CN104611262B - A kind of electricity production bacterium of degraded cellulose and its application in a fuel cell - Google Patents
A kind of electricity production bacterium of degraded cellulose and its application in a fuel cell Download PDFInfo
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- 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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Abstract
Application the invention discloses a kind of electricity production bacterium of degraded cellulose and its in a fuel cell, its bacterial strain preservation title is citrobacter freundii（Citrobacter freundii）HBUZL 1, depositary institution CGMCC, preserving number is CGMCC No.10335, preservation date on January 9th, 2015；The 16S rDNA gene orders of its bacterial strain are SEQ ID NO:1.The electricity production bacterium that the present invention is provided is easy to culture, can not only be using gas chromatography as substrate, it can more importantly be generated electricity using resourceful cellulose as substrate, it is applied in microbiological fuel cell, its strong adaptability, electricity production voltage are big, with higher economic value and wide prospect of the application.
Description
Technical field
The present invention relates to technical field of microbial fuel battery, the electricity production bacterium of specifically a kind of degraded cellulose and its
Application in a fuel cell.
Background technology
Microbiological fuel cell（MFC）It is, using microorganism as reactive agent, to be urged by the use of enzyme or microorganism as anode
Oxidation operation, the device of electric energy is converted into by its metabolism by agent.The life that it belongs in biomass energy utilization technologies
Thing chemistry transformation technology, it is not only pollution-free, efficiency high, reaction condition gentle, and fuel source is extensive.Therefore, microorganism
Fuel cell has become the problem that research is fallen over each other in countries in the world.
China is large agricultural country, and biomass resource very abundant, various crops produce more than 600,000,000 tons of stalk every year, wherein
Can be used as the energy about 400,000,000 tons, if can in this, as electricity generation by microorganism substrate raw material, China's electricity generation by microorganism
With very wide development space.
At present, separated electricity-producing microorganism majority is bacterium, amphimicrobian.From the final electron acceptor of anaerobic respiration
Type analysis, they belong to different function bacteriums, for example：With Fe (III) or Mn（Ⅳ）For the metal of the final electron acceptor of respiratory chain
The Pseudomonas such as reducing bacteria Shewanella, Geobacter and Geopsychrobacter；Using sulfate as the sulfate of electron acceptor
The Pseudomonas such as reducing bacteria desulfoblbus, desulfovibrio and desulfuromon；Using nitrate as the nitre of electron acceptor
The Pseudomonas such as hydrochlorate reducing bacteria pseudomonsa, ochrobactrum and comamonas；In addition, the Escherichia coli of anaerobic fermentation,
The saccharomycete for carrying out photosynthetic rhodopseudomona, green alga chlamydomonas and eucaryon shows electricity production spy
Property.But, electricity production bacterium report that can be by substrate of cellulose in existing strain is few.As CN101728544A discloses one kind
Application and electricity-generating method of the citrobacter freundii in electricity generation by microorganism are protected there is provided a kind of citrobacter freundii LY-3
It is CGMCC No.3246 to hide numbering, and the bacterial strain can be sent out as the anode catalyst of microbiological fuel cell applied to microorganism
Electricity, but the fuel that the new strains of the technology are utilized is glucose and sodium acetate, its required cost of material is higher.And for example
CN100588019C discloses microbiological fuel cell and the method using straw power generation, is with cellulose degradation mixed bacterial pair
Stalk carries out processing generating, and the technology is pollution-free, cost is low, alleviates energy crisis, straw from village has been obtained effectively
Utilize, still, its processing step is cumbersome, and mixed-culture medium can not fully meet the optimal training of all strains in mixed bacterial
The demand of supporting, its degradation capability to stalk is limited, and its electric conversion ratio is relatively low.It can be seen that, can develop it is a kind of using cellulose as
Degrade the efficient electricity production bacterium of substrate, and be applied to microbiological fuel cell, be the heat subject studied in the industry of current line it
One.
