CN114410617A - Immobilization method for improving hydrogen production bacteria biological hydrogen synthesis and application - Google Patents
Immobilization method for improving hydrogen production bacteria biological hydrogen synthesis and application Download PDFInfo
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- 239000001257 hydrogen Substances 0.000 title claims abstract description 116
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- 238000004519 manufacturing process Methods 0.000 title claims abstract description 49
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- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- B—PERFORMING OPERATIONS; TRANSPORTING
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Abstract
The invention discloses an immobilization method for improving hydrogen production bacteria biological hydrogen synthesis and application thereof, and relates to a bacterial agent solidThe technical field of customization mainly comprises: synthesizing nano magnetite by using raw peach gum as a raw material; preparing the peach gum into a solution, adding 0.5 times of sodium alginate by mass, uniformly mixing the sodium alginate with the nano magnetite and hydrogen-producing bacteria suspension synthesized by peach gum with certain concentration, and dropwise adding the mixture to CaCl2In the solution, immobilized gel beads are formed. The immobilized gel beads are applied to a lignocellulose hydrolysate fermentation hydrogen production system, so that the number of viable bacteria of the system can be increased, the accumulated hydrogen production of 120h fermentation can be improved by about 60%, and the utilization rate of glucose and xylose in hydrolysate can be obviously improved. According to the invention, the nano magnetite prepared by taking the raw peach gum as the raw material is used as the additive, and the raw peach gum is used as the main immobilized carrier to prepare the high-efficiency immobilized bacterial agent for hydrogen-producing bacteria, so that the method is simple and convenient, the conditions are mild, the stability is good, the survival rate of the bacteria is high, and the hydrogen production promoting effect is obvious.
Description
Technical Field
The invention relates to a preparation method and application field of a novel coupled nano material immobilizing agent, in particular to a preparation method and application of a coupled peach gum immobilizing agent for synthesizing nano magnetite by taking raw peach gum as a raw material, and especially relates to the effect of the novel immobilizing agent in regulating and controlling the synthesis of hydrogen-producing bacteria biological hydrogen, and the novel coupled nano material immobilizing agent is applied to the field of efficient synthesis and regulation of biological hydrogen energy.
Background
The immobilized hydrogen-producing bacteria cell technology has become an important technology for improving the hydrogen-producing capability of bacteria by increasing the system stability, expanding the activity range of hydrogen-producing bacteria, improving the pH change tolerance capability of the system and the like. The method of immobilizing cells can be classified into an embedding method, a system entrapment method, an adsorption method and a cross-linking method according to the difference of an immobilization carrier and an action mode. The embedding method is the most commonly used method for cell immobilization, and the embedding carrier mainly utilizes natural high molecular polysaccharides and synthetic high molecular compounds. The agar, calcium alginate and carrageenan of natural macromolecular polysaccharide are most applied, and have the advantages of convenient immobilization and formation, low toxicity to microorganisms, high immobilization density and the like, but have poor antimicrobial decomposition performance and lower mechanical strength. The synthetic high molecular compound mainly utilizes polyacrylamide, polyvinyl alcohol, photohardening resin and the like, and has the outstanding advantages of good antimicrobial decomposition performance, high mechanical strength and stable chemical performance, but the formation condition of a polymer network is severe, the damage to microbial cells is large, and the forming diversity and controllability are poor. For this reason, the development of novel immobilizing agents is crucial to effectively increase the bio-hydrogen yield of hydrogen-producing bacteria.
Peach gum is a colloidal substance secreted from bark of Prunus persica or Prunus davidiana belonging to Rosaceae. Peach gum without any treatment is generally called raw peach gum, which is peach red or a translucent solid block from light yellow to yellow brown and has smooth appearance. The original peach gum has large molecular weight and better adsorption performance, and is generally used as a natural capsule wall material to prepare hydrogel for embedding medicaments because the original peach gum is not decomposed by gastric juice in medicine; in the food industry, the microcapsule can be prepared to be used for embedding health care products such as fish oil and the like. Therefore, the original peach gum has the basic characteristic of being used as an embedding carrier, and is expected to be used as a novel immobilization carrier.
