CN104611262A - Electricity-producing bacterium capable of degrading cellulose and application of electricity producing bacterium in fuel cells - Google Patents

Electricity-producing bacterium capable of degrading cellulose and application of electricity producing bacterium in fuel cells Download PDF

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CN104611262A
CN104611262A CN201510046842.5A CN201510046842A CN104611262A CN 104611262 A CN104611262 A CN 104611262A CN 201510046842 A CN201510046842 A CN 201510046842A CN 104611262 A CN104611262 A CN 104611262A
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citrobacter freundii
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赵丽坤
李景晨
李红梅
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Hebei University
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Abstract

The invention discloses an electricity-producing bacterium capable of degrading cellulose and an application of the electricity-producing bacterium in fuel cells. The strain of the electricity-producing bacteria is collected as citrobacter freundii HBUZL-1 in CGMCC with the collection number of CGMCC No.10335 on January 9, 2015; the 16SrDNA gene sequence of the strain is SEQ ID NO: 1. The electricity-producing bacteria are easy to culture; a plurality of organic matters can be taken as a substrate, and more importantly, the rich cellulose can be taken as the substrate for generating electricity; if being applied to the microbial fuel cells, the electricity-producing bacteria are high in adaptability and high in electricity-producing voltage, and have relatively high economic value and wide application prospect.

