CN114032253A - Hydrogen production method based on hydrogel coated bacterium aggregation - Google Patents
Hydrogen production method based on hydrogel coated bacterium aggregation Download PDFInfo
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- CYDQOEWLBCCFJZ-UHFFFAOYSA-N 4-(4-fluorophenyl)oxane-4-carboxylic acid Chemical compound C=1C=C(F)C=CC=1C1(C(=O)O)CCOCC1 CYDQOEWLBCCFJZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000001540 sodium lactate Substances 0.000 claims description 10
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- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
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- 239000007864 aqueous solution Substances 0.000 claims description 6
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- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 6
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Abstract
A method for producing hydrogen based on hydrogel-coated bacteria aggregation, belonging to the field of biological hydrogen production. The invention particularly relates to a method for gathering bacteria containing hydrogenase by using a sodium alginate hydrogel model and applying the bacteria to long-term hydrogen production. The present invention solves two key problems: the invention provides a simple and universal preparation method of bacterial aggregates; secondly, the long-term hydrogen production of the hydrogenase-containing bacteria is realized. The method comprises the following steps: firstly, preparing Shewanella aggregates; secondly, preparing an escherichia coli aggregate; thirdly, preparing a mixed bacterial aggregate; fourthly, long-time hydrogen production of the aggregate. The invention combines the structural characteristics of the sodium alginate hydrogel with the hydrogen production capability of hydrogenase-containing bacteria to prepare the hydrogel bacteria aggregate, and the aggregate has the advantages of simple preparation, convenient operation and the like. The bacteria related to the bacterial aggregate include but are not limited to Shewanella, Escherichia coli, and other bacteria containing hydrogenase can also be used for preparing the aggregate.
Description
Technical Field
The invention belongs to the field of biological hydrogen production, and particularly relates to a hydrogen production method based on hydrogel-coated bacteria aggregation.
Background
The emission of greenhouse gases inevitably occurs during the use of fossil energy, and the reserves of fossil fuels on earth are limited. For this reason, it is necessary to find clean fuels that can replace fossil energy, which not only meets the national carbon neutralization goal, but also is a requirement of sustainable development strategy. Hydrogen is a recognized clean energy source, and is emerging as a new energy source. The hydrogen has many advantages as energy, the heat value of the hydrogen is high, which is more than 3 times of the heat value of the petroleum, the hydrogen has light weight, high storage capacity, environmental protection and good heat conduction. The main industrial hydrogen production methods at present are: hydrogen production by fossil fuel, industrial byproduct hydrogen production, biological raw material hydrogen production, electrolysis hydrogen production and the like. These hydrogen production processes mentioned above are all energy intensive and unsustainable. In contrast, biological hydrogen production is a sustainable, relatively simple method of producing hydrogen. The biological hydrogen production mainly utilizes hydrogenase in organisms. At present, biological hydrogen production is usually achieved by using microorganisms expressing hydrogenase, such as green algae, blue algae, purple photosynthetic bacteria and some heterotrophic bacteria with hydrogenase, or hydrogenase isolated directly. The method for synthesizing hydrogen by using the microorganisms as the whole-cell biocatalyst is simpler and more stable, thereby having more practical application prospect. Because the bacteria are widely distributed, widely available and fast propagated, the bacteria can be used as an ideal main body for hydrogen production. Gram-negative bacteria such as Shewanella, Escherichia coli, Clostridium butyricum, Mourella thermoacetica, and Desulfurovibrio have hydrogenase therein, and hydrogen can be produced under anaerobic fermentation condition.
