CN1332466C - Method for preparing bactermiorhodopsin proton exchange film of fuel battery - Google Patents
Method for preparing bactermiorhodopsin proton exchange film of fuel battery Download PDFInfo
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- CN1332466C CN1332466C CNB2005101142138A CN200510114213A CN1332466C CN 1332466 C CN1332466 C CN 1332466C CN B2005101142138 A CNB2005101142138 A CN B2005101142138A CN 200510114213 A CN200510114213 A CN 200510114213A CN 1332466 C CN1332466 C CN 1332466C
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- bacteriorhodopsin
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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
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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- Y02E60/30—Hydrogen technology
- Y02E60/50—Fuel cells
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Abstract
The present invention discloses a preparing method of using microbes as proton exchanging membranes in fuel batteries. The preparing method is characterized in that thalli are firstly separated from halophilic bacteria and are treated by differential centrifugation to obtain purple membrane raw materials for preparing proton exchanging membranes. After purple membranes are treated by together mixing, membranes are manufactured on a base plate by using the membrane manufacturing technology of flow extension. The proton conducting rate of prepared bacteriorhodopsin proton exchanging membranes is from 0.0012 S/cm to 0.00521 S/cm. The preparing method of bacteriorhodopsin proton exchanging membranes has the advantages of simple and convenient technology and easy material acquisition, has no pollution to environment, effectively reduces the production cost of proton exchanging membranes and promotes the industrial production of microbe fuel batteries.
Description
Technical field
The present invention relates to a kind of act as a fuel preparation method of proton exchange membrane in the battery of microbe that adopts, more particularly, be meant a kind of from the Halophiles thalline the act as a fuel preparation method of the bacteriorhodopsin,BR proton exchange membrane that battery uses of isolated protein.
Background technology
Fuel cell is the electrochemical reaction appts that a kind of chemical energy with fuel is directly changed into electric energy.Wherein Proton Exchange Membrane Fuel Cells be owing to can use at normal temperatures, simultaneously with polymer as electrolyte, thereby can be used as the power supply of electric automobile, movable electrical appliances and stationary electric power plant.The main effect of proton exchange membrane is the passage that proton conduction is provided as electrolyte, isolates the gas of polarization response simultaneously as barrier film.In the oxidized process produce power of anode, show as the power supply effect of fuel cell by proton on the macroscopic view.Proton exchange membrane is the key factor of decision proton exchange film fuel battery performance, in order further to improve the performance of battery, the commercialization process of accelerating proton exchange film fuel battery, need improve existing proton exchange membrane, comprise the ion exchange capacity that improves film, reduce film thickness to reduce film resistance, reduce the cost of manufacture of film etc.
Natrinema altunense sp (Halobacterium Halobium) is a kind of of extreme microorganism, and it has idioadaptation power to the sodium chloride more than 15% (NaCl).Different with the salt tolerant bacterium, it is inviable that Halophiles leaves hypersaline environment.Halophiles has unique cell membrane, and wall construction is kept with ionic bond.At high concentration Na
+Keep structure and cell integrity between cell wall protein subunit under the condition.Halophiles also has unique cell membrane simultaneously.Cell membrane has formed this structure of phytanol in long-term the adaptation under the hypersaline environment.And purple membrane (Purple Membrane PM) is specific function film on the Halophiles cell membrane, studies show that purple membrane is the cell membrane of part alienation.Unique protein-bacteria rhodopsin on the film, (bacteriorhodopsin BR), is a kind of transform light energy chromoprotein to be called for short bacteriorhodopsin,BR.When being subjected to radiation of visible light, photochromic can take place and have the proton pump function.Carry out the light circulation behind the bacteriorhodopsin,BR molecule absorption photon, produce a series of light intercycle bodies that different absorption characteristics and dynamic characteristic are arranged, in this process bacteriorhodopsin,BR with proton from pump in the film outside film, produce electro chemical proton gradient in the film both sides, Halophiles can utilize this gradient carry out atriphos (Adenosine Triphosphate ATP) synthetic, promote a series of physiological activities such as cell movement and amino acid absorption.Outside organism, the excellent stability of bacteriorhodopsin,BR can utilize certain method to go up bacteriorhodopsin,BR film or the chemical reinforcing membrane that forms single or multiple lift at transparent carrier (as electro-conductive glass).The pigmented film system of bacteriorhodopsin,BR is simple and stable very, be enough to control photosignal by the change of its structure, bacteriorhodopsin,BR not only is easy to cultivate and separate, and can carries out the reorganization of genetic modification and molecules align form as the green bio-functional material of pure natural simultaneously; Bigger ratio absorption area and good two-photon characteristic more help the absorption of the low light level and the conversion of energy.Thereby bacteriorhodopsin,BR becomes the novel focus material of biological electronic technology and optical information processing in the world.
