CN111040964A - Method for culturing mixed bacteria of hydroxide bacteria by electrolyzing water - Google Patents
Method for culturing mixed bacteria of hydroxide bacteria by electrolyzing water Download PDFInfo
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- CN111040964A CN111040964A CN201911282622.7A CN201911282622A CN111040964A CN 111040964 A CN111040964 A CN 111040964A CN 201911282622 A CN201911282622 A CN 201911282622A CN 111040964 A CN111040964 A CN 111040964A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
- C25B—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES FOR THE PRODUCTION OF COMPOUNDS OR NON-METALS; APPARATUS THEREFOR
- C25B1/00—Electrolytic production of inorganic compounds or non-metals
- C25B1/01—Products
- C25B1/02—Hydrogen or oxygen
- C25B1/04—Hydrogen or oxygen by electrolysis of water
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
- Y02E60/30—Hydrogen technology
- Y02E60/36—Hydrogen production from non-carbon containing sources, e.g. by water electrolysis
Abstract
The invention provides a method for culturing mixed bacteria of hydroxide bacteria by electrolyzing water, which comprises the following steps: s1: filling a culture medium into an electrolytic cell, inserting an anode and a cathode of an electrolytic device, electrifying the electrolytic device, and introducing air into the culture medium; s2: the pre-enriched mixed bacteria of the hydroxide bacteria are inoculated in the culture medium for culture, hydrogen generated by water in the electrolytic culture medium can be directly used as a hydrogen source for the mixed bacteria growth of the hydroxide bacteria, the danger caused by using a gas cylinder is avoided, and the advantages of low energy consumption and environmental protection are realized.
Description
Technical Field
The invention relates to a method for culturing mixed bacteria of hydroxide bacteria by using electrolyzed water, belonging to the technical field of microorganisms.
Background
Hydrogen Oxidizing Bacteria (HOB) are a class of bacteria that can utilize hydrogen, oxygen and carbon dioxide. The value-added products of HOB include Polyhydroxyalkanoates (PHA), proteins or plant growth promoters. The nitrogen source of HOB is ammonia or nitrate, unlike ammonia oxidizing bacteria and hydrogenophilic denitrifying bacteria. It has been reported that Cupriavidus eutrophus B-10646(HOB pure bacterium) can synthesize Polyhydroxyalkanoate (PHA) in a dry cell weight of 85%. The promotion effect of Burkholderia spp (HOB pure culture) on the root system elongation of spring wheat seedlings is improved from 57% to 254%. However, few studies have been made on the hybrid HOB, and recently it has been reported that hybrid HOB cultures can produce up to 71% of the protein by weight of their stem cells. It has also been reported that Achromobacter vetteriservae sp.for protein production was isolated from HOB mixed culture. The mixed bacteria is superior to pure bacteria, and has the features of no disinfection, simple operation and low cost. Therefore, the mixed bacteria have more potential in the aspect of production of value-added products than pure bacteria.
The hydrogen required by the mixed bacteria growth of the hydroxide bacteria is usually sourced from a gas cylinder, but the solubility of the hydrogen is low, and the hydrogen exceeds a threshold concentration to cause explosion, so that a new hydrogen supply mode for the mixed bacteria growth of the hydroxide bacteria is urgently required.
Disclosure of Invention
The invention provides a method for culturing mixed bacteria of hydroxide bacteria by using electrolyzed water, which can effectively solve the problems.
The invention is realized by the following steps:
the invention provides a method for culturing mixed bacteria of hydroxide bacteria by electrolyzing water, which comprises the following steps:
s1: filling a culture medium into an electrolytic cell, inserting an anode and a cathode of an electrolytic device, electrifying the electrolytic device, and introducing air into the culture medium;
s2: inoculating the previously enriched mixed bacteria of the hydroxide bacteria into the culture medium for culture.
As a further improvement, the electrolytic cell is a glass container, and the working volume is 400-500 mL.
As a further improvement, the cathode is a circular carbon felt with the area of 6-8 cm2, and the anode is a titanium mesh coated with iridium and ruthenium.
As a further improvement, the distance between the anode and the cathode is 3-5 cm.
As a further improvement, the method also comprises the step of continuously stirring the culture medium in the process of culturing the mixed bacteria of the hydroxide bacteria, wherein the stirring speed is 110-130 r/min.