In recent years, the addition of nanoparticles at a certain concentration to a fermentative hydrogen production system is effective in promoting the increase of the yield of biohydrogen, and it has been reported that the surface effect and quantum size effect of nanoparticles can improve the enzymatic activity thereof by accelerating the transfer of electrons from NADPH to catalase, thereby promoting the synthesis of biohydrogen. For this reason, the addition of nanoparticles has become an important regulatory means for promoting the biosynthesis of biohydrogen. Especially, the green synthesized nanoparticles with natural plants and extracts thereof as raw materials are added into a fermentation hydrogen production system, so that a good hydrogen production promoting effect can be achieved, and the green method for synthesizing the nanoparticles has the advantages of mild conditions, simple preparation process, low cost and the like, and is gradually an important nanoparticle synthesis mode at present. The peach gum is a natural raw material, and the chemical components of the peach gum mainly comprise peach gum polysaccharide, common amino acids such as threonine and histidine, and trace elements such as K, Ca, Mg, Fe and Mn. Therefore, the peach gum is used as a raw material, can also be used for green synthesis of nano particles, and is used as a hydrogen production promoter to be added into a fermentation hydrogen production system.
In conclusion, the adoption of the immobilization technology and the addition of the nano particles are effective measures for promoting the biological hydrogen synthesis of the hydrogen-producing bacteria. The immobilized coupling nano-particle addition based on the raw peach gum as the raw material is expected to develop a hydrogen-producing bacteria immobilization mode which has low cost and high efficiency and promotes the synthesis of the biological hydrogen, and aims to solve the key technical problem of low biological hydrogen production efficiency.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: the invention aims to provide an immobilization method for immobilizing hydrogen-producing bacteria cells, which has the characteristics of high immobilization efficiency, strong activity of hydrogen-producing bacteria microbial inoculum, good stability, convenient and controllable formation and the like, and can improve the biological hydrogen synthesis amount, the bacterial survival rate and the utilization rate of lignocellulose hydrolysate glucose and xylose of the hydrogen-producing bacteria.
In order to solve the technical problems, the invention provides the following technical scheme:
an immobilization method for improving hydrogen production bacteria biological hydrogen synthesis comprises the following specific steps:
(A) weighing 4g of peach gum powder, adding into 100mL of distilled water, and placing in a water bath kettle at 80 ℃ for constant temperature treatment for 8h to ensure that the peach gum powder is completely swelled to form peach gum liquid; properly cooling the obtained peach gum solution, adding 2g of sodium alginate, fully and uniformly mixing, and sterilizing to obtain an original peach gum/sodium alginate solution;
(B) taking hydrogen-producing bacteria growing to logarithmic phase, and mixing according to 0.5gThallus/10mLRaw peach gum/sodium alginate solutionAdding the mixture into an original peach gum/sodium alginate solution in proportion, uniformly mixing, adding sterile green synthetic nano magnetite with a certain concentration (0-50 mg/L), uniformly dropping the nano magnetite into a sterile calcium chloride solution by using a sterile medical syringe, calcifying for 1-2 hours after gel beads are formed, discarding redundant calcium chloride solution, and washing by using sterile normal saline to obtain the immobilized gel beads.
Preferably, the hydrogen-producing bacteria is one or more of enterobacter cloacae, klebsiella hydrogen-producing and klebsiella hydrogen-producing engineering bacteria.