Description

A kind of electrogenesis bacterium of degraded cellulose and application in a fuel cell thereof
Technical field
The present invention relates to technical field of microbial fuel battery, specifically a kind of electrogenesis bacterium of degraded cellulose and application in a fuel cell thereof.
Background technology
Microbiological fuel cell (MFC) is using microorganism as reactive agent, utilizes enzyme or microorganism as anode catalyst, by its metabolism, oxidation operation is converted into the device of electric energy.It belongs to the biochemical conversion technology in biomass energy utilization technologies, its not only pollution-free, efficiency is high, reaction conditions is gentle, and fuel source is extensive.Therefore, microbiological fuel cell has become the problem that countries in the world fall over each other to study.
China is large agricultural country, biomass resource is very abundant, and various farm crop produce stalk more than 600,000,000 ton every year, about 400,000,000 tons that wherein can use as the energy, if can in this, as the substrate raw material of electricity generation by microorganism, then China's electricity generation by microorganism has very wide development space.
At present, the electrogenesis microorganism majority be separated is bacterium, amphimicrobian.From the type analysis of the final electron acceptor(EA) of anaerobic respiration, they belong to different function yeast, such as: with Fe (III) or Mn(IV) be the Pseudomonas such as metal reducing miroorganisms Shewanella, Geobacter and Geopsychrobacter of the final electron acceptor(EA) of respiratory chain; Take vitriol as Pseudomonas such as sulphate reducing bacteria desulfoblbus, desulfovibrio and desulfuromon of electron acceptor(EA); Take nitrate as Pseudomonas such as nitrate reduction bacterium pseudomonsa, ochrobactrum and comamonas of electron acceptor(EA); In addition, the intestinal bacteria of anaerobically fermenting, the yeast carrying out photosynthetic rhodopseudomona, green alga chlamydomonas and eucaryon all shows electrogenesis characteristic.But, can be that the electrogenesis bacterium report of substrate is few with Mierocrystalline cellulose in existing bacterial classification.As CN101728544A discloses the application of a kind of citrobacter freundii in electricity generation by microorganism and electricity-generating method, provide a kind of citrobacter freundii LY-3, deposit number is CGMCC No.3246, this bacterial strain can be applied to electricity generation by microorganism as the anode catalyst of microbiological fuel cell, but the fuel that the new strains of this technology utilizes is glucose and sodium acetate, and its desired raw material cost is higher.And for example CN100588019C discloses microbiological fuel cell and utilizes the method for straw power generation; with cellulose degradation mixed bacterial, process generating is carried out to stalk; this technology is pollution-free, cost is low; alleviate energy dilemma, make straw from village obtain effective utilization, but; its processing step is loaded down with trivial details; the best that mixed-culture medium can not meet all bacterial classifications in mixed bacterial completely cultivates demand, and it is limited to the degradation capability of stalk, and its electric transformation efficiency is relatively low.Visible, can develop a kind of take Mierocrystalline cellulose as the efficient electrogenesis bacterium of degraded substrate, and is applied to microbiological fuel cell, is one of heat subject of studying in the industry of current line.
Summary of the invention
Object of the present invention is just to provide a kind of electrogenesis bacterium and application in a fuel cell thereof of degraded cellulose, a kind of new bacterial strain is provided to select to utilizing Mierocrystalline cellulose electrogenesis for industrial community, also for industrial community utilizes Mierocrystalline cellulose electrogenesis to provide a kind of new application method, also solve bacterial strain that existing microbial battery adopts simultaneously and cannot utilize Mierocrystalline cellulose electrogenesis or the poor problem of its electricity generation performance.
The object of the present invention is achieved like this:
The invention provides a kind of electrogenesis bacterium of degraded cellulose, described bacterial strain preservation title be citrobacter freundii ( citrobacter freundii) HBUZL-1, depositary institution CGMCC, preserving number is CGMCC No.10335, preservation date on January 9th, 2015; Depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
The citrobacter freundii of degraded cellulose provided by the present invention ( citrobacter freundii) HBUZL-1, its biological property is as follows: be gram negative bacillus, bacterium colony shows stick-slip, and protuberance, translucent, diameter 3 about mm, edge is irregular.
Citrobacter freundii of the present invention ( citrobacter freundii) the 16S rDNA sequence of HBUZL-1 is as shown in SEQ ID NO: 1.
Invention also provides citrobacter freundii ( citrobacter freundii) HBUZL-1 application in a fuel cell.
More particularly, citrobacter freundii of the present invention ( citrobacter freundii) HBUZL-1 application method in a fuel cell comprises the following steps:
A, aseptically, citrobacter freundii HBUZL-1 to be inoculated in cellulosic pulp substratum, to cultivate 6-8 h, be cultured to logarithmic phase, must bacterium be inoculated, save backup for 30 DEG C; Consisting of of described Mierocrystalline cellulose substratum: Mierocrystalline cellulose 20 parts, Na 2hPO 41.5 parts, peptone 2.5 parts, yeast extract paste 0.5 part and distilled water 1000 parts, the pH value of described Mierocrystalline cellulose substratum is 7.0-7.2;
B, structure microbial fuel cells system, this system comprises cathode compartment, anolyte compartment, proton exchange membrane, external resistance case and data acquisition unit; In described anolyte compartment, anolyte is housed, cathode compartment is equipped with catholyte, and described anolyte consists of: Mierocrystalline cellulose 276mg/L, NaHCO 33.13g/L, NH 4cl 0.31g/L, Na 2hPO 42.75g/L, (NH 4) 2sO 40.56g/L, MgSO 40.2g/L, KCl 0.13g/L, CaCl 215mg/L, MnSO 420mg/L, FeCl 31mg/L; Described catholyte consists of: the K of 25mmol/L 3[Fe (CN) 6], volume ratio is the NaH of 1:1 2pO 4and Na 2hPO 4each 50 mmol/L;
C, get described inoculation bacterium 5mL, collected by centrifugation under aseptic condition, is inoculated in anolyte compartment, carries out electrogenesis.
