation layer 11 in the reaction process, so that an enzymolysis reaction solution is obtained;
3) After the enzymolysis reaction is finished, the top cover 2 is opened, the enzymolysis reaction liquid is taken out, the enzymolysis supernatant is centrifugally taken, the photosynthetic bacteria HAU-M1 initial culture liquid is added, then 5mL of hydrogen production culture medium is added, and the mixture is placed in a photo-fermentation hydrogen production reactor (the photo-fermentation hydrogen production reactor adopts a micro speed control photo-biological hydrogen production reaction device disclosed in China patent CN 113373051A), and photo-fermentation biological hydrogen production reaction is carried out under the condition that the temperature is set to 30 ℃ and the illumination intensity is set to 3000Lux.
The biomass raw material added in the step 1) is arundo donax, wherein the Total Solid (TS) content of the arundo donax is 5g, the added buffer solution is 0.05M, the pH value is 4.8 citric acid-sodium citrate buffer solution, the volume of the buffer solution is 100mL, the added enzyme is cellulase with the enzyme activity of more than or equal to 1800U/mg, and the total volume of the initial reaction solution in the step 1) is 100mL.
The total solid-to-liquid ratio of the solid material and the initial reaction liquid in the enzymolysis reaction process in the step 2) is 1g to 20mL.
In the step 2), the stirring speed of the inner spiral stirring rod 5 and the outer spiral stirring rod 6 is 150rpm, the enzymolysis temperature is adjusted to 30 ℃, the enzymolysis time is 72 hours, and different enzyme loads (10-50 FPU) are set, so that the substrate enzymolysis rate is changed.
In the step 3), specifically, when the photo-fermentation biological hydrogen production is performed, the pH value of the enzymolysis supernatant after centrifugation is regulated to 7, and then 30% (v/v) of photosynthetic bacteria HAU-M1 initial culture solution of the volume of the enzymolysis supernatant is added for performing the photo-fermentation biological hydrogen production reaction, wherein the working volume of the fermentation solution in the photo-fermentation biological hydrogen production reaction process is 150mL.
In the step 3), the culture method of the initial culture solution of the photosynthetic bacteria HAU-M1 comprises the following steps: and placing the photosynthetic bacteria HAU-M1 in a growth medium, and fermenting and culturing for 72 hours at the temperature of 30 ℃ and the illumination intensity of 3000Lux to obtain an initial culture solution of the photosynthetic bacteria HAU-M1 for later use.
Specifically, the total duration of the hydrogen production experiment is 96 hours, and hydrogen production, air extraction and sample measurement are carried out every 12 hours.
The strain used in hydrogen production is photosynthetic bacteria HAU-M1, wherein the HAU-M1 photosynthetic bacteria group is obtained by adopting a method in a literature (Han Binxu. Separation and identification of photosynthetic hydrogen producing bacteria group and hydrogen production characteristic analysis [ D ]. Henan agricultural university, 2011), and can decompose organic matters to produce hydrogen under illumination conditions, and the HAU-M1 photosynthetic bacteria group mainly comprises rhodospirillum (R.hot spiral rubrum), rhodopseudomonas capsulata (R.capsule), rhodopseudomonas palustris (R.pulassis), rhodobacter sphaeroides (R.hot sphaeroides) and rhodobacter capsulatus (Rhodobacter capsulatus).
In HAU-M1 photosynthetic bacteria liquid, rhodospirillum profundum bacterial liquid, rhodopseudomonas capsulata bacterial liquid, rhodopseudomonas palustris bacterial liquid, rhodococcus pseudolaris bacterial liquid and rhodobacter capsulatus bacterial liquid are respectively 27 in volume ratio: 25:28:9:11; the number of viable bacteria in the rhodospirillum rubrum bacterial liquid is 12.0 multiplied by 10 8 Each mL, rhodopseudomonas capsulata 11.0X10 8 Each mL, rhodopseudomonas palustris 12.5X10 8 Rhodobacter sphaeroides at 4.0X10 g/mL 8 Each mL, rhodobacter capsulatus was 5.0X10 8 And each mL.