Preferably, the preparation method of the hydrogen-producing klebsiella bacterial cells growing to logarithmic phase comprises the following steps: picking activityThe method comprises the following steps of (1) overnight culturing a single hydrogen-producing Klebsiella in a liquid culture medium, further centrifuging at a low speed, removing a supernatant, collecting thalli, washing for 2-3 times by using normal saline, removing the supernatant, collecting the thalli, and preparing immobilized gel beads; the formula of the liquid culture medium is as follows: 10g of D-xylose, 10g of glucose, 5g of beef extract, 10g of peptone, 5g of NaCl and KH2PO4 0.5g，MgSO4·7H2O1 g, pH7.5, 1000mL of water.
Preferably, the sterile green synthetic nano magnetite is prepared by sterilizing nano magnetite synthesized by raw peach gum at 121 ℃ for 20 min; the sterile calcium chloride solution is prepared by sterilizing 10g/L calcium chloride aqueous solution at 121 ℃ for 20 min.
Preferably, the nano magnetite synthesized from the raw peach gum is prepared by the following method:
(1) weighing 1g of peach gum powder, adding into 100mL of distilled water, and placing in a water bath kettle at 80 ℃ for constant temperature treatment for 8h to ensure that the peach gum powder is completely swelled to form peach gum liquid; further adjusting the pH value to 10-11, placing the mixture on a constant-temperature magnetic stirrer, heating and stirring the mixture for 3 hours at 85 ℃, then cooling the mixture to 50 ℃, and adding 0.5mol/L Fe with a certain volume into the peach gum solution3+And 0.75mol/L Fe2+Solution of Fe in the mixed solution3+：Fe2+2: 1, placing the mixture on a constant-temperature magnetic stirrer, and continuously stirring the mixture for 4 hours at the temperature of 60 ℃;
(2) cooling the reaction liquid to room temperature, slowly adding a newly prepared 2mol/L NaOH solution while stirring until all precipitates are changed into black, and continuously stirring at 80 ℃ for reaction for 1 h;
(3) after the reaction is finished, standing and cooling the reaction solution obtained in the step (2), separating the obtained nano particles by using a permanent magnet, washing the nano particles for 3 times by using absolute ethyl alcohol and distilled water respectively, centrifuging, collecting precipitates, and drying the precipitates in a constant-temperature drying box at 70 ℃ to constant weight; and taking out the dried substances, grinding and sieving by a 200-mesh sieve to obtain the green peach gum synthesized nano magnetite.
Preferably, the Fe3+The salt is ferric chloride; said Fe2+The salt is ferrous sulfate.
Application of the immobilized gel beads in hydrogen production by fermentation, wherein the immobilized gel beads are used for preparing hydrogenThe gel beads are quantified according to a certain inoculation amount (0.1 g)Thallus/100mLCulture mediumAccording to the amount of thalli) is inoculated into a straw hydrolysate fermentation medium, the hydrogen volume, the glucose and xylose concentrations and the number of live cells of hydrogen-producing bacteria are detected regularly, and the total fermentation time is 120 h; the straw hydrolysate fermentation medium comprises the following components in percentage by weight: 1000mL of straw hydrolysate, 5g of beef extract, 10g of peptone, 5g of NaCl and KH2PO4 0.5g，MgSO4·7H2O0.5 g, sugar concentration 50g/L, pH 8.0.
The invention has the following beneficial effects:
1) the method adopts the original peach gum as the raw material to synthesize the nano magnetite and takes the nano magnetite as the main immobilized carrier to be used for the bacterial fermentation hydrogen production system, and has the advantages of high raw material yield, low cost, simple operation steps, mild reaction conditions, easy control, safety and environmental protection.
2) The immobilized gel beads added by green synthetic nano magnetite are coupled by taking the original peach gum as a main immobilized carrier, and have the advantages of convenient formation, good stability, high immobilization efficiency, strong activity of hydrogen-producing bacterial agent, high bacterial survival rate and high hydrogen-producing efficiency.