Summary of the invention
The objective of the invention is to propose a kind of method that adopts high speed centrifugation, differential centrifugation, blending technology to prepare the bacteriorhodopsin,BR proton exchange membrane, realize that by the rotating speed of regulating centrifugation apparatus thalline separates with purple membrane with protein, red film among this preparation method, its separating technology is simple.
The present invention is a kind of method for preparing bactermiorhodopsin proton exchange film of fuel battery, and it may further comprise the steps:
First step high speed centrifugation:
Adopt centrifugation centrifugal 10～20min under the condition of rotating speed 11000～15000g that the thalline in the Halophiles culture medium is isolated; Then, isolated thalline is mixed stirring 10～18h with distilled water, broken ribozyme, make the fragmentation of thalline swelling make the thalline mixed liquor, wherein, the volume ratio of thalline and distilled water is 1.5～3: 10, and the volume ratio of thalline and broken ribozyme is 1.5～3: 0.01;
The second step differential centrifugation:
(A) adopt centrifugation with the thalline mixed liquor centrifugal 5～10min under the condition of rotating speed 8000～12000g that makes in the first step, the reject precipitated impurities keeps supernatant;
(B) with centrifugal 25～40min under the condition of above-mentioned supernatant with rotating speed 35000～45000g, reject supernatant part keeps the precipitation part; Then,
(C) adding concentration in the precipitation part is sodium chloride centrifugal 5～10min under the condition of rotating speed 8000～12000g of 0.05～1mol/L, and the reject precipitated impurities keeps supernatant for the second time; Again with centrifugal 25～40min under the condition of the supernatant second time with rotating speed 35000～45000g, reject supernatant part, keep and precipitate part for the second time, repeat this step to supernatant part water white transparency and keep should last precipitation part, the precipitation part is 1.5～3: 10 with the volume ratio of sodium chloride;
(D) with the part of the last precipitation in (C) and distilled water by volume proportioning 1.5～3: 10 mix, make the purple membrane aqueous solution;
The 3rd step blend is handled:
(A) polyvinyl alcohol and buffer are added in the distilled water, and be cooled to room temperature after being heated to 80 ℃～160 ℃ and make blend solvent, the volume ratio of polyvinyl alcohol and distilled water is 0.5～1: 10, and the volume ratio of buffer and distilled water is 0.0 1～1: 10;
(B) the purple membrane aqueous solution that makes in second step is added in the blend solvent, through stirring along wall, ultrasonicly go to leave standstill 10～30min behind the bubble and promptly make bacteriorhodopsin,BR proton exchange coating solution, wherein, the volume ratio of the purple membrane aqueous solution and blend solvent is 1.5～3: 10;
The 4th step preparation bacteriorhodopsin,BR proton exchange membrane:
Adopt the casting film method that the bacteriorhodopsin,BR proton exchange coating solution that makes in the third step is sprawled on substrate,, promptly obtain being used for the bacteriorhodopsin,BR proton exchange membrane that proton fuel cell is used through 10～24 hours rear demouldings of drying at room temperature.
The preparation method of described bacteriorhodopsin,BR exchange membrane, the bacteriorhodopsin,BR proton exchange film thickness of sprawling in the 4th step on substrate is 30～80 microns.
The preparation method of described bacteriorhodopsin,BR exchange membrane, the bacteriorhodopsin,BR exchange membrane for preparing in the 4th step is kept in the deionized water of normal temperature.