The content of the invention
Application it is an object of the invention to provide a kind of electricity production bacterium of degraded cellulose and its in a fuel cell, to
Produced electricity for industrial circle using cellulose and a kind of new bacterial strain selection is provided, also provide a kind of new using cellulose electricity production for industrial circle
Application process, can not be using cellulose electricity production or its electricity production property while also solving bacterial strain that existing microorganism battery used
Can be poor the problem of.
The object of the present invention is achieved like this：
The invention provides a kind of electricity production bacterium of degraded cellulose, the bacterial strain preservation title is citrobacter freundii
（Citrobacter freundii）HBUZL-1, depositary institution CGMCC, preserving number are CGMCC No.10335, preservation date
On January 9th, 2015；Depositary institution address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microbe research
Institute.
The citrobacter freundii of degraded cellulose provided by the present invention（Citrobacter freundii）HBUZL-
1, its biological property is as follows：For gram-Negative bacillus, bacterium colony shows stick-slip, protuberance, translucent, mm of diameter 3 or so, side
Edge is irregular.
Citrobacter freundii of the present invention（Citrobacter freundii）HBUZL-1 16S rDNA sequences are such as
SEQ ID NO :Shown in 1.
Invention also provides citrobacter freundii（Citrobacter freundii）HBUZL-1 is in fuel cell
In application.
More specifically, citrobacter freundii of the present invention（Citrobacter freundii）HBUZL-1 is in combustion
Application process in material battery comprises the following steps：
A, aseptically, citrobacter freundii HBUZL-1 is inoculated in cellulosic pulp culture medium, 30 DEG C of trainings
6-8 h are supported, cultivates to exponential phase, bacterium must be inoculated with, saved backup；The composition of the cellulose culture medium is：Cellulose 20
Part, Na2HPO41000 parts of 1.5 parts, 2.5 parts of peptone, 0.5 part of yeast extract and distilled water, the pH of the cellulose culture medium
It is worth for 7.0-7.2；
B, structure microbial fuel cells system, the system include cathode chamber, anode chamber, PEM, external circuit electricity
Hinder case and data acquisition unit；Anolyte is housed in the anode chamber, cathode chamber is equipped with catholyte, and the anolyte composition is：
Cellulose 276mg/L, NaHCO3 3.13g/L、NH4Cl 0.31g/L、Na2HPO42.75g/L、（NH4）2SO4 0.56g/L、
MgSO4 0.2g/L、KCl 0.13g/L 、CaCl2 15mg/L、MnSO4 20mg/L、FeCl31mg/L；The catholyte composition
For：25mmol/L K3[Fe(CN)6], volume ratio is 1:1 NaH2PO4And Na2HPO4Each 50 mmol/L；
C, take and be collected by centrifugation under the inoculation bacterium 5mL, aseptic condition, be inoculated in anode chamber, produced electricity.
The invention provides one plant of electricity production new strains-citrobacter freundii（Citrobacter freundii）
HBUZL-1, the bacterial strain is easy to culture, can not only be using gas chromatography as fuel, more importantly can be with resourceful
Cellulose is that fuel is generated electricity, and is applied in microbiological fuel cell, and its adaptability is strong, electricity production voltage is big, has
Higher use value.
The electricity production new strains that the present invention is provided are applied in microbiological fuel cell, and its processing step is simple, easy to operate, only
Single culture need to be added can just be generated electricity by substrate of cellulose, and it produces electricity big voltage, electric high conversion rate, practical, tool
There is higher economic value and vast potential for future development.
Citrobacter freundii HBUZL-1 disclosed by the invention suggestion method for preserving：Lyophil preservation method.
Brief description of the drawings
Fig. 1 is glucose standard curve figure.
Fig. 2 is that citrobacter freundii HBUZL-1 Congo Red tests observe figure.
Fig. 3 is that citrobacter freundii HBUZL-1 is cultivated to the electron microscope of logarithmic phase.
Fig. 4 is citrobacter freundii HBUZL-1 and associated germline system development tree.
Fig. 5 is citrobacter freundii HBUZL-1 cyclic voltammetry curve.
Fig. 6 is that citrobacter freundii HBUZL-1 produces voltage versus time curve figure using cellulose.
Embodiment
Example below is used to the present invention is further described, but the invention is not limited in any way.