3) The immobilized gel beads prepared by the invention are inoculated in a hydrogen-producing bacteria fermentation hydrogen-producing system, and have the effects of remarkably promoting the synthesis of biological hydrogen and the utilization of reducing sugar in straw hydrolysate, so that the accumulated hydrogen yield, the utilization rate of glucose and the utilization rate of xylose obtained under the optimal immobilization condition are respectively and remarkably improved compared with the control treatment.
Drawings
FIG. 1 is a diagram showing the results of the characterization of the original peach gum of the present invention into nano-magnetite (a: XRD; b: FTIR; c: SEM; d: TEM).
FIG. 2 is a Scanning Electron Microscope (SEM) image of the immobilized gel beads of the present invention.
FIG. 3 is a graph of the cumulative hydrogen production of a single addition of nano-magnetite according to the present invention.
FIG. 4 shows the results of glucose and xylose utilization with single addition of nano-magnetite according to the present invention.
FIG. 5 is a graph of the cumulative hydrogen production of immobilized coupled nano-magnetite addition according to the present invention.
FIG. 6 shows the results of glucose and xylose utilization by immobilized coupled nano-magnetite addition according to the present invention.
FIG. 7 shows the results of monitoring the number of living cells by immobilization coupled with addition of nano-magnetite according to the present invention (Im: immobilization; NP: nanoparticles).
FIG. 8 is a graph showing the cumulative hydrogen production of Enterobacter cloacae with the addition of immobilized coupled nano-magnetite according to the present invention.
FIG. 9 is a cumulative hydrogen production curve of hycG-pET-28a-Klebsiella sp. (RT-H) engineering bacteria with the addition of immobilized coupled nano-magnetite according to the present invention.
Detailed Description
The following examples are included to provide further detailed description of the present invention and to provide those skilled in the art with a more complete, concise, and exact understanding of the principles and spirit of the invention.
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Example 1: the preparation of green synthetic nano magnetite from original peach gum comprises the following steps:
weighing 1g of peach gum powder, adding into 100mL of distilled water, and placing in a water bath kettle at 80 ℃ for constant temperature treatment for 8h to ensure that the peach gum powder is completely swelled to form peach gum liquid; further adjusting the pH value to 10-11, placing the mixture on a constant-temperature magnetic stirrer, heating and stirring the mixture for 3 hours at 85 ℃, then cooling the mixture to 50 ℃, and adding 0.5mol/L Fe with a certain volume into the peach gum solution3+And 0.75mol/L Fe2+Solution of Fe in the mixed solution3+：Fe2+2: 1, placing on a constant-temperature magnetic stirrer, and continuously stirring for 4 hours at the temperature of 60 ℃. Said Fe3+The salt is ferric chloride; said Fe2+The salt is ferrous sulfate. The reaction solution is cooled to room temperature, a newly prepared 2mol/L NaOH solution is slowly added while stirring until all the precipitate turns black, and then the reaction is continuously stirred at 80 ℃ for 1 hour. After the reaction is completed, the reaction solution is stood still and cooled, and the obtained nano particles are aligned by a permanent magnetSeparating the granules, washing with anhydrous ethanol and distilled water for 3 times respectively, centrifuging, collecting precipitate, and oven drying in a constant temperature drying oven at 70 deg.C to constant weight; taking out the dried substance, grinding, and sieving with 200 mesh sieve to obtain green synthetic nano magnetite from original peach gum. The characterization results of the nano magnetite are shown in fig. 1, and the diffraction peaks of the obtained nano magnetite at 2 theta, 30.2, 35.5, 43.2,57.3 and 62.8, corresponding to crystal faces (220), (311), (400), (511) and (440), are consistent with standard cards JCPDS No.19-0629, and are in spinel structure (fig. 1 (a)). The diffraction peak in the sample was sharper and there were no other diffraction peaks, indicating that it was purer. FTIR plots (FIG. 1(b)) show 637 and 472cm-1The peaks indicate the formation of Fe-O bonds, confirming the formation of nano-magnetite, and 3408 and 1626cm-1The peak of (a) indicates the presence of OH, possibly as a water residue indicated by the nano-magnetite; as can be seen from the SEM image (fig. 1(c)), the surface morphology of the nanoparticles is more consistent and the morphology is more regular; the TEM image (FIG. 1(d)) shows that the nanoparticles have a spherical structure with an average particle size of about 22 nm.