At present, most researches on hydrogen production by using the bacteria are combined with inorganic photosensitive substances, such as titanium dioxide, cadmium sulfide, quantum dots and the like. The photosensitive substances are modified inside or outside the bacteria, and then under the condition of illumination, the photosensitive substances can generate photoelectrons which are further utilized by hydrogenase, so that hydrogen production can be realized. Although this method can combine the light-sensitive property of inorganic light-sensitive materials with the hydrogen-producing ability of bacteria to produce hydrogen, these light-sensitive materials produce photoelectrons and active oxygen under light conditions. Active oxygen has been proved to have a harmful effect on bacteria, so the method can damage the activity of the bacteria while producing hydrogen, so that the method cannot produce hydrogen for a long time, usually tens of hours, thereby limiting the practical application of the method. In addition, strict anaerobic environment is required for producing hydrogen by bacterial fermentation. The hydrogenase is not activated by the rapid establishment of an anaerobic environment due to the respiratory action of bacteria alone, so that no hydrogen is produced during the initial period of anaerobic fermentation. Currently, the anaerobic environment is usually accelerated by pumping inert gas such as nitrogen, but this also increases the operation cost and difficulty.
Disclosure of Invention
The invention aims to solve the problems of rapid establishment of anaerobic conditions and incapability of producing hydrogen for a long time by bacteria in the hydrogen production process by bacteria, and provides a method for producing hydrogen based on hydrogel-coated bacteria aggregation. After the substrate of the hydrogel bacterial aggregate is exhausted, the hydrogel bacterial aggregate can be subjected to long-term hydrogen production by supplementing the substrate and compacting again in a calcium chloride solution, so that the hydrogel bacterial aggregate can continuously produce hydrogen for 400 hours. The aggregate has the advantages of simple preparation, good biocompatibility, long-term use, convenient operation, universality and the like. The bacteria related to the bacterial aggregate include but are not limited to Shewanella, Escherichia coli, and other bacteria containing hydrogenase can also be used for preparing the aggregate.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a hydrogen production method based on hydrogel coated bacteria aggregation comprises the following specific steps:
step one, preparation of Shewanella aggregates:
(1) dissolving 100-150 mul of span 80 into 40ml of liquid paraffin to be used as an oil phase;
(2) dissolving 15-30 mg of calcium chloride in 200 mul of deionized water to serve as a water phase A;
(3) mixing 1-2% of sodium alginate solution with bacterial liquid containing hydrogenase to serve as a water phase B, wherein the volume ratio of the sodium alginate solution to the bacterial liquid is 1: 1-1: 5;
(4) mixing 200 mu l of the water phase A and 6ml of the oil phase in a centrifuge tube, shaking uniformly to obtain a water-in-oil emulsion, standing for 1-5 minutes, adding the water phase B into the centrifuge tube, shaking uniformly, standing for 30min, and obtaining a bacterial aggregate at the bottom of the centrifuge tube, wherein the volume ratio of the water phase B to the oil phase is 1: 1-1: 5;
(5) centrifuging, sequentially washing 3 times by using 1-3% of Tween 80 aqueous solution and deionized water, completely transferring the aggregate to a water phase to obtain an aggregate precipitate, dispersing the aggregate precipitate into 10ml of 2-5 mg/ml calcium chloride solution, compacting by compaction, wherein the density of thalli in the aggregate is controlled by controlling the concentration of bacterial liquid;
step two, long-time hydrogen production of aggregate
Centrifuging the aggregate, washing for 2 times by using deionized water to obtain an aggregate precipitate, adding a solution containing a corresponding substrate, transferring the solution into a thick-walled mouth small bottle with an air suction joint, sealing by using a sealing film for long-time hydrogen production, centrifuging and washing the aggregate, compacting and compacting in a calcium chloride solution again and supplementing the corresponding substrate when the substrate is exhausted and the aggregate stops producing hydrogen, wherein the compacting conditions are as follows: dispersing the bacterial aggregates into 10ml of 2.5mg/ml calcium chloride solution, and reacting for 2-5 h under magnetic stirring at the rotating speed of 100-250 rpm; the ratio of aggregate precipitate to substrate solution was 310 mg: 3 mL.
Compared with the prior art, the invention has the beneficial effects that: the invention realizes long-term biological hydrogen production by using the hydrogel bacterial aggregate, and the prepared hydrogel bacterial aggregate does not contain any volatile or corrosive substance and has good biocompatibility. The preparation method is simple, can be used for a long time, is convenient to operate, can continuously produce hydrogen for 400 hours, and has certain commercial value.