The acquisition of the bacterial strain of embodiment 1
（1）The separation and purifying of bacterial strain：Bipolar chamber using carboxymethyl cellulose as fuel of the source from stable operation 6 months
MFC anode is circulated, a small amount of carbon cloth is cut, is put into the mass percent concentration added with bead in 1% NaCl solution, to shake
Shake uniform rear gradient dilution to be applied on using carboxymethyl cellulose as the flat board of sole carbon source, be placed in anaerobic box（YQX- II, Shanghai
New talent medicine equipment Manufacturing Co., Ltd）In at 30 DEG C cultivate 3 days；Continue separation of ruling, preserve；
（2）The screening of bacterial strain：By step（1）The strains tested of preservation is inoculated into 37 DEG C of concussion and cultivate 48h of culture medium,
Take and centrifuge 10 min under zymotic fluid 10 mL, 4000 r/min, supernatant is used as crude enzyme liquid；The acetic acid containing CMC-Na is pipetted to delay
Fliud flushing (0.05 mol/L, pH 4.4) 1.5 mL add the mL of enzyme liquid 0.5 in test tube, are incubated in 50 DEG C after 30 min, by DNS
Method surveys sugar.In the basic conditions, amino-compound of the DNS solution with being reduced into brownish red after reduction sugar juice altogether heat, one
Determine in scope, the amount of reduced sugar and the shade of brownish red thing are in certain proportion relation.Determine reddish brown under 540 nm wavelength
The absorbance of color substance, by standard curve, calculates the content of reduced sugar in sample.Define catalyzing cellulose hydrolysis per minute
It is an enzyme activity unit U to generate the enzyme amount of 1g glucose.Defined according to international enzyme activity, 1g solid enzymes（Or 1mL liquid enzymes）,
（50±1）DEG C, under the conditions of specific pH, 1h hydrolysis substrates produce the reduction sugar amount equivalent to 1mg glucose, are a vigor
Unit, with μ/g（Or μ/mL）Represent.With reference to national standard, the 1 μ g glucose amounts that this research defines enzyme generation thick per 1mL per minute are
One enzyme activity unit, is designated as μ/mL or U.
Glucose standard curve is as shown in figure 1, y=0.91x-0.025, R2=0.997, enzyme activity calculation formula is：
Enzyme activity（U）=（OD+b）/a×1000×2/30
Wherein：b：Curve indulges intercept, 0.025；a：The slope of curve：0.91；1000：Mg is converted into μ g
According to measurement result, OD values are 0.115, are calculated by standard curve and enzyme activity formula and obtain bacterium disclosed by the invention
The enzyme activity of strain is up to 10.32 U.
It can be seen that, strains tested carboxymethylcelluloenzyme enzyme (CMCase) vigor is determined through DNS methods, enzyme activity is up to 10.32 U
Bacterial strain be the citrobacter freundii that provides of the present invention（Citrobacter freundii）HBUZL-1, and in 2015 1
The moon is preserved in CGMCC on the 9th, and preserving number is CGMCC No.10335.
The identification of the bacterial strain of embodiment 2
Carry out Morphological Identification, Physiology and biochemistry identification and Molecular Identification respectively to gained citrobacter freundii HBUZL-1, its
Experimentation and result are as follows.
（1）Strain morphology is identified
Reference《The conventional authentication method of general bacterium》Bacterial strain identification is carried out, using the micro- Microscopic observation of Gram's stain
Thalli morphology.As a result show, by culture, obtain that transparent circle can be produced on the flat board of sole carbon source in carboxymethyl cellulose
Single bacterium colony it is some, its Congo Red test result is shown in Fig. 2.Learnt by the strain morphology identification after culture：It is provided by the present invention
Degraded cellulose citrobacter freundii（Citrobacter freundii）HBUZL-1 is gram-Negative bacillus, bacterium
Fall and show stick-slip, swell, translucent, mm of diameter 3 or so, edge is irregular；By isolated strains liquid medium within（Carboxymethyl
Cellulose 20 g, Na2HPO41.5 g, peptone 2.5 g, yeast extract 0.5g, distilled water 1000ml, pH value is 7.0-7.2）
12 h of middle culture were observed and shot with transmission electron microscope, see Fig. 3 to exponential phase of growth.
