CN106047754B - A kind of method and its application improving Clostridium beijerinckii electricity production - Google Patents
A kind of method and its application improving Clostridium beijerinckii electricity production Download PDFInfo
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- CN106047754B CN106047754B CN201610399488.9A CN201610399488A CN106047754B CN 106047754 B CN106047754 B CN 106047754B CN 201610399488 A CN201610399488 A CN 201610399488A CN 106047754 B CN106047754 B CN 106047754B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
- H01M8/00—Fuel cells; Manufacture thereof
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P70/00—Climate change mitigation technologies in the production process for final industrial or consumer products
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Abstract
The invention discloses a kind of method and its application of raising Clostridium beijerinckii electricity production, this method is after the gene for encoding memebrane protein Cbei_3304 is carried out insertion inactivation, this gene to be made to be unable to normal expression in Clostridium beijerinckii.In the resulting high electricity production recombinant bacterial strain glucose seed liquor of the present invention, when using methyl viologen as electron mediator, maximum output voltage is up to 409mV, and peak power output density is 144.4mW/m2.Simple process of the invention, easy to operate, pollution-free, at low cost, fuel utilization is general relatively wide, and electron recovery efficiency is higher, alleviates energy crisis；High electricity production Clostridium beijerinckii of the invention is one plant of strain excellent for being suitable for microbiological fuel cell research and application.
Description
Technical field
The invention belongs to environment and new energy technology fields, and in particular to it is a kind of improve Clostridium beijerinckii electricity production method and its
Using.
Background technique
With the fast development of economic globalization, various countries are growing energy demand, and lack of energy becomes the difficulty in the world
Topic, when facing this problem, eye is put into a cyclopean family --- microorganism by scientist.It is produced using microorganism
Electricity, to solve the problems, such as energy shortage.Currently, microbiological fuel cell (Microbial Fuel Cell, MFC) technology increasingly at
It is ripe, and paid attention to.
However, the electricity generation ability of microorganism differs greatly, electricity-producing microorganism but determine in microbiological fuel cell function
And application.Through reporting, height electricity production microorganism still be concentrated mainly on genus Shewanella (Shewanella) and the thin end of the scroll Pseudomonas
(Geobacter) some Gram-negative bacterias such as (Energy Environ. Sci., 2011,4,4366-4379).And
Very few, Sophie Peguin et al. (Biotechnology and is reported in the gram-positive bacterias such as Clostridium beijerinckii
Bioengineering, Vol. 51, Pp. 342-348 (1996)) using three-electrode system have studied clostridium acetobutylicum
Microbiological fuel cell in the case where methyl viologen is as electron mediator produces electricity situation；Liu Jun et al.
(Biotechnology Lett, 2015,37:95-100) is also using 0.15g/L methyl viologen as electron mediator, the Portugal 1g/L
Maximum voltage 230mV, 79.2 mW/m of peak power output density are obtained when grape sugar is as carbon source2.As it can be seen that Clostridium beijerinckii etc. is removed from office
Application of the Lan Shi positive bacteria in microbiological fuel cell increasingly causes attention.
Itself electricity generation ability of known microorganisms seriously hinders the development of MFC technology, however is transformed by genetic engineering
Thallus can speed up the metabolism of cell, improve electron transport rate and quantity.Based on this, genetic engineering means are to the micro- life of promotion
The development of object fuel cell and industrial application will have considerable prospect.
Summary of the invention
The purpose of the present invention is to provide a kind of methods of raising Clostridium beijerinckii electricity production.
Another object of the present invention is to provide a kind of Clostridium beijerinckiis of high activity electricity generation ability to prepare Microbial fuel
Application in battery.
The technical solution used in the present invention is:
A method of Clostridium beijerinckii electricity production is improved, the function of Clostridium beijerinckii PROTEIN C bei_3304 is lacked.
Further, method described above prevents to encode the gene of PROTEIN C bei_3304 in Clostridium beijerinckii from normal table
It reaches.
Further, the gene that PROTEIN C bei_3304 is encoded in Clostridium beijerinckii is carried out insertion mistake by method described above
It is living.