Drawings
FIG. 1 is an optical microscope photograph of Shewanella aggregates obtained in example 1;
FIG. 2 is an optical microscope photograph of Shewanella aggregates obtained in example 1, further compacted by compaction in a calcium chloride solution;
FIG. 3 is an optical microscope photograph of Shewanella aggregates after 400 hours of hydrogen production obtained in example 1;
FIG. 4 is a fluorescence microscope photograph of Shewanella aggregates obtained in example 1;
FIG. 5 is a graph showing hydrogen production of Shewanella aggregates obtained in example 1;
FIG. 6 is a graph showing hydrogen production curves of Shewanella aggregates prepared using different concentrations of bacterial solutions obtained in examples 1 and 2;
FIG. 7 is a graph of hydrogen production of Shewanella aggregates obtained in examples 1 and 3 under different sodium lactate concentrations;
FIG. 8 is an optical microscope photograph of the Escherichia coli aggregate obtained in example 1;
FIG. 9 is an optical microscope photograph of the E.coli aggregates obtained in example 1, further compacted in a calcium chloride solution;
FIG. 10 is a graph of hydrogen production of the Escherichia coli aggregates obtained in example 1, example 6 and example 7 under different glucose concentrations;
Detailed Description
The technical solutions of the present invention are further described below with reference to the drawings and the embodiments, but the present invention is not limited thereto, and modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
The first embodiment is as follows: the embodiment describes a method for producing hydrogen based on hydrogel-coated bacteria aggregation, which comprises the following specific steps:
step one, preparation of Shewanella aggregates:
(1) dissolving 100-150 mul of span 80 into 40ml of liquid paraffin to be used as an oil phase;
(2) dissolving 15-30 mg of calcium chloride in 200 mul of deionized water to serve as a water phase A;
(3) mixing 1-2% of sodium alginate solution with bacterial liquid containing hydrogenase to serve as a water phase B, wherein the volume ratio of the sodium alginate solution to the bacterial liquid is 1: 1-1: 5;
(4) mixing 200 mu l of the water phase A and 6ml of the oil phase in a centrifuge tube, shaking uniformly to obtain a water-in-oil emulsion, standing for 1-5 minutes, adding the water phase B into the centrifuge tube, shaking uniformly, standing for 30min, and obtaining a bacterial aggregate at the bottom of the centrifuge tube, wherein the volume ratio of the water phase B to the oil phase is 1: 1-1: 5;
(5) centrifuging, sequentially washing 3 times by using 1-3% of Tween 80 aqueous solution and deionized water, completely transferring the aggregate to a water phase to obtain an aggregate precipitate, dispersing the aggregate precipitate into 10ml of 2-5 mg/ml calcium chloride solution, compacting, namely further crosslinking calcium ions in the solution with sodium alginate, and improving the compactness of the aggregate, wherein the density of thalli in the aggregate is controlled by controlling the concentration of bacterial liquid;
step two, long-time hydrogen production of aggregate
Centrifuging the aggregate, washing for 2 times by using deionized water to obtain an aggregate precipitate, adding a solution containing a corresponding substrate, transferring the solution into a thick-walled mouth small bottle with an air suction joint, sealing by using a sealing film for long-time hydrogen production, centrifuging and washing the aggregate, compacting and compacting in a calcium chloride solution again and supplementing the corresponding substrate when the substrate is exhausted and the aggregate stops producing hydrogen, wherein the compacting conditions are as follows: dispersing the bacterial aggregates into 10ml of 2.5mg/ml calcium chloride solution, and reacting for 2-5 h under magnetic stirring at the rotating speed of 100-250 rpm; the ratio of aggregate precipitate to substrate solution was 310 mg: 3 mL.
The second embodiment is as follows: in the method for producing hydrogen based on aggregation of hydrogel-coated bacteria, in the first step, the bacterial liquid is shewanella bacterial liquid, escherichia coli bacterial liquid or mixed bacterial liquid of escherichia coli and shewanella.