Example 2: preparing the original peach gum immobilized gel beads:
removing impurities such as bark from original peach gum collected from peach trees, air drying, and pulverizing into peach gum powder with a traditional Chinese medicine pulverizer for later use. Weighing 4g of peach gum powder, adding into 100mL of distilled water, and placing in a water bath kettle at 80 ℃ for constant temperature treatment for 8h to ensure that the peach gum powder is completely swelled to form peach gum liquid; and (3) properly cooling the obtained peach gum solution, adding 2g of sodium alginate, fully and uniformly mixing, and placing in a high-pressure steam sterilizer for sterilization at 121 ℃ for 20 min. Simultaneously picking activated Klebsiella oxytoca single colony in a liquid culture medium (formula: 10g of D-xylose, 10g of glucose, 5g of beef extract, 10g of peptone, 5g of NaCl and KH)2PO4 0.5g，MgSO4·7H2O1 g, pH7.5, water 1000mL) overnight, further centrifuging at low speed (4000r/min for 10min), discarding the supernatant, collecting the thallus, washing with normal saline for 2-3 times, discarding the supernatant, collecting the thallus, and using the thallus for preparing immobilized gel beads. The cell of the hydrogen-producing Klebsiella grows to logarithmic phase according to 0.5gThallus/10mLRaw peach gum/sodium alginate solutionAdding into original peach gum/sodium alginate solution at a certain proportion, mixing, and adding 121Adding the original green peach gum synthetic nano magnetite sterilized for 20min into the solution according to the concentration of 0, 10, 20, 30, 40 and 50mg/L, fully and uniformly mixing, dripping into a sterile 10g/L calcium chloride solution at a constant speed by using a sterilized medical injector, calcifying for 1-2 h after gel beads are formed, discarding the redundant calcium chloride solution, washing for 2-3 times by using sterilized normal saline, and using the washed immobilized gel beads for a subsequent fermentation hydrogen production test. After the obtained immobilized gel beads are sliced, the Scanning Electron Microscope (SEM) is used for observation, and the immobilized gel beads form a net structure with the aperture of 1.0-2.0 microns (average aperture of 1.56 microns), so that hydrogen-producing bacteria can be embedded in the net structure, and as shown in figure 2, the immobilized gel beads are rod-shaped Klebsiella.
Example 3: the green synthesis of the nano magnetite regulates and controls hydrogen production and reducing sugar utilization of the straw hydrolysate fermented by the Klebsiella:
the nano magnetite prepared from green peach gum is added into a straw hydrolysate fermentation hydrogen production system according to the concentration of 0, 10, 20, 30, 40 and 50mg/L, wherein the treatment of 0mg/L is control treatment (CK). Accurately weighing nano magnetite according to different treatments, adding into fermentation medium (formula: straw hydrolysate 1000mL, beef extract 5g, peptone 10g, NaCl 5g, KH)2PO4 0.5g，MgSO4·7H2O0.5 g, sugar concentration 50g/L, pH 8.0), effectively dispersing the nanoparticles in a fermentation medium by adopting a mode of fully stirring and ultrasonic treatment, inoculating Klebsiella seed liquid according to the inoculum size of 10 percent after sterilization and cooling, periodically detecting the hydrogen production by adopting a sodium hydroxide solution discharging method, accumulating day by day, detecting the accumulated hydrogen production within 120h, simultaneously monitoring the concentration change of glucose and xylose, and converting the utilization rate of reducing sugar, wherein the result is shown in attached figure 3 and attached figure 4. As can be seen from the attached figure 3, when the nano magnetite is added at a concentration of 20mg/L, the accumulated hydrogen production of the strain in the whole fermentation period is significantly higher than that of the control treatment (0mg/L) for 120 hours, the accumulated hydrogen production can reach 4470.8 +/-97.1 mL/L, and is improved by about 20% compared with that of the control treatment (0mg/L), namely the addition concentration treatment can promote the synthesis of the biological hydrogen of the strain to a certain extent. The utilization rates of glucose and xylose are respectively increased by 11.9% and 6.1% (figure 4).