Further, method described above will encode the base sequence of PROTEIN C bei_3304 as shown in SEQ ID No:1.
Further, above-mentioned Clostridium beijerinckii isClostridium beijerinckii NCIMB 8052。
A kind of high application for producing electricity Clostridium beijerinckii in electricity production, the high electricity production Clostridium beijerinckii is to lack normal albumen
The Clostridium beijerinckii of Cbei_3304 function.
A kind of high electricity production Clostridium beijerinckii is preparing the application in microbiological fuel cell, and the high electricity production Clostridium beijerinckii is scarce
Lose the Clostridium beijerinckii of normal PROTEIN C bei_3304 function.
Further, the high electricity production Clostridium beijerinckii is the Clostridium beijerinckii that method described above is prepared.
Further, above-mentioned high electricity production Clostridium beijerinckii is using glucose, xylose, sucrose, fructose or starch as electron donor,
Further, above-mentioned high electricity production Clostridium beijerinckii is with methyl viologen, methylene blue or anthraquinone -2,6- sodium disulfonate work
For electron mediator.
The beneficial effects of the present invention are:
The present invention is by carrying out insertion inactivation for the Cbei_3304 gene for encoding memebrane protein in Clostridium beijerinckii, so that this base
Because being unable to normal expression, to achieve the purpose that improve Clostridium beijerinckii electricity generation ability.The present invention provides a kind of simple, efficient
The method for improving Clostridium beijerinckii electricity production, the recombinant bacterial strain obtained by the method is in 1 g/L glucose seed liquor, 0.4 g/L
When (optium concentration) methyl viologen (MV) is electron mediator, maximum output voltage is up to 409mV, compares initial strainsClostridium beijerinckiiNCIMB 8052 improves 3.13 times, and peak power output density is 144.4mW/m2,
Compare initial strainsClostridium beijerinckiiNCIMB 8052 improves 1.71 times of (best methyl in 8052-MFC
Purpurine concentration is 0.1g/L).
Simple process of the invention, easy to operate, pollution-free, at low cost, fuel utilization is general relatively wide, electron recovery efficiency compared with
Height, alleviates energy crisis, and the high electricity production Clostridium beijerinckii of the present invention is one plant and is suitable for microbiological fuel cell research and application
Strain excellent.
Detailed description of the invention
Fig. 1 is the plasmid map of present invention insertion inactivating vectors pWJ；
Fig. 2 is the mechanism figure that the present invention is inactivated using two type Intron insertions；
Fig. 3 is conversion daughter colony PCR electrophoretogram；
Fig. 4 is electricity production activity figure of the Clostridium beijerinckii 3304 of the present invention in 1g/L glucose；
Fig. 5 is electricity production activity figure of the initial strains NCIMB 8052 in 1g/L glucose；
Fig. 6 is the production that electricity production Clostridium beijerinckii of the invention high and initial strains NCIMB 8052 are fuel using 1g/L glucose
Electric situation.
Specific embodiment
A method of Clostridium beijerinckii electricity production is improved, the function of Clostridium beijerinckii PROTEIN C bei_3304 is lacked.
Preferably, method described above prevents to encode the gene of PROTEIN C bei_3304 in Clostridium beijerinckii from normal expression.
Preferably, the gene that PROTEIN C bei_3304 is encoded in Clostridium beijerinckii is carried out insertion inactivation by method described above.
Preferably, method described above will encode the base sequence of PROTEIN C bei_3304 as shown in SEQ ID No:1.
Preferably, above-mentioned Clostridium beijerinckii isClostridium beijerinckii NCIMB 8052。
A kind of high application for producing electricity Clostridium beijerinckii in electricity production, the high electricity production Clostridium beijerinckii is to lack normal albumen
The Clostridium beijerinckii of Cbei_3304 function.
Preferably, the high electricity production Clostridium beijerinckii is the Clostridium beijerinckii that method described above is prepared.
A kind of high electricity production Clostridium beijerinckii is preparing the application in microbiological fuel cell, and the high electricity production Clostridium beijerinckii is scarce
Lose the Clostridium beijerinckii of normal PROTEIN C bei_3304 function.