The third concrete implementation mode: in the second step, the substrate components and the concentrations of the three aggregates are different, and for the Shewanella aggregate, the substrate solution comprises 10-40 mM sodium lactate and 10-40 mM sodium thiosulfate; for the Escherichia coli aggregate, the substrate solution contains 10-40 mM of glucose and 0.5-2 mM of cysteine; for the aggregate of mixed bacteria, the substrate solution contains 10-40 mM of glucose, 0.5-2 mM of cysteine, 10-40 mM of sodium formate and 10-40 mM of sodium thiosulfate.
The fourth concrete implementation mode: in the second step of the method for preparing a hydrogel-based bacterial aggregate, the substrate solution comprises 20mM sodium lactate and 10mM sodium thiosulfate for the Shewanella aggregates; for E.coli aggregates, the substrate solution contained 20mM glucose and 1mM cysteine; for aggregates of mixed bacteria, the substrate solution contained 20mM glucose, 1mM cysteine, 20mM sodium formate, 10mM sodium thiosulfate.
The fifth concrete implementation mode: in the preparation method of the hydrogel-based bacterial aggregate according to the first embodiment, in the first step, the amount of span 80 is 110 μ l; the dosage of the calcium chloride is 25 mg; the concentration of the sodium alginate solution is 1 percent.
The sixth specific implementation mode: in the preparation method of the hydrogel-based bacterial aggregate according to the first embodiment, in the first step, the volume ratio of the sodium alginate solution to the bacterial liquid is 1: 1.
the seventh embodiment: in the preparation method of the hydrogel-based bacterial aggregate according to the first embodiment, in the first step (4), firstly, the water phase A and 6ml of oil phase are mixed in a centrifuge tube and are shaken up to obtain a water-in-oil emulsion, and the emulsion is kept still for 3 minutes; adding the water phase B into the mixture and shaking up, wherein the volume ratio of the water phase B to the oil phase is 1: 3; in the step one (5), the rotating speed of the centrifugation is 2000-5000 rpm, and the time of each centrifugation is 2-5 minutes.
The specific implementation mode is eight: in the first step, the bacterial aggregate is centrifuged, and then washed 3 times with 1% tween 80 aqueous solution and deionized water, wherein the rotation speed of each centrifugation is controlled at 3000rpm, and the time of each centrifugation is controlled at 3 minutes.
The specific implementation method nine: in the first step of the preparation method of the hydrogel-based bacterial aggregate, the aggregate transferred to the water phase is further compacted in 10ml of 2.5mg/ml calcium chloride solution.
The detailed implementation mode is ten: in the second step, the compacting conditions are as follows: the bacterial aggregates were dispersed in 10ml of 2.5mg/ml calcium chloride solution and reacted for 3h with magnetic stirring at 200 rpm.
Example 1:
the method for producing hydrogen based on hydrogel coated bacteria aggregation specifically comprises the following steps:
firstly, preparation of Shewanella aggregates:
first, a liquid paraffin solution of span 80 (sorbitan oleate) was prepared, and 110. mu.l of span 80 was dissolved in 40ml of liquid paraffin to prepare an oil phase. 25mg of calcium chloride were dissolved in 200. mu.l of deionized water as aqueous phase A. Mixing 1% sodium alginate solution with OD600(absorbance of bacterial liquid at 600 nm) of 12.9 was mixed as aqueous phase B, and the volume ratio of sodium alginate solution to bacterial liquid was 1: 1. 200 mul of calcium chloride solution (water phase A) and 6ml of oil phase are mixed in a centrifuge tube and shaken to obtain water-in-oil emulsion, and the mixture is stood for 3 minutes. And adding the water phase B into the mixture and shaking up the mixture, wherein the volume ratio of the water phase B to the oil phase is 1: 3. Standing for 30min to obtain bacterial aggregate at the bottom of the centrifuge tube. The aggregates can be completely transferred to the aqueous phase by centrifugation and washing 3 times with a 1% volume fraction aqueous solution of tween 80 and then with deionized water, the rotation speed of each centrifugation being controlled at 3000rpm and the time of each centrifugation being controlled at 3 minutes. The aggregate transferred to the aqueous phase can be further compacted in 10ml of 2.5mg/ml calcium chloride solution. Wherein the density of Shewanella in the aggregate can be controlled by controlling the concentration of the bacterial liquid. OD of unaggregated 3ml Shewanella bacteria solution in control group600(absorbance of bacterial liquid at 600 nm) was 4.3, and the total amount of Shewanella aggregates used for hydrogen production was the same as that in the control group.