The advantage that the present invention prepares bacteriorhodopsin,BR proton exchange film method is: (1) just can realize separating of thalline by the rotating speed of control centrifugation apparatus with centrifugation time in preparation process, safe and reliable so technology is simple to operation, and environment is not polluted; (2) the Halophiles microbe raw material is easy to get, as its low production cost of proton exchange membrane of fuel cell; (3) be used for fuel cell exploitation and drop into lessly relatively, the production cycle is shorter; (4) proton conductivity of bacteriorhodopsin,BR proton exchange membrane in 20 ℃～50 ℃ scopes is 1.20 * 10
-2S/cm～5.21 * 10
-2S/cm.
Embodiment
The employing bacterial classification is the halobacterium halobium (Halobacterium halobium bacterium numbering 1.2368) in the Halobacterium among the present invention, and available from Institute of Micro-biology of Chinese Academy of Sciences bacterial classification center, activated, expansion is cultivated, Continuous Cultivation, obtains the Halophiles of requirement.Know partly that by principle the growth of Halophiles growing microorganism and purple membrane is nonsynchronous, first blowing air is bred thalline in a large number, reaches the top of exponential phase, stops oxygen supply again, to promote the purple membrane growth.Halophiles is the special amphimicrobe of a class: produce atriphos (ATP) with oxidative phosphorylation under the condition of oxygen abundance, can realize oneself's growth and flora propagation faster; Utilize luminous energy growth purple membrane under the anoxia condition, by the effect of BR optical drive proton pump, produce film both sides electrochemistry potential energy, every physiological activity is kept in the generation that promotes ATP.Have when studies show that the rayed Halophiles that three kinds of effect: ATP increase, matrix acidifying (expression proton from pump in the cell to the extracellular) and breathing be suppressed.Bacteria rhodopsin is similar to chlorophyll, and under the driving of photon, what produce proton striding the film transportation and formation ATP.If with pH electrode measurement purple membrane suspension, the proton that is easy to observe under optical drive discharges, i.e. optical drive proton pump function.This shows Halophiles only in environment oxygen concentration very low and have under the condition of illumination and could synthesize purple membrane.At this moment normal oxidative phosphorylation can't be satisfied its energy demand, then is provided by the photophosphorylation of purple membrane.Halophiles purple membrane photophosphorylation is another the new photosynthesis type outside the photophosphorylation that carried out of classical chlorophyll and bacteriochlorophyll (bacteriochlorin), and the simplest photophosphorylation reaction that is up to now to be found.
The centrifugation apparatus of Cai Yonging is the AVANTIJ-25 CENTRIFUGE that U.S. BECKMAN COULTER company produces in the present invention.
The present invention is a kind of preparation method of bactermiorhodopsin proton exchange film of fuel battery, and it has the following steps:
First step high speed centrifugation:
Adopt centrifugation centrifugal 10～20min under the condition of rotating speed 11000～15000g that the thalline in the Halophiles culture medium is isolated; Then, isolated thalline is mixed stirring 10～18h with distilled water, broken ribozyme, make the fragmentation of thalline swelling make the thalline mixed liquor, wherein, the volume ratio of thalline and distilled water is 1.5～3: 10, and the volume ratio of thalline and broken ribozyme is 1.5～3: 0.01;
The second step differential centrifugation:
(A) adopt centrifugation with the thalline mixed liquor centrifugal 5～10min under the condition of rotating speed 8000～12000g that makes in the first step, the reject precipitated impurities keeps supernatant;
(B) with centrifugal 25～40min under the condition of above-mentioned supernatant with rotating speed 35000～45000g, reject supernatant part keeps the precipitation part; Then,
(C) adding concentration in the precipitation part is sodium chloride nacl centrifugal 5～10min under the condition of rotating speed 8000～12000g of 0.05～0.2mol/L, and the reject precipitated impurities keeps supernatant for the second time; Again with centrifugal 25～40min under the condition of the supernatant second time with rotating speed 35000～45000g, reject supernatant part, keep and precipitate part for the second time, repeat this step to supernatant part water white transparency and keep should last precipitation part, the precipitation part is 1.5～3: 10 with the volume ratio of sodium chloride;
(D) with the part of the last precipitation in (C) and distilled water by volume proportioning 1.5～3: 10 mix, make the purple membrane aqueous solution;
In the present invention NaCl as a kind of desirable functionally gradient material (FGM) possess following some: 1. not reacting with separated biomaterial is complete inertia, and easily separates with the biomone that is separated; 2. can meet the requirements of density range, and in desired density range, viscosity is low, infiltration is forced down, and ionic strength and pH change less; 3. can be to centrifugation apparatus generation corrosiveness; 4. easy purifying, low price or recovery easily; 5. concentration is convenient to measure; 6. for ultracentrifugal analysis work, its physical property, macroscopic property are known.After the centrifugal beginning, when gradient liquid owing to action of centrifugal force forms the dense rare density gradient in top of managing in the end gradually, the particle that meanwhile was evenly distributed originally also redistributes.When pipe end density of medium greater than Particle Density, i.e. ρ
m＞ρ
PThe time particle come-up; At the curved top ρ of place
P＞ρ
mThe time, then particle precipitation, the density position that final particle enters into an itself is ρ
P=ρ
m, this moment, dx/dt was that Z-zero particle is no longer mobile, particle forms district's band of pure component, relevant with the density of sample particle, and irrelevant with size and other parameters of particle, therefore as long as rotating speed, temperature-resistant then prolongs the one-tenth band position that centrifugation time can not change these particles.