Preferably, the high electricity production Clostridium beijerinckii is the Clostridium beijerinckii that method described above is prepared.
Preferably, above-mentioned high electricity production Clostridium beijerinckii is using glucose, xylose, sucrose, fructose or starch as electron donor,
Preferably, above-mentioned high electricity production Clostridium beijerinckii is with methyl viologen, methylene blue or the sodium disulfonate conduct of anthraquinone -2,6-
Electron mediator.
Preferably, above-mentioned high electricity production Clostridium beijerinckii is produced electricity by dual chamber MFC.
The present invention is further illustrated combined with specific embodiments below, and however, it is not limited to this.
Embodiment 1
The building of Clostridium beijerinckii Cbei_3304 gene disruption mutant strain
The principle of Clostridium beijerinckii Cbei_3304 gene disruption mutant strain is constructed as shown in Fig. 2, specific building process packet
Include following steps:
(1) Cbei_3304 is inserted into the building of inactivating vectors
1) introne is designed
According to the Cbei_3304 gene order (as shown in SEQ ID No:1) for the Clostridium beijerinckii that ncbi database is included, borrow
Software design is helped suitably to be inserted into gene loci (http://www.clostron.com), selection is inserted in the 101-102 alkali
Between base, and generate intron sequences, synthesize its sequence of intron sequences S-304(as shown in SEQ ID NO:2), and design with
Lower primer.
Cloning primer:
PWJ-OSC-304-S:5 '-ggagtgtcgaggatcctcgagAtaattatccttacacttcgcc -3 ',
Sequence is as shown in SEQ ID NO:3；
PWJ-OSC-304-A:5 '-ggttctcctacagattgtacaAatgtggtgataacagataag -3 ', sequence
Column are as shown in SEQ ID NO:4.
Verify primer:
Cbei-3304-T-S:5 '-taaattacctacagcaaaactgtg-3 ', sequence is as shown in SEQ ID NO:5；
Cbei-3304-T-A:5 '-ggaattaagaaccttgaatctatc-3 ', sequence is as shown in SEQ ID NO:6.
Primer pWJ-OSC-304-S introduces I restriction enzyme site of Xho (underscore part), and primer pWJ-OSC-304-A is introduced
I restriction enzyme site of BsrG (underscore part).
2) Cbei_3304-pWJ-304 construction of recombinant vector
With Xho I and I double digestion carrier pWJ, pWJ plasmid map of BsrG as shown in Figure 1, its sequence such as SEQ ID NO:7
It is shown.The purified kit of digestion products (Takara) passes through one-step cloning after purification, with intron sequences S-304
(ClonExpress) it connects.By the recombinant plasmid transformed of one-step cloning connection to Escherichia coli E.coliDH5a is applied to and contains
There are 50 μ g/ml ammonia benzyl chloramphenicol resistance LB plates, 37 DEG C of 12~16h of culture, picking transformant is connected to liquid and contains 50 μ g/ml
In ammonia benzyl mycin LB culture medium, 37 DEG C, 200rpm culture 12h extract recombinant plasmid (AXYGEN), sequence verification, obtain Cbei_
3304-pWJ-304 recombinant vector.
3) methylation of Cbei_3304-pWJ-304 recombinant vector
Prepare E.coliSuccessful Cbei_3304-pWJ-304 recombination will be sequenced in the Competent of Top 10/pAN2
Carrier is transformed into Escherichia coli E.coliTop 10 since pAN2 plasmid has tetracyclin resistance, therefore is applied to containing 50 μ g/
Ml ammonia benzyl mycin and the 10 Double LB plates of μ g/ml tetracycline, 37 DEG C of 12~16h of culture, picking transformant are connected to liquid and contain
In 50 μ g/ml ammonia benzyl mycins and 10 μ g/ml tetracycline LB culture mediums, 37 DEG C, 200rpm culture 12h extract methylation
(pAN2 plasmid contains a bacillus subtilis phage gene to Cbei_3304-pWJ-304 recombinant vector, can encode methyl and turn
Enzyme is moved, methylation of the exogenous plasmid in Escherichia coli is able to achieve), i.e. Cbei_3304 is inserted into inactivating vectors.