Preparation of Escherichia coli aggregates
The preparation method of the escherichia coli aggregate is approximately the same as that of the Shewanella aggregate. The difference is that the used bacterial liquid is the bacterial liquid of escherichia coli.
Preparation of mixed bacterial aggregates
The method of preparing the mixed bacterial aggregate is substantially the same as the method of preparing the Shewanella aggregate. The difference is that the used bacterial liquid is mixed bacterial liquid of escherichia coli and shewanella.
Fourthly, long-time hydrogen production of aggregate
The compacted aggregate was centrifuged and washed 2 times with deionized water, 3ml of the solution containing the corresponding substrate was added, transferred to a 5ml thick-walled vial with an air-bleed adapter and sealed with a sealing membrane for long-term hydrogen production. The substrate composition and concentration of the three aggregates varied. For Shewanella aggregates, the substrate solution contained 20mM sodium lactate and 10mM sodium thiosulfate; for E.coli aggregates, the substrate solution contained 20mM glucose and 1mM cysteine; for aggregates of mixed bacteria, the substrate solution contained 20mM glucose, 1mM cysteine, 20mM sodium formate, 10mM sodium thiosulfate. When the substrate is exhausted and the hydrogen production of the aggregate is stopped, after the aggregate is centrifugally washed, the corresponding substrate is continuously compacted in a calcium chloride solution and supplemented, wherein the compacting conditions are as follows: the bacterial aggregates were dispersed in 10ml of 2.5mg/ml calcium chloride solution and reacted for 3h with magnetic stirring at 200 rpm.
Example 2:
this example differs from example 1 in that: the concentration of the used bacterial liquid is 1.5 times of that of the step one. The other steps and parameters were the same as in example 1.
Example 3:
this example differs from example 1 in that: in step four, the substrate solution comprises 40mM sodium lactate and 20mM sodium thiosulfate for Shewanella aggregates. The other steps and parameters were the same as in example 1.
Example 4:
this example differs from example 1 in that: in step four, the substrate solution contains 100mM glucose and 5mM cysteine for the E.coli aggregates. The other steps and parameters were the same as in example 1.
Example 5:
this example differs from example 1 in that: in step four, the substrate solution contains 50mM glucose and 2.5mM cysteine for the E.coli aggregates. The other steps and parameters were the same as in example 1.
FIG. 1 is an optical microscope photograph of Shewanella aggregates obtained in example 1, and it can be seen that the overall morphology of the aggregates appears to be circular and exists independently of each other, and Shewanella is uniformly entrapped in hydrogel microspheres.
FIG. 2 is an optical microscope photograph of the Shewanella aggregates compacted further in the calcium chloride solution obtained in example 1, compared with the uncompacted compacted aggregates, the Shewanella aggregates compacted further in the calcium chloride solution are darker in color, the bacteria inside the aggregates are more closely contacted, and the stability of the whole aggregates is improved due to the further cross-linking of calcium ions, so that the aggregates can be used for long-term hydrogen production.
FIG. 3 is an optical microscope image of the Shewanella aggregate obtained in example 1 after 400 hours of hydrogen production, and it can be seen that the Shewanella aggregate still maintains an intact structure after 400 hours of hydrogen production. The top right inset is an enlarged view of a single aggregate.