Because the Halophiles cell membrane mainly is made up of red film and purple membrane, the separation in (C) step is in order to remove the purple membrane that red film obtains being used as proton exchange membrane.
The 3rd step blend is handled:
(A) polyvinyl alcohol and buffer are added in the distilled water, and be cooled to room temperature after being heated to 80 ℃～160 ℃ and make blend solvent, the volume ratio of polyvinyl alcohol and distilled water is 0.5～1: 10, and the volume ratio of buffer and distilled water is 0.01～1: 10;
(B) the purple membrane aqueous solution that makes in second step is added in the blend solvent, through stirring along wall, ultrasonicly go to leave standstill 10～30min behind the bubble and promptly make bacteriorhodopsin,BR proton exchange coating solution, wherein, the volume ratio of the purple membrane aqueous solution and blend solvent is 1.5～3: 10;
In the present invention, consider that the film forming of protein self is relatively poor, so with bacteriorhodopsin,BR (BR) and polyvinyl alcohol (PVA) blend.PVA is a kind of polarity high polymer, has water-soluble and excellent biological compatibility, to the molecular structure of BR and not influence of spectrochemical property, the film forming homogeneous, transparent, mechanical strength good; And PVA has anisotropy at glass surface, just finished the orientation works to BR in the time of film forming, so that the proton stream that illumination produces down has certain directivity.
The 4th step preparation bacteriorhodopsin,BR proton exchange membrane:
Adopt the casting film method that the bacteriorhodopsin,BR proton exchange coating solution that makes in the third step is sprawled on substrate (as quartz glass plate), sprawl 30～80 microns of thickness, through 10～24 hours rear demouldings of drying at room temperature, promptly obtain being used for the bacteriorhodopsin,BR proton exchange membrane that proton fuel cell is used.Adopting the Britain SI of Sa1artron company 1260 electric impedance analyzers to test its conductivity in 20 ℃～50 ℃ scopes to described bacteriorhodopsin,BR proton exchange membrane is 1.20 * 10
-2S/cm～5.21 * 10
-2S/cm.
In the present invention, adopting the casting film method is to have thick compounding substances (bacteriorhodopsin,BR proton exchange coating solution) and evenly be layered on the level and smooth quartz glass plate a kind of, and after drying, thick material just forms a very thin membrane structure.Because prepared bacteriorhodopsin,BR proton exchange membrane is thinner, should be kept in the deionized water of room temperature.