Example 4: the hydrogen production and reducing sugar utilization of straw hydrolysate by adding immobilized coupling nano magnetite and regulating and controlling Klebsiella fermentation are as follows:
the immobilized gel beads prepared in example 2 were inoculated into a straw hydrolysate hydrogen production system by fermentation (same as in example 3), and the treatment without nanoparticles added and immobilized was selected as a control treatment (CK). And (3) periodically detecting the hydrogen production by adopting a sodium hydroxide solution discharging method, accumulating day by day, detecting the accumulated hydrogen production within 120h, simultaneously monitoring the concentration change of glucose and xylose, and converting the utilization rate of reducing sugar, wherein the result is shown in an attached figure 5 and an attached figure 6. As can be seen from figure 5, on the premise of immobilization, the addition concentration of the original green peach gum synthesized nano magnetite is 50mg/L, and the accumulated hydrogen production under the other addition concentrations is higher than that of the control treatment (CK), particularly when the addition concentration of the nano magnetite is 10mg/L, the accumulated hydrogen production of the strain fermented for 120h can reach 5898.4 +/-121.5 mL/L, which is improved by about 60% compared with that of the control group (CK), namely, the treatment can remarkably promote the synthesis of the biological hydrogen of the strain, and the utilization rates of glucose and xylose under the condition are respectively improved by 14.3% and 7.4% (figure 6). The present example also shows that immobilization is an effective hydrogen production promoting measure, and on the premise of immobilization, the added nano magnetite only needs a lower concentration (10mg/L) to achieve a significant hydrogen production promoting effect, and has a higher hydrogen production promoting potential than that of the nano magnetite added alone at an optimal concentration (20mg/L, example 3). And when the fermentation is finished within 120h, almost all the glucose and xylose in the straw hydrolysate fermentation medium are digested, and the utilization rates of the glucose and the xylose are respectively as high as 96.1 percent and 96.9 percent.
Example 5: the survival rate of the Klebsiella in the fermentation process can be improved by adding the immobilized coupling nano magnetite:
the treatment without nanoparticles added and without immobilization was selected as the control treatment (CK), the treatment with the optimal concentration of added nano magnetite (20mg/L) in example 3 was selected as the control treatment, the treatment with the optimal concentration of nano magnetite (immobilization +10mg/L nanoparticles) and the treatment with only immobilization (Im +0mg/L NP) in example 4 were selected, and the straw fermentation hydrolysate hydrogen production system was inoculated in the same manner as the immobilized gel bead inoculation method in example 4. The number of viable bacterial cells during the whole fermentation was monitored and the results are shown in FIG. 7. As can be seen from FIG. 7, the number of viable cells treated with Im +10mg/L was significantly higher than that of the Control (CK) during the whole fermentation period, and even though the number of viable cells decreased significantly to the middle and late stages of fermentation (72-96), the number of viable cells remained above the initial (0h) inoculated viable cell number. While the CK, 20mg/L NP treatment was lower than the initial number of viable cells at 96 hours from fermentation, and Im +0mg/L NP treatment was lower than the initial number of viable cells at 120 hours. Therefore, the immobilization treatment is beneficial to maintaining the activity of hydrogen-producing bacteria cells, so that the hydrogen-producing bacteria cells can maintain a higher living cell number level under the anaerobic fermentation condition, and particularly, the treatment effect of adding nanoparticles with proper concentration (10mg/L) is optimal.