The photosynthetic bacteria HAU-M1 is obtained from renewable energy new materials and equipment key laboratories of agricultural rural areas of Henan university.
The culture medium and the reagent used in the hydrogen production process are as follows:
growth medium: NH (NH) 4 Cl 0.5g/L；NaHCO 3 1g/L; yeast extract 0.5g/L; k (K) 2 HPO 4 0.1g/L；CH 3 COONa2g/L；MgSO 4 0.1g/L；NaCl 1g/L。
Hydrogen-producing medium: NH (NH) 4 Cl 0.4g/L；MgCl 2 0.2g/L; yeast extract 0.1g/L; k (K) 2 HPO 4 0.5g/L; naCl 2g/L; sodium glutamate 3.56g/L.
Cellulase: enzyme activity is more than or equal to 1800U/mg, and the enzyme is purchased from Shanghai source leaf biotechnology Co., ltd, plays a biological catalysis role in decomposing cellulose, and can decompose the cellulose into monosaccharide.
The buffer solution is as follows: pH4.8 citrate-sodium citrate buffer.
The preparation method of the citric acid-sodium citrate buffer solution comprises the following steps: and (3) solution A: accurately weigh C 6 H 8 O 7 .H 2 O21.014 g is dissolved in a 500mL beaker with a small amount of deionized water, and the volume is fixed to 1000mL to obtain a 0.1mol/L citric acid solution; and (2) liquid B: accurately weigh Na 3 C 6 H 5 O 7 .2H 2 O29.412 g is dissolved in a 500mL beaker with a small amount of deionized water, and the volume is fixed to 1000mL to obtain a 0.1mol/L sodium citrate solution; taking 230mL of A solution and 270mL of B solution, fully mixing, transferring into a 1000mL volumetric flask, fixing the volume to 1000mL by using deionized water, fully mixing, and refrigerating and preserving in a refrigerator at 4 ℃.
The specific hydrogen production test method comprises the following steps: OD measurement of photosynthetic bacteria liquid using 721 spectrophotometer (Shanghai essence science and technology instruments Co., ltd.) 660nm The value, photosynthetic bacteria are put into a 50mL centrifuge tube, centrifuged at 6000r/min, then the supernatant is poured off, sediment is dried to constant weight at 65 ℃, then the sediment is weighed, and then the dry weight of the photosynthetic bacteria is plotted against OD 660nm Standard curve of values. By testing OD during growth of photosynthetic bacteria 660nm The values were used to calculate the photosynthetic bacteria dry weight change.
The gas was collected using a gas collection bag and the hydrogen concentration was measured using a 7890B type gas chromatograph. Experimental data were measured every 12 hours.
Experimental results
The invention takes arundo donax as a photosynthetic organism hydrogen production substrate, researches hydrogen production performance by adjusting different enzyme loads, wherein accumulated hydrogen production is taken as an evaluation index, and the following conclusion is obtained.
When the enzyme load is 20FPU (FPU refers to the enzyme amount consumed for producing unit glucose in unit time, namely 1FPU is the enzyme amount used for producing 1 mu mol glucose in 1 min), the hydrogen production delay period of the whole fermentation hydrogen production process is the shortest and is 11.19h, under the condition, sugar and acid substances in fermentation liquid have no inhibition effect on the growth metabolism of photosynthetic bacteria, under the condition, the content of the acid substances in fermentation liquid is always kept stable, so that the bacteria can utilize the sugar acid substances to simultaneously metabolize and produce hydrogen, the fermentation environment is relatively stable, the hydrogen production delay period is shorter, and meanwhile, photosynthetic bacteria can adapt to hydrogen production conditions more quickly to synthesize enzymes related to hydrogen production. The experimental results are shown in table 1, fig. 3 and fig. 4:
TABLE 1 analysis of hydrogen production kinetics for different enzyme loads
As can be seen from Table 1, in the experimental group with the shortest hydrogen production delay period, the enzyme load was 20FPU, the hydrogen production delay period under this condition was 11.19 hours, and at 24.09 hours, the hydrogen production rate reached the maximum value (7.38 mL/h), and the hydrogen production delay period was shortened by 40% compared with the experimental group with the enzyme load of 50FPU. The reason is probably that the content of sugar and acid substances in the fermentation liquid has no inhibition effect on the growth metabolism of photosynthetic bacteria, and the photosynthetic bacteria can adapt to hydrogen production conditions more quickly, synthesize enzymes related to hydrogen production, and the light and bacteria mainly perform hydrogen production metabolism under the enzyme loading condition. And too high or too low enzyme load can inhibit or limit the growth and metabolism of photosynthetic bacteria, and simultaneously lead the photosynthetic bacteria to reach the time delay of the state with relatively optimal physiological activity, so the hydrogen production delay period is higher.