（2）Physiology and biochemistry is identified
To citrobacter freundii（Citrobacter freundii）HBUZL-1 has carried out sugar fermentating test, methyl red
Experiment, acetonyl formic acid experiment (V.P. experiments), indole test, nitrate reduction test, gelatin hydrolysis experiment, H2O2Enzyme is surveyed
The Physiology and biochemistry identification such as fixed experiment.Its qualification result is shown in Table 1.
The bacterial strain bio-chemical characteristics result of table 1
Note:"+" experimental result is positive, and "-" experimental result is negative.
（3）Molecular Identification
Using general 27F/ 1492R primers（Sense primer：5′-AGAGTTTGATCMTGGCT2CAG-3′；Anti-sense primer：
5′-GGYTACCTTGT2TACGACTT-3′）Carry out 16SrDNA PCR amplifications.PCR reaction systems：Template DNA 50 ng, Taq
Archaeal dna polymerase 0.6 μ L, Mg2+(1.3 mmol/ L) 3.2 μ L, dNTP 4 μ L, Buffer 3.2 μ L, each 4 μ of upstream and downstream primer
L, 19 μ L water.Pcr amplification reaction program：94 DEG C of min of pre-degeneration 5；94 DEG C of denaturation 1 min, 55 DEG C of 30 s of annealing, 72 DEG C of extensions
1.5 min, 29 circulations；72 DEG C terminate 10 min of extension.Pcr amplification product cuts gel band after agarose gel electrophoresis；
Operating procedure according to PCR primer purification kit carries out after purification, sending Beijing three to win the sequencing of polygala bioengineering Co., Ltd,
Measure the 16S rDNA sequences such as SEQ ID NO of the bacterial strain:Shown in 1；The sequence of acquisition is submitted into NCBI
（www.ncbi.nlm.gov）Blast comparisons are carried out, the higher correlated series of homology is transferred, is compared with clustalX softwares
It is right, Phylogenetic Analysis is carried out using Mega softwares, using adjacent method constructing system chadogram, Fig. 4 is seen.Can from Fig. 4
Go out, performing PCR is entered to bacterial strain 16SrDNA genes and expands and is sequenced, its sequence is entered with known sequence in Gen-bank
Row compares, and as a result shows bacterial strain 16SrRNA genes and citrobacter freundii（Citrobacter freuudii）16SrDNA
It is KM272633 that genetic homology, which is more than 99%, GenBank receptions number,.According to the morphosis, physiological and biochemical property and bacterium of bacterial strain
Strain 16S rDNA the sequencing results, determine that the bacterial strain belongs to citric acid Pseudomonas.
Embodiment 3 produces electricity application of the bacterium in biological fuel cell
Microbiological fuel cell is built by existing method（MFC）System, the system includes cathode chamber, anode chamber, resistance box
And PEM, its electrode material is carbon fiber felt；Two interpolars are connected by copper conductor with a precision resister case
（ZX21,0-100000 Ω）, pass through short side pipe between two Room and connect a piece of PEM（Ultrex CMI7000, MI Mem.
Int. Inc.）.
（1）Citrobacter freundii HBUZL-1 electroactive detection
By MFC reactors and anolyte after 121 DEG C of sterilizing 20min, citrobacter freundii is inoculated with anode chamber
HBUZL-1, brings into operation and uses data collecting system（DAM-3058R voltage acquisition modules, the limited public affairs of Beijing Altay science and technology
Department）, 1min record primary voltages, when output voltage reduction is to below 0.3V, replacing anode fuel, temperature is room temperature（About 25
℃）.It is investigated after stable operation and produces electricity characteristic, peak power is obtained by polarization curve, when voltage output is stable, by changing
Become the resistance of external circuit（0-90000Ω）, and record the burning voltage in different external resistances.