The formula of the culture medium is further improved to be KH2PO 42200-2400 mg/L, Na2HPO412H2O 2800-3000 mg/L, MgSO47H 2O-550 mg/L, CaCl 26-10 mg/L, MnCl 24H 2O 0.004-0.006 mg/L, NaVO 3H 2O-6 mg/L, NaHCO 3450-550 mg/L, ZnSO 47H2O 0.4-0.6 mg/L, H3BO 23-2 mg/L, CoCl 26H 2O.5-1.5 mg/L, CuCl 22H 2O 0.04-0.06 mg/L, NiCl 26H2O0.05-0.15 mg/L, Na2MoO 42H 2O.1-0.2 mg/L, riboflavin 0.4-0.6 mg/L, NiCl 26H2O0.05-0.15 mg/L, Ca 2 Btoxin 3-0.0005-0.003 mg/L, Ca 2 Btoxin 3-0.3-0.5 mg/L, and Ca 2 Btoxin 3-0.3 mg/L.
As a further improvement, the temperature of the culture medium is 18-22 ℃, and the pH value is 6.8-7.2.
As a further improvement, the flow rate of the air is 140-160 mL/min.
As a further improvement, the electrified current density is 1.0-5.0 mA/cm 2.
As a further improvement, the enrichment method of the previously enriched bacterial hydroxide mixture comprises the following steps:
a: taking sludge, sieving with an iron sieve, suspending with a culture medium, uniformly mixing by oscillation, centrifuging to remove supernatant, and repeating the above operations for a plurality of times;
b: inoculating the sludge obtained in the step S1 into a culture bottle, adding a culture medium, sealing the culture bottle, vacuumizing, introducing a mixed gas of hydrogen, oxygen and carbon dioxide into the culture bottle under the vacuum degree of-0.1 Mpa, putting the culture bottle into a constant-temperature incubator, culturing for 15-30 days at the temperature of 34-36 ℃ and the rotating speed of 110-130 rpm, and carrying out mixed bacteria enrichment on the hydroxide bacteria.
The invention has the beneficial effects that:
the method for culturing the mixed bacteria of the hydroxide bacteria by electrolyzing the water directly uses the hydrogen generated by the water in the electrolysis culture medium as the hydrogen source for the growth of the mixed bacteria of the hydroxide bacteria, avoids the danger caused by using a gas cylinder, and has the advantages of low energy consumption and environmental protection.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic view of an apparatus for performing an embodiment of the present invention to provide a method for culturing a mixed bacterial strain by electrolyzing water.
FIG. 2 is a statistical chart of the growth of the mixed bacteria of the hydroxide bacteria provided by the embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
Construction of an electrolytic water reactor: the electrolytic cell reactor is a 600mL glass bottle with a working volume of 450 mL. The cathode is a circular carbon felt with an area of 7cm2The anode is a titanium mesh coated with iridium and ruthenium. The distance between the two electrodes was 4 cm. Hydrogen was from the cathode, oxygen from the anode, and air (inlet flow 150 mL/min). The reactor was stirred with a magnetic stirrer at 120rpm, the temperature was controlled at 20. + -. 0.5 ℃ with a water bath and the pH was set to 7.0. + -. 0.2 by 1m NaOH and 1m HCl.
By using the above-mentioned electrolytic cellReactor, set different current densities: 1.43mA/cm2,2.14mA/cm2,2.86mA/cm2,and 4.29mA/cm2The quantity of the generated hydrogen is determined by gas chromatography, and the smooth operation of hydrogen production by water electrolysis is ensured.
Adding pre-enriched mixed bacteria of hydroxide bacteria into the electrolytic water reactor at 20 + -0.5 deg.C and 120rpm to obtain KH culture medium2PO42300mg/L,Na2HPO412H2O 2900mg/L,MgSO47H2O 500mg/L,CaCl28mg/L,MnCl24H2O 0.005mg/L,NaVO3H2O 5mg/L,NaHCO3500mg/L,ZnSO47H2O 0.5mg/L,H3BO31.5mg/L,CoCl26H2O 1mg/L,CuCl22H2O 0.05mg/L,NiCl26H2O 0.1mg/L,Na2MoO42H2O 0.15mg/L,riboflavin 0.5mg/L,thiamine-HCl 2H2O 2.5mg/L,nicotinic acid2.5mg/L,pyridoxine-HCl 2.5mg/L,Ca-pantothenate 2.5mg/L,biotin 0.001mg/L,folicacid 0.002mg/L,vitaminB120.002mg/L。
Adding 1mol/L hydrochloric acid into a closed column type container to adjust the pH value of the culture medium to about 7.0, determining the amount of generated hydrogen by gas chromatography during the operation of the process, detecting the concentration of a nitrogen source by an ultraviolet spectrophotometer, and determining the activity of mixed bacteria of the hydroxide bacteria by ATP enzyme activity.