However, as photosynthetic bacteria adapt to the fermentation environment of higher enzyme load, sufficient nutrients allow the photosynthetic bacteria to metabolize more hydrogen, so that the cumulative hydrogen production (445.56) is highest when the enzyme load reaches 50FPU.
According to the light fermentation hydrogen production process using the portable mixing and stirring enzymolysis device, the enzyme load is changed in the hydrogen production process, so that the enzymolysis degree of biomass raw materials is improved, the contact degree between microorganisms and the biomass raw materials is accelerated, the hydrogen production rate can be improved, and the utilization rate of the biomass raw materials can be improved. The application provides a new strengthening way for improving the hydrogen production efficiency of microorganisms.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (9)
1. A method for producing hydrogen by light fermentation by utilizing a portable light fermentation hydrogen production mixing stirring enzymolysis device is characterized in that,
the portable mixing and stirring enzymolysis device comprises a cylindrical shell with an open top end and a top cover; the top cover is detachably connected with the shell;
the inner cavity of the shell is provided with a baffle plate, the baffle plate divides the inner cavity of the shell into an upper cavity and a lower cavity, and a control motor is arranged in the lower cavity;
a turntable is arranged on the upper end surface of the partition plate, and an inner spiral stirring rod and an outer spiral stirring rod are arranged at the top end of the turntable; the inner spiral stirring rod and the outer spiral stirring rod are both spiral;
one end of the external spiral stirring rod is fixedly arranged on the upper end surface of the turntable, and the other end of the external spiral stirring rod extends upwards in a spiral manner along the vertical direction;
the motor shaft of the control motor extends upwards to pass through the partition plate and is fixedly connected with the center of the turntable, the motor shaft of the control motor rotates and can drive the turntable to rotate, and the turntable drives the inner spiral stirring rod and the outer spiral stirring rod to rotate by taking the axial center line of the turntable as the center;
the inner spiral stirring rod extends spirally along the horizontal direction, and the side walls of the inner spiral stirring rod are fixed on the upper end surface of the turntable;
the two tail end parts of the inner spiral stirring rod are respectively provided with a vortex stirring block, and the vortex stirring blocks are respectively in a pointed tower shape extending vertically upwards;
the inner spiral stirring rod and the outer spiral stirring rod are arranged in opposite extending directions;
the method comprises the following steps:
1) Adding biomass raw materials, buffer solution and enzyme into the enzymolysis device to obtain initial reaction solution, and regulating the initial reaction temperature of the initial reaction solution in the enzymolysis device to be 45-50 ℃;
2) Then, a top cover is covered for sealing enzymolysis reaction, different enzyme loads are set by changing the addition amount of enzyme, the enzymolysis rate of a substrate is changed, the rotation speed of a motor is controlled in the reaction process, the stirring speeds of an inner spiral stirring rod and an outer spiral stirring rod are set, and the temperature of a reaction solution is ensured through a heat preservation layer in the reaction process, so that an enzymolysis reaction solution is obtained;
3) After the enzymolysis reaction is finished, opening a top cover, taking out enzymolysis reaction liquid, centrifuging to obtain enzymolysis supernatant, adding photosynthetic bacteria HAU-M1 initial culture solution, adding hydrogen production culture medium, and placing in a photo-fermentation hydrogen production reactor to perform photo-fermentation biological hydrogen production reaction;