Example 6 (comparative test example): the addition of the immobilized coupling nano magnetite can also promote the fermentation of enterobacter cloacae to produce hydrogen:
the immobilized gel beads are prepared by the same method as the example 2, the same treatment mode as the example 5 is selected, and the regulation and control effect of the addition of the immobilized coupling nano magnetite on the fermentation hydrogen production process of enterobacter cloacae, another type of hydrogen production bacteria, is detected, the result is shown in the attached figure 8, the result shows that the addition of the immobilized coupling nano magnetite can also promote the fermentation hydrogen production of the enterobacter cloacae, the accumulated hydrogen production processed by Im +10mg/L is the highest, and the accumulated hydrogen production of the fermentation 120h is improved by about 40 percent compared with the comparison processing (CK). The enterobacter cloacae for producing hydrogen in the embodiment is reported in the literature, "influence of segmented pH value regulation on the hydrogen production of the hydrolysis sugar solution of cotton stalk fermented by enterobacter cloacae WL 1318" (Daowei et al, 2018).
Example 7 (comparative test example): the addition of the immobilized coupling nano magnetite can also promote the biological hydrogen synthesis of hydrogen-producing engineering bacteria:
the immobilized gel beads are prepared by the same method as that of the embodiment 2, the same treatment mode as that of the embodiment 5 is selected, the regulation and control effect of the addition of the immobilized coupled nano magnetite on the fermentation hydrogen production process of a strain of hydrogen production engineering bacteria, hycG-pET-28a-Klebsiella sp (RT-H) is detected, the result is shown in the attached figure 8, the change of the curve of the accumulated hydrogen production amount in the figure shows that the addition of the immobilized coupled nano magnetite can also promote the biological hydrogen synthesis of the hydrogen production engineering bacteria, the accumulated hydrogen production amount treated by Im +10mg/L is the highest, and the accumulated hydrogen production amount fermented for 120H is improved by about 52 percent compared with that treated by Contrast (CK). The hydrogen-producing engineering bacteria hycG-pET-28a-Klebsiella sp (RT-H) in this example are reported in the literature "Klebsiella sp" optimization of metabolic pathways for synthesizing biological hydrogen by fermenting lignocellulose hydrolysate (Youngin, 2020).
The above embodiments are only for illustrating the technical idea of the present invention, and the protection scope of the present invention cannot be limited thereby, and any modification made on the basis of the technical scheme according to the technical idea proposed by the present invention falls within the protection scope of the present invention; the technology not related to the invention can be realized by the prior art.
Claims (7)
1. An immobilization method for improving the biological hydrogen synthesis of hydrogen-producing bacteria is characterized by comprising the following specific steps:
(A) weighing peach gum powder, adding into distilled water, and placing in a water bath kettle at 80 deg.C for constant temperature treatment for 8 hr to make the peach gum powder fully swell to form peach gum solution; properly cooling the obtained peach gum solution, adding sodium alginate, fully and uniformly mixing, and sterilizing to obtain an original peach gum/sodium alginate solution;
(B) adding hydrogen-producing bacteria growing to logarithmic phase into the original peach gum/sodium alginate solution according to the proportion of 0.5g of thallus to 10mL of the original peach gum/sodium alginate solution, uniformly mixing, adding sterile green synthetic nano magnetite with a certain concentration, dripping into the sterile calcium chloride solution at a constant speed by using a sterile medical injector, calcifying for 1-2 h after the gel beads are formed, discarding the redundant calcium chloride solution, and washing by using sterilized normal saline to obtain the immobilized gel beads.
2. The immobilization method for enhancing the biological hydrogen synthesis of hydrogen-producing bacteria according to claim 1, wherein: the hydrogen-producing bacteria are one or more of enterobacter cloacae, hydrogen-producing klebsiella and hydrogen-producing klebsiella engineering bacteria.