The 5th step assembling fuel cell:
Use heat pressing forming machines at 45 ℃ the bacteriorhodopsin,BR proton exchange membrane, pressure is 1Mpa, and the membrane electrode that 60 seconds aftershapings of hot pressing are fuel cell feeds hydrogen, and it carried out battery performance detect as shown in the table.
| H 2Flow (ml/min) | 30 | 50 | 60 | 70 |
| Burning voltage (V) | 0.85 | 0.86 | 0.88 | 0.87 |
The present invention adopts universal instrument that the bacteriorhodopsin,BR proton exchange membrane is carried out performance test, and the bacteriorhodopsin,BR proton exchange membrane that the data declaration the present invention through recording prepares has good battery performance.Because the existence of optical drive proton pump is strengthened proton conductivity.Under field conditions (factors), Halophiles can impel the generation of ATP with the optical drive proton pump.Therefore with certain density NaCl solutions simulate the growing environment of Halophiles.When the adding diluted acid provides proton source in above-mentioned salting liquid, can measure unsettled voltage.The experimental record recording voltage reaches 0.12V under solar radiation.Bacteriorhodopsin,BR optical drive proton pump characteristics makes its function that can realize proton conduction, promptly under the effect of " proton pump ", proton by the BR film by the side " pump " of film to opposite side, thereby promote proton by anode to the negative electrode transmission; By making its film forming with the polyvinyl alcohol blend, reach the effect of isolated gas, just can be used as proton exchange membrane and be applied in the fuel cell.Experimental result shows that the effect of this proton pump efficiently helps to improve the proton transfer efficient of fuel cell, has realized the improvement to fuel battery performance.
Embodiment 1:
First step high speed centrifugation:
Adopt centrifugation centrifugal 15min under the condition of rotating speed 13000g that the thalline in the Halophiles culture medium is isolated; Then, isolated thalline is mixed stirring 12h with distilled water and broken ribozyme, make the fragmentation of thalline swelling make the thalline mixed liquor, wherein, the volume ratio of thalline and distilled water is 1.5: 10, and the volume ratio of thalline and broken ribozyme is 3: 0.01;
The second step differential centrifugation:
(A) adopt centrifugation with the thalline mixed liquor centrifugal 7min under the condition of rotating speed 10000g that makes in the first step, the reject precipitated impurities keeps the supernatant that contains cell membrane;
(B) with centrifugal 35min under the condition of above-mentioned supernatant with rotating speed 40000g, reject supernatant part keeps the precipitation part; Then,
(C) adding concentration in the precipitation part is sodium chloride centrifugal 7min under the condition of rotating speed 10000g of 0.1mol/L, and the reject precipitated impurities keeps supernatant for the second time; Again with centrifugal 35min under the condition of the supernatant second time with rotating speed 740000g, reject supernatant part, keep and to precipitate part for the second time, repeat this step to supernatant part water white transparency and keep should last precipitation part, the precipitation part is 3: 10 with the volume ratio of sodium chloride;
(D) with the part of the last precipitation in (C) and distilled water by volume proportioning mix at 3: 10, make the purple membrane aqueous solution;
The 3rd step blend is handled:
(A) polyvinyl alcohol and N-2-ethoxy-piperazine-N-2-ethyl sulfonic acid (buffer) are added in the distilled water, and be cooled to room temperature after being heated to 120 ℃ and make blend solvent, the volume ratio of described polyvinyl alcohol and distilled water is 1: 10, and the volume ratio of described N-2-ethoxy-piperazine-N-2-ethyl sulfonic acid and distilled water is 1: 10;
(B) the purple membrane aqueous solution that makes in second step is added in the blend solvent, through stirring along wall, ultrasonicly go to leave standstill 30min behind the bubble and promptly make bacteriorhodopsin,BR proton exchange coating solution, wherein, the volume ratio of the purple membrane aqueous solution and blend solvent is 3: 10
The 4th step preparation bacteriorhodopsin,BR proton exchange membrane:
Adopt the casting film method that the bacteriorhodopsin,BR proton exchange coating solution that makes in the third step is sprawled on quartz glass plate, sprawling thickness is 50 microns, through drying at room temperature 18h rear demoulding, promptly obtains being used for the bacteriorhodopsin,BR proton exchange membrane that proton fuel cell is used.Its conductivity at 20 ℃ is 2.73 * 10
-2S/cm.