(2) Cbei_3304 be inserted into inactivating vectors conversion Clostridium beijerinckii (Clostridium beijerinckii NCIMB
8052)
1) willClostridium beijerinckiiNCIMB 8052 be seeded to YPS culture medium (yeast powder 3g/L,
Peptone 5g/L, glucose 1g/L, ammonium acetate 2g/L, sodium chloride 3g/L, bitter salt 3g/L, potassium dihydrogen phosphate 1g/
L, dipotassium hydrogen phosphate 1g/L, green vitriol 0.1g/L, pH=6.) 37 DEG C be incubated overnight, next day is inoculated into 5% ratio
YPS culture medium, 37 DEG C of culture 6-8h are inoculated into 2 × YTG culture medium (16 g/L of yeast powder, 10 g/L of peptone, Portugal with 10%
Grape sugar 5 g/L, 5 g/L of sodium chloride) 37 DEG C of culture 3h, OD600nm=1；
2) 50ml Clostridium beijerinckii bacterium solution is taken, 5000rpm, 4 DEG C of 10 min of centrifugation abandon supernatant.With ETM buffer (270mM
Sucrose, 0.6mM Na2HPO4, 4.4mM Na2HPO4, 10mM MgCl2) be resuspended；It is ibid centrifuged, removes supernatant, buffered again with ETM
Liquid is resuspended, and is ibid centrifuged, thoroughly takes supernatant；
3) with 1ml ET buffer (270mM sucrose, 0.6mM Na2HPO4, 4.4mM NaH2PO4It is resuspended, takes 200 μ l, add
Enter 1ug Cbei_3304 insertion inactivating vectors, the electric revolving cup of 0.2cm pre-cooling is added, mixes gently；
4) turned using MicroPulserTM electroporation electricity, condition is voltage 1.8kV, 200 Ω of resistance, 2.5 μ F of capacitor, electricity
It is added immediately 1mL 2 × YTG culture medium after hitting, is transferred to 2~3h of recovery in sterile centrifugation tube；
5) the 200 above-mentioned bacterium solutions of μ l are taken, the YPS solid medium containing 10 μ g/ml erythromycin, culture 2~3 are applied to
It.
(3) Cbei_3304 is inserted into the screening of Inactivating mutations strain
Picking above-mentioned steps 5) in 2~3 days transformants of culture, use primer Cbei-3304-T-S and Cbei-3304-
T-A carries out bacterium colony PCR verifying to transformant, and filtering out the mutant strain of Intron insertion genome, (after insertion, PCR amplification goes out base
Because on band electrophoretogram than wild type about 1Kbp), as shown in figure 3, by the mutant strain being correctly inserted into passage three times, be coated with simultaneously
The mutant strain for knocking out plasmid loss containing Erythromycinresistant and not on the YPS solid medium of Erythromycinresistant, is not being filtered out
(mutant strain that cannot be grown on Erythromycinresistant plate) is to get high electricity production Clostridium beijerinckii of the invention.
Embodiment 2
This example demonstrates that the high electricity production Clostridium beijerinckii of above-mentioned building utilizes the production of 1g/L glucose as anode catalyst
Electricity experiment.
(1) microbiological fuel cell is constructed
The present embodiment establishes micro- life using Clostridium beijerinckii for anode catalyst power generation according to existing technology and methods
Object fuel cell, including four anode chamber, cathode chamber, proton exchange membrane and external circuit parts.Anode electrode and cathode electrode are equal
For PAN base graphite soft felt (5 × 5cm), using titanium silk as external circuits, outer connecting resistance is 1000 Ω, and proton exchange membrane is Du Pont
NafionN117 uses data collector for Keithley series.