FIG. 4 is a fluorescence microscopic image of Shewanella aggregates obtained in example 1, in which bacteria are labeled with blue fluorescent Dye (DAPI), and aggregates are prepared from the dye-labeled bacteria, so that the distribution of Shewanella in the aggregates can be seen more clearly. It can be seen from the figure that Shewanella is uniformly distributed in the aggregate and that there is no bacteria outside the aggregate. The top right inset is an enlarged view of a single aggregate.
FIG. 5 is a graph showing hydrogen production by Shewanella aggregates obtained in example 1, and it can be seen that Shewanella aggregates can continuously produce hydrogen for 400 hours under the conditions of 20mM of sodium lactate and 10mM of sodium thiosulfate compared with Shewanella where no aggregates are formed, and the ordinate represents the accumulated amount of hydrogen (the same below). The arrow indicates that after centrifugation, hydrogen production can continue with substrate addition.
FIG. 6 is a graph showing hydrogen production curves of Shewanella aggregates prepared using bacterial solutions of different concentrations obtained in examples 1 and 2, and it can be seen that the cumulative hydrogen production amount of the aggregates increases as the concentration of the bacterial solution for preparing the bacterial aggregates increases.
FIG. 7 is a graph of hydrogen production curves of Shewanella aggregates obtained in examples 1 and 3 under different sodium lactate concentrations, and it can be seen that the cumulative hydrogen production of the Shewanella aggregates in 16 days (384 hours) increases from 6.18. mu. mol to 8.26. mu. mol as the sodium lactate concentration increases (from 20mM to 40 mM).
FIG. 8 is an optical microscopic image of the Escherichia coli aggregate obtained in example 1, and in order to verify the versatility of the method, a bacterial aggregate was prepared using Escherichia coli. The overall morphology of the aggregates is circular, and the figures show that the overall morphology of the aggregates is circular and independent of each other, and escherichia coli is uniformly encapsulated in the hydrogel microspheres.
FIG. 9 is an optical microscopic image of the E.coli aggregates compacted further in calcium chloride solution obtained in example 1, compared to the uncompacted compacted aggregates, the E.coli aggregates compacted further in calcium chloride solution are darker in color, the bacteria contact inside the aggregates is tighter, and the stability of the whole aggregates is improved due to the further cross-linking of calcium ions, so as to be used for long-term hydrogen production.
FIG. 10 is a graph showing hydrogen production of the Escherichia coli aggregates obtained in example 1, example 4 and example 5 under different glucose concentrations, and it can be seen that the cumulative hydrogen production of the large intestine straw aggregates in 11 days (264 hours) is further increased from 10.20. mu. mol to 10.90. mu. mol to 21.19. mu. mol as the glucose concentration is increased (from 20mM, 50mM further to 100 mM).
Claims (10)
1. A hydrogen production method based on hydrogel-coated bacteria aggregation is characterized in that: the method comprises the following specific steps:
step one, preparation of Shewanella aggregates:
(1) dissolving 100-150 mul of span 80 into 40ml of liquid paraffin to be used as an oil phase;
(2) dissolving 15-30 mg of calcium chloride in 200 mul of deionized water to serve as a water phase A;
(3) mixing 1-2% of sodium alginate solution with bacterial liquid containing hydrogenase to serve as a water phase B, wherein the volume ratio of the sodium alginate solution to the bacterial liquid is 1: 1-1: 5;
(4) mixing 200 mu l of the water phase A and 6ml of the oil phase in a centrifuge tube, shaking uniformly to obtain a water-in-oil emulsion, standing for 1-5 minutes, adding the water phase B into the centrifuge tube, shaking uniformly, standing for 30min, and obtaining a bacterial aggregate at the bottom of the centrifuge tube, wherein the volume ratio of the water phase B to the oil phase is 1: 1-1: 5;
(5) centrifuging, sequentially washing 3 times by using 1-3% of Tween 80 aqueous solution and deionized water, completely transferring the aggregate to a water phase to obtain an aggregate precipitate, dispersing the aggregate precipitate into 10ml of 2-5 mg/ml calcium chloride solution, compacting by compaction, wherein the density of thalli in the aggregate is controlled by controlling the concentration of bacterial liquid;
step two, long-time hydrogen production of aggregate
Centrifuging the aggregate, washing for 2 times by using deionized water to obtain an aggregate precipitate, adding a solution containing a corresponding substrate, transferring the solution into a thick-walled mouth small bottle with an air suction joint, sealing by using a sealing film for long-time hydrogen production, centrifuging and washing the aggregate, compacting and compacting in a calcium chloride solution again and supplementing the corresponding substrate when the substrate is exhausted and the aggregate stops producing hydrogen, wherein the compacting conditions are as follows: dispersing the bacterial aggregates into 10ml of 2.5mg/ml calcium chloride solution, and reacting for 2-5 h under magnetic stirring at the rotating speed of 100-250 rpm; the ratio of aggregate precipitate to substrate solution was 310 mg: 3 mL.