Anolyte：Glucose（2g/L）、NaHCO3(3.13g/L)、NH4Cl（0.31g/L）、Na2HPO4(2.75g/L)、
（NH4）2SO4(0.56g/L) 、MgSO4(0.2g/L)、KCl（0.13g/L）、CaCl2（15mg/L）、MnSO4(20mg/L)、
FeCl3（1mg/L）；Anolyte can also be：Sodium acetate（3g/L）、NaHCO3(3.13g/L)、NH4Cl（0.31g/L）、Na2HPO4
(2.75g/L)、（NH4）2SO4(0.56g/L) 、MgSO4(0.2g/L)、KCl（0.13g/L）、CaCl2（15mg/L）、MnSO4
(20mg/L)、FeCl3（1mg/L）.
Catholyte：25mmol/L K3[Fe(CN)6], volume ratio is 1:1 NaH2PO4And Na2HPO4Each 50mmol/L.
Battery operation is to voltage plateau, and using anode carbon felt electrode as to electrode, cathode electrode is working electrode, Ag/
AgCl is reference electrode, adjusts scanning range, sweep speed etc., uses electrochemical workstation（Shanghai Chen Hua, CHI660C）Determine electricity
The cyclic voltammetry curve of pole（CV）.Adjust initial potential E0（V）：0.5, peak potential E1（V）：0.5, peak potential E2（V）：-
0.7, sampling interval（V）：0.001, sweep speed (V/s) 0.002.
Experimental result is shown in that Fig. 5, Fig. 5 are the cyclic voltammetry curves determined using glucose by substrate, and it is using sodium acetate the bottom of as
Thing acquired results are similar therewith, and cyclic voltammetry curve shows obvious redox peaks as seen from the figure.
（2）It is that substrate electricity generation performance is determined using citrobacter freundii HBUZL-1 as electricity production bacterium, carboxymethyl cellulose
Experimental procedure is：
A, aseptically, cellulosic pulp culture medium is inoculated in by citrobacter freundii HBUZL-1（Carboxymethyl is fine
Tie up plain 20 g, Na2HPO41.5 g, peptone 2.5 g, yeast extract 0.5g, distilled water 1000ml, pH value is 7.0-7.2）In,
30 DEG C of culture 6-8 h, make cell be in exponential phase, must be inoculated with bacterium, save backup；
B, anode chamber load anolyte, cathode chamber load catholyte, anolyte composition is：Carboxymethyl cellulose 276
mg/L、NaHCO3 3.13g/L、NH4Cl 0.31g/L、Na2HPO42.75g/L、（NH4）2SO4 0.56g/L、MgSO4 0.2g/
L、KCl 0.13g/L、CaCl2 15mg/L 、MnSO4 20mg/L、FeCl31mg/L；The step of catholyte is with this example（1）In
Catholyte it is identical；
C, take inoculation bacterium 5mL, it is sterile under be collected by centrifugation, addition is inoculated into the anode chamber of microbiological fuel cell and produced electricity；
D, resistance box connected into external circuit, data acquisition is carried out using the data collecting system.
The experimental result of gained is shown in that Fig. 6, Fig. 6 show, using carboxymethyl cellulose as substrate, citrobacter freundii HBUZL-
1 has good electricity generation performance, and through domestication after a while, ceiling voltage is up to 600mV.