(2) culture medium prescription:
YPS seed culture medium: yeast powder 3g/L, peptone 5g/L, glucose 1g/L, ammonium acetate 2g/L, sodium chloride
3g/L, bitter salt 3g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, green vitriol 0.1g/L, pH=
6。
Dual chamber MFC Anodic liquid: yeast powder 3g/L, peptone 5g/L, glucose 1g/L, ammonium acetate 2g/L, sodium chloride
3g/L, bitter salt 3g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, green vitriol 0.1g/L, methyl
Purpurine (MV concentration is 0.4g/L in 3304-MFC, and MV concentration is 0.1g/L in 8052-MFC), pH=6.
Catholyte in dual chamber MFC: sodium dihydrogen phosphate dihydrate 2.772g/L, disodium hydrogen phosphate dodecahydrate 11.5g/L, chlorine
Change potassium 0.13g//l, potassium ferricyanide 40mM, pH=7.
(3) the high electricity production Bai Shi shuttle that the high electricity production recombinant bacterial strain 3304(embodiment 1 of activation constructs respectively in YPS seed liquor
Bacterium) and initial strains 8052(Clostridium beijerinckiiNCIMB 8052), 37 DEG C of cultivation temperature, Anaerobic culturel
Time 12h.Then 250mL dual chamber MFC technology is carried out, anolyte 225mL is accessed into anode chamber, methyl viologen (MV) is passed through
N2 3 minutes, then access the Clostridium beijerinckii (inoculum concentration 10%(v/v) of 25mL), it is passed through N2 3 minutes, the oxygen in solution is discharged, rapidly
Anode chamber is sealed, catholyte is added to cathode chamber, cathode chamber is sealed rapidly, 30 DEG C of cultivation temperature, produces electricity detection time 33h, most
Result is obtained eventually as shown in 1 table and Fig. 4 ~ 6.
According to output voltage data, calculates electron recovery efficiency and obtain the polarization curve of electricity production, experimental result such as 1 He of table
Shown in Fig. 4 ~ 6；Wherein, Fig. 4 is electricity production activity figure of the high electricity production Clostridium beijerinckii of the present invention in 1g/L glucose；Fig. 5 is initial
Electricity production activity figure of the bacterial strain NCIMB 8052 in 1g/L glucose；Fig. 6 is the high electricity production Clostridium beijerinckii of the present invention and initial strains
NCIMB 8052 is the electricity production situation of fuel using 1g/L glucose.It can be seen that the high electricity production Clostridium beijerinckii of the present invention is maximum
Output voltage is up to 409mV, improves 3.13 times than initial strains NCIMB 8052, peak power output density is 144.4mW/
m2, 1.71 times are improved than initial strains NCIMB 8052.
The present invention is in the course of the research, it was found that glucose is replaced with xylose, sucrose, fructose or starch as electronics and is supplied
Body, the high electricity production Clostridium beijerinckii of the present invention equally can also generate the maximum output voltage height of similar effect, peak power output density；
Methyl viologen is replaced with into methylene blue, anthraquinone -2,6- sodium disulfonate as electron mediator, the present invention is high, and electricity production Clostridium beijerinckii is same
Sample can also play the electricity generation performance of similar effect.