2. The method for producing hydrogen based on hydrogel-coated bacteria aggregation as claimed in claim 1, wherein: in the first step, the bacterium liquid is a Shewanella bacterium liquid, an Escherichia coli bacterium liquid or a mixed bacterium liquid of Escherichia coli and Shewanella.
3. The method for producing hydrogen based on hydrogel-coated bacteria aggregation as claimed in claim 1 or 2, wherein: in the second step, substrate components and concentrations of the three aggregates are different, and for the Shewanella aggregates, a substrate solution contains 10-40 mM of sodium lactate and 10-40 mM of sodium thiosulfate; for the Escherichia coli aggregate, the substrate solution contains 10-40 mM of glucose and 0.5-2 mM of cysteine; for the aggregate of mixed bacteria, the substrate solution contains 10-40 mM of glucose, 0.5-2 mM of cysteine, 10-40 mM of sodium formate and 10-40 mM of sodium thiosulfate.
4. The method for preparing hydrogel-based bacterial aggregates according to claim 3, wherein: in the second step, for Shewanella aggregates, the substrate solution contains 20mM sodium lactate and 10mM sodium thiosulfate; for E.coli aggregates, the substrate solution contained 20mM glucose and 1mM cysteine; for aggregates of mixed bacteria, the substrate solution contained 20mM glucose, 1mM cysteine, 20mM sodium formate, 10mM sodium thiosulfate.
5. The method for preparing hydrogel-based bacterial aggregates according to claim 1, wherein: in the first step, the dosage of the span 80 is 110 mu l; the dosage of the calcium chloride is 25 mg; the concentration of the sodium alginate solution is 1 percent.
6. The method for preparing hydrogel-based bacterial aggregates according to claim 1, wherein: in the first step, the volume ratio of the sodium alginate solution to the bacterial liquid is 1: 1.
7. the method for preparing hydrogel-based bacterial aggregates according to claim 1, wherein: in the step one (4), firstly, mixing the water phase A and 6ml of oil phase in a centrifuge tube, shaking uniformly to obtain a water-in-oil emulsion, and standing for 3 minutes; adding the water phase B into the mixture and shaking up, wherein the volume ratio of the water phase B to the oil phase is 1: 3; in the step one (5), the rotating speed of the centrifugation is 2000-5000 rpm, and the time of each centrifugation is 2-5 minutes.
8. The method for preparing hydrogel-based bacterial aggregates according to claim 1 or 7, wherein: in the first step, through centrifugation, respectively washing 3 times with 1% Tween 80 aqueous solution and deionized water, the rotating speed of each centrifugation is controlled at 3000rpm, and the time of each centrifugation is controlled at 3 minutes.
9. The method for preparing hydrogel-based bacterial aggregates according to claim 1, wherein: in step one, the aggregate transferred to the aqueous phase is further compacted in 10ml of 2.5mg/ml calcium chloride solution.
10. The method for preparing hydrogel-based bacterial aggregates according to claim 1, wherein: in the second step, the compaction conditions are as follows: the bacterial aggregates were dispersed in 10ml of 2.5mg/ml calcium chloride solution and reacted for 3h with magnetic stirring at 200 rpm.
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