Claims (5)
1, a kind of preparation method of bactermiorhodopsin proton exchange film of fuel battery is characterized in that may further comprise the steps:
First step high speed centrifugation:
Adopt centrifugation centrifugal 10～20min under the condition of rotating speed 11000～15000g that the thalline in the Halophiles culture medium is isolated; Then, isolated thalline is mixed stirring 10～18h with distilled water, broken ribozyme, make the fragmentation of thalline swelling make the thalline mixed liquor, wherein, the volume ratio of thalline and distilled water is 1.5～3: 10, and the volume ratio of thalline and broken ribozyme is 1.5～3: 0.01;
Described centrifugation apparatus is the AVANTI J-25CENTRIFUGE that U.S. BECKMAN COULTER company produces;
The second step differential centrifugation:
(A) adopt centrifugation with the thalline mixed liquor centrifugal 5～10min under the condition of rotating speed 8000～12000g that makes in the first step, the reject precipitated impurities keeps supernatant;
(B) with centrifugal 25～40min under the condition of above-mentioned supernatant with rotating speed 35000～45000g, reject supernatant part keeps the precipitation part; Then,
(C) adding concentration in the precipitation part is sodium chloride centrifugal 5～10min under the condition of rotating speed 8000～12000g of 0.05～1mol/L, and the reject precipitated impurities keeps supernatant for the second time; Again with centrifugal 25～40min under the condition of the supernatant second time with rotating speed 35000～45000g, reject supernatant part, keep and precipitate part for the second time, repeat this step to supernatant part water white transparency and keep should last precipitation part, the precipitation part is 1.5～3: 10 with the volume ratio of sodium chloride;
(D) with the part of the last precipitation in (C) and distilled water by volume proportioning 1.5～3: 10 mix, make the purple membrane aqueous solution;
The 3rd step blend is handled:
(A) polyvinyl alcohol and buffer are added in the distilled water, and be cooled to room temperature after being heated to 80 ℃～160 ℃ and make blend solvent, the volume ratio of polyvinyl alcohol and distilled water is 0.5～1: 10, and the volume ratio of buffer and distilled water is 0.01～1: 10;
Described buffer is N-2-ethoxy-piperazine-N-2-ethyl sulfonic acid;
(B) the purple membrane aqueous solution that makes in second step is added in the blend solvent, through stirring along wall, ultrasonicly go to leave standstill 10～30min behind the bubble and promptly make bacteriorhodopsin,BR proton exchange coating solution, wherein, the volume ratio of the purple membrane aqueous solution and blend solvent is 1.5～3: 10;
The 4th step preparation bacteriorhodopsin,BR proton exchange membrane:
Adopt the casting film method that the bacteriorhodopsin,BR proton exchange coating solution that makes in the third step is sprawled on substrate,, promptly obtain being used for the bacteriorhodopsin,BR proton exchange membrane that proton fuel cell is used through 10～24 hours rear demouldings of drying at room temperature.
2, the preparation method of bacteriorhodopsin,BR exchange membrane according to claim 1 is characterized in that: the bacteriorhodopsin,BR proton exchange film thickness of sprawling in the 4th step on substrate is 30～80 microns.
3, the preparation method of bacteriorhodopsin,BR exchange membrane according to claim 1 is characterized in that: substrate is a quartz glass plate in the 4th step.
4, the preparation method of bacteriorhodopsin,BR exchange membrane according to claim 1 is characterized in that: the bacteriorhodopsin,BR exchange membrane for preparing in the 4th step is kept in the deionized water of normal temperature.
5, the preparation method of bacteriorhodopsin,BR exchange membrane according to claim 1 is characterized in that: the proton conductivity of preparation-obtained bacteriorhodopsin,BR proton exchange membrane in 20 ℃～50 ℃ scopes is 1.20 * 10
-2S/cm～5.21 * 10
-2S/cm.
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| CN102024957B (en) * | 2009-09-17 | 2014-11-26 | 北京航空航天大学 | Biological material-based direct methanol fuel cell proton exchange membrane and preparation method thereof |
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| 生物燃料电池 贾鸿飞，谢阳，王宇新，电池，第30卷第2期 2000 * |
| 生物燃料电池 贾鸿飞，谢阳，王宇新，电池，第30卷第2期 2000;生物燃料电池进展 康峰，伍艳辉，李佟茗，电源技术，第28卷第11期 2004 * |
| 生物燃料电池进展 康峰，伍艳辉，李佟茗，电源技术，第28卷第11期 2004 * |
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