SEQUENCE LISTING
<110>University Of Hebei
<120>A kind of electricity production bacterium of degraded cellulose and its application in a fuel cell
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1409
<212> DNA
<213>Citrobacter freundii（Citrobacter freundii）HBUZL-1
<400> 1
gagcacaaag gagctggctc cttgtgtgac gagtggcgga cgggtgagta atgtctggga 60
aactgcccga tggaggggga taactactgg aaacggtatc taataccgca taacgtcgca 120
agaccaaaga gggggacctt cgggcctctt gccatcggat gtgcccagat gggattagct 180
agtaggtggg gtaacggctc acctaggcga cgatccctag ctggtctgag aggatgacca 240
cccacactgg aactgagaca cggtccacac tcctacggga ggcagcagtg gggaatattg 300
cacaatgggc gcaagcctga tgcagccatg ccgcgtgtat gaaaaaggcc ttcgggttgt 360
aaagtacttt cagcgaggaa gaaggcttta gggttaataa ccttagtgat tgacgttact 420
cgcaaaagaa gcaccggcta actccgtgcc agcagccgcg gtaatacgga gggtgcaagc 480
gttaatcgga attactgggc gtaaagcgca cgcaggcggt ctgtcaagtc agatgtgaaa 540
tccccggtct caccctggaa tctgcatccg aatctggcag gctagagtct tgtagagggg 600
ggtagaattc cgggtgtagc ggagaaatgc gtagagatct ggaggaatac cggtggcgaa 660
ggcggccccc tggacaaaga ctgacgctca ggtgcgaaag cgtggggagc aaacaggatt 720
agataccctg gtagtccacg ccgtaaacga tgtcgacttg gaggttgtgc ccttgaggcg 780
tggcttccgg agctaacgcg ttaagtcgac cgcctgggga gtacggccgc aaggttaaaa 840
ctcaaatgaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgatgcaa 900
cgcgaagaac cttacctact cttgacatcc agagaactta gcagagatgc tttggtgcct 960
tcgggaactc tgagacaggt gctgcatggc tgtcgtcagc tcgtgttgtg aaatgttggg 1020
ttaagtcccg caacgagcgc aacccttatc ctttgttgcc agcgattcgg tcgggaactc 1080
aaaggagact gccagtgata aactggagga aggtggggat gacgtcaagt catcatggcc 1140
cttacgagta gggctacaca cgtgctacaa tggcatatac aaagagaagc gacctcgcga 1200
gagcaagcgg acctcataaa gtatgtcgta gtccggattg gagtctgcaa ctcgactcca 1260
tgaagtcgga atcgctagta atcgtggatc agaatgccac ggtgaatacg ttcccgggcc 1320
ttgtacacac cgcccgtcac accatgggag tgggttgcaa aagaagtagg tagcttaacc 1380
ttcgggaggg cgcttaccac tttgtgatc 1409
Claims (2)
1. the electricity production bacterium of a kind of degraded cellulose, it is characterised in that the bacterial strain preservation title is citrobacter freundii
（Citrobacter freundii）HBUZL-1, depositary institution CGMCC, preserving number are CGMCC No.10335, preservation date
On January 9th, 2015.
2. application of the electricity production bacterium of a kind of degraded cellulose in microbiological fuel cell, it is characterised in that comprise the following steps：
A, aseptically, citrobacter freundii HBUZL-1 is inoculated in cellulosic pulp culture medium, 30 DEG C of culture 6-
8 h, cultivate to exponential phase, must be inoculated with bacterium, save backup；The composition of the cellulose culture medium is：20 parts of cellulose,
Na2HPO41000 parts of 1.5 parts, 2.5 parts of peptone, 0.5 part of yeast extract and distilled water, the pH value of the cellulose culture medium is
7.0-7.2；Citrobacter freundii HBUZL-1, depositary institution CGMCC, preserving number are CGMCC No.10335, preservation date
On January 9th, 2015；
B, structure microbial fuel cells system, the system include cathode chamber, anode chamber, PEM and external circuit electricity
Resistance；Anolyte is housed in the anode chamber, cathode chamber is equipped with catholyte, and the anolyte composition is：Cellulose 276mg/L,
NaHCO3 3.13g/L、NH4Cl 0.31g/L、Na2HPO42.75g/L、（NH4）2SO4 0.56g/L、MgSO4 0.2g/L、KCl
0.13g/L 、CaCl2 15mg/L、MnSO4 20mg/L、FeCl31mg/L；The catholyte is constituted：25mmol/L K3
[Fe(CN)6], volume ratio is 1:1 NaH2PO4And Na2HPO4Each 50 mmol/L；
C, take and be collected by centrifugation under the inoculation bacterium 5mL, aseptic condition, be inoculated in anode chamber, produced electricity.
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| CN201510046842.5A CN104611262B (en) | 2015-01-30 | 2015-01-30 | A kind of electricity production bacterium of degraded cellulose and its application in a fuel cell |
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