the biomass raw material in the step 1) is arundo donax;
in the step 2), the stirring speed of the inner spiral stirring rod and the outer spiral stirring rod is 120-150rpm, the enzymolysis temperature is 30-32 ℃, the enzymolysis time is 0-72h, and different enzyme loads are set to be 10-50FPU;
in the step 1), the volume of the buffer solution is 80-100mL;
the total volume of the initial reaction liquid in the step 1) is 100-120mL;
the total solid-liquid ratio of the solid material and the initial reaction liquid in the enzymolysis reaction process in the step 2) is 1g (20-30 mL;
the volume of the hydrogen-producing culture medium added in the step 3) is 5-10mL;
the reaction conditions of the photo-fermentation biological hydrogen production in the step 3) are as follows: the temperature is 30-32 ℃, and the illumination intensity is 1000-3000Lux;
in the step 3), the volume of the initial culture solution of the added photosynthetic bacteria HAU-M1 is 20-30% of the volume of the enzymolysis supernatant; the working volume of the fermentation liquid in the process of the photo-fermentation biological hydrogen production reaction is 120-150mL;
the growth medium is as follows: NH (NH) 4 Cl 0.5g/L；NaHCO 3 1g/L; yeast extract 0.5g/L; k (K) 2 HPO 4 0.1g/L；CH 3 COONa 2g/L；MgSO 4 0.1g/L；NaCl 1g/L；
The enzyme is cellulase, and the enzyme activity is more than or equal to 1800U/mg.
2. The method of claim 1, wherein the top cover comprises an arched top wall and a cylindrical barrel, the top wall is fixedly connected with the top end of the barrel, internal threads are arranged in the barrel, external threads matched with the internal threads of the barrel are arranged on the outer wall of the top end of the shell, and the top cover is detachably connected with the top end of the shell through the matched connection of the internal threads and the external threads.
3. The method of claim 1, wherein in step 1) the buffer is a ph4.8 citric acid-sodium citrate buffer.
4. The method according to claim 1, characterized in that in step 1) the total solids content of the biomass feedstock is 5-8g.
5. The method according to claim 1, wherein the enzyme load in step 2) is set to 10FPU, 20FPU, 30FPU, 40FPU, 50FPU.
6. The method according to claim 1, wherein in the step 3), the initial culture solution of photosynthetic bacteria HAU-M1 is cultured by the following method: placing photosynthetic bacteria HAU-M1 into a growth medium, fermenting and culturing for 48-72h at the temperature of 30-32 ℃ and the illumination intensity of 2000-3000Lux to obtain initial culture solution of the photosynthetic bacteria HAU-M1 for later use.
7. The method according to claim 1, wherein the photosynthetic bacteria HAU-M1 used in the hydrogen production in step 3) consist essentially of rhodospirillum rubrum, rhodopseudomonas capsulata, rhodopseudomonas palustris, rhodococcus globosum, rhodobacter capsulatus.
8. The method according to claim 7, wherein the volume ratio of rhodospirillum profundum bacterial liquid, rhodopseudomonas capsulata bacterial liquid, rhodopseudomonas palustris bacterial liquid, rhodococcus pseudolaris bacterial liquid and rhodobacter capsulatus bacterial liquid in HAU-M1 photosynthetic bacterial liquid is 27:25:28:9:11; the number of viable bacteria in the rhodospirillum rubrum bacterial liquid is 12.0 multiplied by 10 8 Each mL, rhodopseudomonas capsulata 11.0X10 8 Each mL, rhodopseudomonas palustris 12.5X10 8 Rhodobacter sphaeroides at 4.0X10 g/mL 8 Each mL, rhodobacter capsulatus was 5.0X10 8 And each mL.
9. The method according to claim 1, wherein the hydrogen-producing medium in step 3) is: NH (NH) 4 Cl 0.4g/L；MgCl 2 0.2g/L; yeast extract 0.1g/L; k (K) 2 HPO 4 0.5g/L; naCl 2g/L; sodium glutamate 3.56g/L.
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