The enrichment method of the mixed bacteria of the hydroxide bacteria comprises the following steps:
sludge from a secondary sedimentation tank of a sewage treatment plant of a synthetic manure hope pond is used as an inoculation source, and the sludge is sieved by a 200-mesh iron sieve to remove non-biological impurities. Suspending the sludge by using a culture medium, uniformly mixing by vortex oscillation, placing the mixture in a centrifuge at 8000rpm for 5 minutes, centrifuging, and removing supernatant; the steps are repeated for 3 times, and organic matters and other interference result factors are removed. The washed sludge was inoculated into a 120mL serum bottle while 20mL of medium was added to ensure that the amount of added microorganisms was around 1-2 g/L. The serum bottle was sealed with a butyl rubber stopper and an aluminum cap. The air at the top of the serum bottle is pumped out by a vacuum pump, so that the air pressure of the headspace is-0.1 Mpa. 100mL (65%/20%/15% hydrogen/oxygen/carbon dioxide) of a mixed gas was introduced. The serum bottle is placed in a constant temperature incubator (temperature: 30 ℃, rotating speed: 120rpm) for culture, and the bacterial mixture of the hydroxide is enriched for 25 days.
The hydrogen test method comprises the following steps:
the hydrogen gas was detected by a gas chromatograph (Lunan, model: sp7890) thermal conductivity detector. The optimal working conditions of the gas chromatography are that a 5A molecular sieve packed column and a Thermal Conductivity Detector (TCD) are adopted, the flow rate of carrier gas is 35-42 m L/m in, the temperature of the chromatographic column is 100 ℃, the temperature of a vaporization chamber is 120 ℃, the temperature of a detection chamber is 100 ℃, and the bridge current is set to be +50 mA. The sample injector is used for injecting 0.5 mL. And (3) preparing a standard curve: respectively collecting 0.5mL, 0.4mL, 0.3mL, 0.2mL and 0.1mL of 100% pure hydrogen by using a sample injector, injecting the pure hydrogen into a gas chromatograph, enabling the gas chromatograph to generate a hydrogen peak within 1min, and recording corresponding peak areas. The peak area corresponds to the proportion of hydrogen. Sample detection: and injecting 0.5mL of sample into the sample injector, recording the peak area within 1min, substituting the peak area into a standard curve, and calculating the hydrogen ratio.
The testing method of the nitrate nitrogen comprises the following steps:
1. test range: 0.08-2mg/L
2. Preparing nitrate standard stock solution (100mg/L nitrate solution 100ml)
(1) 0.0607g of sodium nitrate (NaNO3) were weighed out and the four digit balance was turned on for 30 minutes. And folding the weighing paper in half, putting the weighing paper into a balance, closing all glass windows of the balance, and keeping the scale display stable and pressing to 0. Then, the window on the right side is opened, sodium nitrate is slowly added to the weighing paper by using an iron spoon, and the scale reading is observed until the required value is displayed after the window is closed.
(2) Adding pure water to a clean beaker to be close to 30ml, adding weighed reagents into the beaker, stirring by using a glass rod to uniformly mix the reagents (the reagents cannot be seen at the bottom of the beaker), washing by using the pure water for three times, pouring the reagents into a 100ml volumetric flask in sequence, and adding the pure water to a constant volume of 100 ml.
3. Preparing nitric acid solutions with different concentrations
(1) Preparing 6 10ml colorimetric tubes, marking each colorimetric tube, and then respectively adding 0, 0.025, 0.05, 0.10, 0.15 and 0.2ml nitrate nitrogen standard stock solutions.
(2) Dilute to the reticle with pure water (achieve head-up level until the lowest point of the page is at the reticle). The concentrations of the solutions at this time were 0, 0.25, 0.50, 1, 1.5 and 2mg/L nitric acid solutions, respectively. (0.25 × 10 ═ n × 100 → n ═ 0.025; 0.5 × 10 ═ n × 100 → n ═ 0.05).
4. Color developing liquid
(1) To each cuvette, 0.2ml (200. mu.l) of 1mol/L hydrochloric acid and 0.02ml (20. mu.l) of a 0.8% sulfamic acid solution were added, respectively; the cuvette lid was covered and shaken evenly.
(3) The absorbance was measured at wavelengths of 220 and 275nm using quartz cuvettes.
5. Making a standard curve
6. And (4) measuring the absorbance of the sample according to the same method, and calculating the content of the nitrate nitrogen.
The ATPase assay was determined using Solarbio's Na + K + -ATPase activity detection kit, cat # BC 0065.
As shown in FIG. 2, it can be seen from FIG. 2 that the method of the present invention can directly use hydrogen generated by electrolyzing water in the culture medium to culture mixed bacteria of hydroxide bacteria, the nitrate nitrogen utilization rate, energy efficiency and ATPase activity of the mixed bacteria of hydroxide bacteria vary with different current densities, and the optimal current density is 2.14mA/cm2。
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for culturing mixed bacteria of hydroxide bacteria by electrolyzing water is characterized in that: the method comprises the following steps:
s1: filling a culture medium into an electrolytic cell, inserting an anode and a cathode of an electrolytic device, electrifying the electrolytic device, and introducing air into the culture medium;
s2: inoculating the previously enriched mixed bacteria of the hydroxide bacteria into the culture medium for culture.