3. The immobilization method for enhancing the bio-hydrogen synthesis of hydrogen-producing bacteria according to claim 2, wherein: said raw material isThe preparation method of the hydrogen-producing Klebsiella cell with long log phase comprises the following steps: selecting an activated Klebsiella pneumoniae single colony to be cultured in a liquid culture medium overnight, further centrifuging at a low speed, removing supernate, collecting thalli, washing for 2-3 times by using normal saline, removing supernate, collecting thalli, and preparing immobilized gel beads; the formula of the liquid culture medium is as follows: 10g of D-xylose, 10g of glucose, 5g of beef extract, 10g of peptone, 5g of NaCl and KH2PO4 0.5g，MgSO4·7H2O1 g, pH7.5, 1000mL of water.
4. The immobilization method for enhancing the biological hydrogen synthesis of hydrogen-producing bacteria according to claim 1, wherein: the sterile green synthetic nano magnetite is prepared by sterilizing nano magnetite synthesized by raw peach gum at 121 ℃ for 20 min; the sterile calcium chloride solution is prepared by sterilizing 10g/L calcium chloride aqueous solution at 121 ℃ for 20 min.
5. The immobilization method for improving the biological hydrogen synthesis of hydrogen-producing bacteria according to claim 4, wherein the nano magnetite synthesized from the raw peach gum is prepared by the following method:
(1) weighing peach gum powder, adding into distilled water, and placing in a water bath kettle at 80 deg.C for constant temperature treatment for 8 hr to make the peach gum powder fully swell to form peach gum solution; further adjusting the pH value to 10-11, placing the mixture on a constant-temperature magnetic stirrer, heating and stirring the mixture for 3 hours at 85 ℃, then cooling the mixture to 50 ℃, and adding 0.5mol/L Fe with a certain volume into the peach gum solution3+And 0.75mol/L Fe2+Solution of Fe in the mixed solution3+：Fe2+2: 1, placing the mixture on a constant-temperature magnetic stirrer, and continuously stirring the mixture for 4 hours at the temperature of 60 ℃;
(2) cooling the reaction liquid to room temperature, slowly adding a newly prepared 2mol/L NaOH solution while stirring until all precipitates are changed into black, and continuously stirring at 80 ℃ for reaction for 1 h;
(3) after the reaction is finished, standing and cooling the reaction solution obtained in the step (2), separating the obtained nano particles by using a permanent magnet, washing the nano particles for 3 times by using absolute ethyl alcohol and distilled water respectively, centrifuging, collecting precipitates, and drying the precipitates in a constant-temperature drying box at 70 ℃ to constant weight; and taking out the dried substances, grinding and sieving by a 200-mesh sieve to obtain the green peach gum synthesized nano magnetite.
6. The immobilization method for enhancing the biosynthesis of hydrogen-producing bacteria according to claim 5, wherein the Fe is Fe3+The salt is ferric chloride; said Fe2+The salt is ferrous sulfate.
7. The application of the immobilized gel beads of any one of claims 1 to 6 in hydrogen production by fermentation, which is characterized in that: inoculating the immobilized gel beads into a straw hydrolysate fermentation medium according to a certain inoculation amount, periodically detecting the volume of hydrogen, the concentrations of glucose and xylose and the number of living cells of hydrogen-producing bacteria, and fermenting for 120 hours; the straw hydrolysate fermentation medium comprises the following components in percentage by weight: 1000mL of straw hydrolysate, 5g of beef extract, 10g of peptone, 5g of NaCl and KH2PO4 0.5g，MgSO4·7H2O0.5 g, sugar concentration 50g/L, pH 8.0.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113881710A (en) * | 2021-10-29 | 2022-01-04 | 安徽工程大学 | Method for co-producing biological hydrogen and microalgae grease by utilizing lignocellulose fermentation |
| CN113881710B (en) * | 2021-10-29 | 2023-09-22 | 安徽工程大学 | Method for co-production of biological hydrogen and microalgae grease by lignocellulose fermentation |
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