The above results illustrate the present invention provides a kind of simple, efficient method for improving Clostridium beijerinckii electricity production, acquired
High electricity production Clostridium beijerinckii can be used for producing electricity, in particular in the electricity production of microbiological fuel cell；Therefore it can be by high yield of the present invention
Electric Clostridium beijerinckii is used for the preparation of microbiological fuel cell.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
<110>Guangzhou Inst of Cane Sugar
<120>a kind of method and its application for improving Clostridium beijerinckii electricity production
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 723
<212> DNA
<213>artificial sequence
<400> 1
atgacaaagg ttaataaatt acctacagca aaactgtgga atccgaaatc atttattata 60
ttttccgtat ttttctcatt tctgccagct ggaatcatgt gtgctttgaa ttatggacga 120
tctggaagtc aaaaaaagaa gtggatattt cttttagcaa gcatcttagt gtttatagca 180
cttatagcac ttctgcctat attatcaatt aatacttcta tcatattttt cagtattaat 240
atagctttag gcataattct tatgtttaca cagttaaagc tgtataatga acatattcaa 300
aatggcgggc aaagtgcttc gtatttattt cctataatca tagggctttt gattttttca 360
ttgtcagctg cttcaattct atactcaatt tacgtgccaa aaaatgctct tgattatggc 420
gaaaatcact tattttatac taataagata acagaatccc aagcgaaaaa acttggagat 480
tatttaaatt cagaaggata cttcactcct agttcaaaag tggacgttaa gattgataaa 540
caagatacac tctatatttt atctctcgtt gtagaaggtg actataaaag tgatacaagc 600
tatgtagaac ctatgaaagc aatatcaaaa gagatttcaa aaaatgtttt tgaaaataat 660
aaagttagaa tagatttatg caatgataga ttcaaggttc ttaattccat caatgttgat 720
taa 723
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<211> 344
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<213>artificial sequence
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ctcgagataa ttatccttac acttcgcccg cgtgcgccca gatagggtgt taagtcaagt 60
agtttaaggt actactctgt aagataacac agaaaacagc caacctaacc gaaaagcgaa 120
agctgatacg ggaacagagc acggttggaa agcgatgagt tacctaaaga caatcgggta 180
cgactgagtc gcaatgttaa tcagatataa ggtataagtt gtgtttactg aacgcaagtt 240
tctaatttcg gttaagtgtc gatagaggaa agtgtctgaa acctctagta caaagaaagg 300
taagttatgg cgggcgactt atctgttatc accacatttg taca 344
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ggagtgtcga ggatcctcga gataattatc cttacacttc gcc 43
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ggttctccta cagattgtac aaatgtggtg ataacagata ag 42
<210> 5
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<210> 6
<211> 24
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<400> 6
ggaattaaga accttgaatc tatc 24
Claims (8)
1. a kind of method for improving Clostridium beijerinckii electricity production, which is characterized in that carry out the function of Clostridium beijerinckii PROTEIN C bei_3304
Missing encodes the base sequence of PROTEIN C bei_3304 as shown in SEQ ID No:1, and the Clostridium beijerinckii isClostridium beijerinckii NCIMB 8052。
2. the method according to claim 1, wherein making the gene for encoding PROTEIN C bei_3304 in Clostridium beijerinckii
It is unable to normal expression.
3. according to the method described in claim 2, it is characterized in that, the gene that PROTEIN C bei_3304 will be encoded in Clostridium beijerinckii
Carry out insertion inactivation.
4. a kind of application of high electricity production Clostridium beijerinckii in electricity production, the high electricity production Clostridium beijerinckii is to lack normal PROTEIN C bei_
The Clostridium beijerinckii of 3304 functionsClostridium beijerinckiiNCIMB 8052 encodes the base of PROTEIN C bei_3304
Sequence is as shown in SEQ ID No:1.
5. a kind of high electricity production Clostridium beijerinckii is preparing the application in microbiological fuel cell, the high electricity production Clostridium beijerinckii is missing
The Clostridium beijerinckii of normal PROTEIN C bei_3304 functionClostridium beijerinckiiNCIMB 8052 encodes albumen
The base sequence of Cbei_3304 is as shown in SEQ ID No:1.
6. application according to claim 5, which is characterized in that the high electricity production Clostridium beijerinckii is that claim 1 ~ 3 is any
The Clostridium beijerinckii that the method is prepared.
7. application according to claim 5, which is characterized in that the high electricity production Clostridium beijerinckii is with glucose, xylose, sugarcane
Sugar, fructose or starch are electron donor.
8. application according to claim 5, which is characterized in that the high electricity production Clostridium beijerinckii is with methyl viologen, methylene
Blue or anthraquinone 2,6 disulfonic acid sodium is as electron mediator.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610399488.9A CN106047754B (en) | 2016-06-07 | 2016-06-07 | A kind of method and its application improving Clostridium beijerinckii electricity production |
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| CN201610399488.9A CN106047754B (en) | 2016-06-07 | 2016-06-07 | A kind of method and its application improving Clostridium beijerinckii electricity production |
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