2. The method for culturing mixed bacteria of hydroxide bacteria by electrolyzing water according to claim 1, wherein: the electrolytic cell is a glass container, and the working volume is 400-500 mL.
3. The method for culturing mixed bacteria of hydroxide bacteria by electrolyzing water according to claim 1, wherein: the cathode is a round carbon felt with an area of 6-8 cm2The anode is a titanium mesh coated with iridium and ruthenium.
4. The method for culturing mixed bacteria of hydroxide bacteria by electrolyzing water according to claim 3, wherein: the distance between the anode and the cathode is 3-5 cm.
5. The method for culturing mixed bacteria of hydroxide bacteria by electrolyzing water according to claim 1, wherein: and continuously stirring the culture medium in the process of mixed culture of the hydroxide bacteria at a stirring speed of 110-130 r/min.
6. The method for culturing mixed bacteria of hydroxide bacteria by electrolyzing water according to claim 1, wherein: the formula of the culture medium is KH2PO42200～2400mg/L，Na2HPO412H2O 2800～3000mg/L，MgSO47H2O 450～550mg/L，CaCl26～10mg/L，MnCl24H2O0.004～0.006mg/L，NaVO3H2O 4～6mg/L，NaHCO3450～550mg/L，ZnSO47H2O 0.4～0.6mg/L，H3BO31～2mg/L，CoCl26H2O 0.5～1.5mg/L，CuCl22H2O0.04～0.06mg/L，NiCl26H2O 0.05～0.15mg/L，Na2MoO42H2O0.1～0.2mg/L，riboflavin 0.4～0.6mg/L，thiamine-HCl 2H2O 2～3mg/L，nicotinic acid 2～3mg/L，pyridoxine-HCl 2～3mg/L，Ca-pantothenate 2～3mg/L，biotin 0.0005～0.0015mg/L，folic acid 0.001～0.003mg/L，vitaminB120.001～0.003mg/L。
7. The method for culturing mixed bacteria of hydroxide bacteria by electrolyzing water according to claim 6, wherein: the temperature of the culture medium is 18-22 ℃, and the pH value is 6.8-7.2.
8. The method for culturing mixed bacteria of hydroxide bacteria by electrolyzing water according to claim 1, wherein: the flow rate of the air is 140-160 mL/min.
9. The method for culturing mixed bacteria of hydroxide bacteria by electrolyzing water according to claim 1, wherein: the electrified current density is 1.0-5.0 mA/cm2。
10. The method for culturing mixed bacteria of hydroxide bacteria by electrolyzing water according to claim 1, wherein: the enrichment method of the pre-enriched mixed bacteria of the hydroxide bacteria comprises the following steps:
a: taking sludge, sieving with an iron sieve, suspending with a culture medium, uniformly mixing by oscillation, centrifuging to remove supernatant, and repeating the above operations for a plurality of times;
b: inoculating the sludge obtained in the step S1 into a culture bottle, adding a culture medium, sealing the culture bottle, vacuumizing, introducing a mixed gas of hydrogen, oxygen and carbon dioxide into the culture bottle under the vacuum degree of-0.1 Mpa, putting the culture bottle into a constant-temperature incubator, culturing for 15-30 days at the temperature of 34-36 ℃ and the rotating speed of 110-130 rpm, and carrying out mixed bacteria enrichment on the hydroxide bacteria.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268391A (en) * | 2011-07-09 | 2011-12-07 | 西北大学 | Hydrogen-oxidizing bacteria WMQ-7, and separation method and application thereof |
| CN109735474A (en) * | 2019-03-04 | 2019-05-10 | 西北大学 | One plant of hydrogen-oxidizing bacterium zw-26 and its separation method and application with growth-promoting functions |
| CN109880757A (en) * | 2019-03-04 | 2019-06-14 | 西北大学 | One plant of hydrogen-oxidizing bacterium and its separation method and application with itself nitrogen fixing capacity |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268391A (en) * | 2011-07-09 | 2011-12-07 | 西北大学 | Hydrogen-oxidizing bacteria WMQ-7, and separation method and application thereof |
| CN109735474A (en) * | 2019-03-04 | 2019-05-10 | 西北大学 | One plant of hydrogen-oxidizing bacterium zw-26 and its separation method and application with growth-promoting functions |
| CN109880757A (en) * | 2019-03-04 | 2019-06-14 | 西北大学 | One plant of hydrogen-oxidizing bacterium and its separation method and application with itself nitrogen fixing capacity |
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| GEORGYGIVIROVSKIY等: "Electrode material studies and cell voltage characteristics of the in situ water electrolysis performed in a pH-neutral electrolyte in bioelectrochemical systems", 《HELIYON》 * |
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Application publication date: 20200421 |