The invention provides a strain electrogenesis new strains-citrobacter freundii ( citrobacter freundii) HBUZL-1, this bacterial strain is easy to cultivate, and can not only take gas chromatography as fuel, more importantly can with resourceful Mierocrystalline cellulose for fuel generates electricity, be applied in microbiological fuel cell, its adaptability is strong, electrogenesis voltage large, has higher use value.
Electrogenesis new strains provided by the invention is applied in microbiological fuel cell, its processing step is simple, easy to operate, only need add single culture can be just that substrate generates electricity with Mierocrystalline cellulose, large, the electric transformation efficiency of its electrogenesis voltage is high, practical, has higher economic worth and vast potential for future development.
The suggestion method for preserving of citrobacter freundii HBUZL-1 disclosed by the invention: lyophil preservation method.
Accompanying drawing explanation
Fig. 1 is glucose standard curve figure.
Fig. 2 is citrobacter freundii HBUZL-1 Congo Red test observation figure.
Fig. 3 is the Electronic Speculum figure that citrobacter freundii HBUZL-1 is cultured to logarithmic phase.
Fig. 4 is that citrobacter freundii HBUZL-1 and associated germline are united to grow and set.
Fig. 5 is the cyclic voltammetry curve of citrobacter freundii HBUZL-1.
Fig. 6 is that citrobacter freundii HBUZL-1 utilizes Mierocrystalline cellulose to produce voltage graphic representation over time.
Embodiment
Embodiment is for further describing the present invention below, but does not limit the present invention in any form.
The acquisition of embodiment 1 bacterial strain
(1) abstraction and purification of bacterial strain: source take carboxymethyl cellulose as the anode of the bipolar chamber circulation MFC of fuel from steady running 6 months, cut a small amount of carbon cloth, being put into the mass percent concentration being added with granulated glass sphere is in the NaCl solution of 1%, rock gradient dilution be evenly applied to carboxymethyl cellulose be sole carbon source flat board on, be placed in anaerobic box (YQX-II, Shanghai new talent medicine equipment Manufacturing Co., Ltd) and cultivate 3 days at 30 DEG C; Continue line to be separated, preserve;
(2) screening of bacterial strain: the strains tested that step (1) is preserved is inoculated into culture medium 37 DEG C concussion and cultivates 48h, get fermented liquid 10 mL, centrifugal 10 min under 4000 r/min, supernatant liquor is as crude enzyme liquid; Pipette acetate buffer solution (0.05 mol/L, pH 4.4) 1.5 mL containing CMC-Na in test tube, add enzyme liquid 0.5 mL, after 50 DEG C of insulation 30 min, survey sugar by DNS method.In the basic conditions, be reduced into henna aminocompound after DNS solution and reducing sugar solution are total to heat, within the specific limits, the amount of reducing sugar and the shade of red-brown thing are certain proportion relation.The absorbance of red-brown material is measured under 540 nm wavelength, by typical curve, the content of reducing sugar in calculation sample.The enzyme amount that definition per minute catalyzing cellulose hydrolysis generates 1g glucose is an enzyme activity unit U.Live according to international enzyme and define, 1g solid enzyme (or 1mL liquid enzymes), in (50 ± 1) DEG C, under specific pH condition, 1h hydrolysis substrate produces the reducing sugar amount being equivalent to 1mg glucose, is a unit of activity, with μ/g(or μ/mL) represent.With reference to GB, the 1 μ g glucose amount that the thick enzyme of the every 1mL of this research definition per minute produces is an enzyme activity unit, is designated as μ/mL or U.
Glucose standard curve as shown in Figure 1, y=0.91x-0.025, R 2=0.997, enzyme activity calculation formula is:
Enzyme activity (U)=(OD+b)/a × 1000 × 2/30
Wherein: b: curve indulges intercept, 0.025; A: rate of curve: 0.91; 1000:mg is converted into μ g
According to measurement result, OD value is 0.115, and the enzyme work being obtained bacterial strain disclosed by the invention by typical curve and enzyme work formulae discovery is up to 10.32 U.
Visible, measure strains tested carboxymethylcelluloenzyme enzyme (CMCase) vigor through DNS method, enzyme live the bacterial strain that is up to 10.32 U be citrobacter freundii provided by the invention ( citrobacter freundii) HBUZL-1, and being preserved in CGMCC on January 9th, 2015, preserving number is CGMCC No.10335.
The qualification of embodiment 2 bacterial strain
To gained citrobacter freundii HBUZL-1 carry out respectively identification of morphology, Physiology and biochemistry qualification and Molecular Identification, its experimentation and result as follows.
(1) strain morphology qualification
Carry out identification of strains with reference to " authentication method that general bacterium is conventional ", adopt gram staining method basis of microscopic observation thalli morphology.Result shows, and through cultivating, the single bacterium colony obtaining to produce on the flat board that carboxymethyl cellulose is sole carbon source transparent circle is some, and its Congo Red test the results are shown in Figure 2.Learnt by the strain morphology qualification after cultivating: the citrobacter freundii of degraded cellulose provided by the present invention ( citrobacter freundii) HBUZL-1 is gram negative bacillus, bacterium colony shows stick-slip, protuberance, translucent, diameter 3 about mm, and edge is irregular; By isolated strains liquid medium within (carboxymethyl cellulose 20 g, Na 2hPO 41 .5 g, peptone 2.5 g, yeast extract paste 0.5g, distilled water 1000ml, pH value is 7.0-7.2) in cultivate 12 h to exponential phase of growth, with transmission electron microscope observation and shooting, see Fig. 3.
(2) Physiology and biochemistry qualification
To citrobacter freundii ( citrobacter freundii) HBUZL-1 has carried out sugar fermentating test, methyl red test, acetylmethyl formic acid tests (V.P. test), indole test, nitrate reduction test, gelatin hydrolysis are tested, H 2o 2the Physiology and biochemistry qualifications such as enzymatic determination test.Its qualification result is in table 1.
Table 1 bacterial strain bio-chemical characteristics result
Note: "+" experimental result is positive, "-" experimental result is negative.
(3) Molecular Identification
Adopt general 27F/ 1492R primer (upstream primer: 5 '-AGAGTTTGATCMTGGCT2CAG-3 '; Downstream primer: 5 '-GGYTACCTTGT2TACGACTT-3 ') carry out the pcr amplification of 16SrDNA.PCR reaction system: template DNA 50 ng, Taq archaeal dna polymerase 0.6 μ L, Mg 2+(1.3 mmol/ L) 3.2 μ L, dNTP 4 μ L, Buffer 3.2 μ L, each 4 μ L of upstream and downstream primer, 19 μ L water.Pcr amplification reaction program: 94 DEG C of denaturation 5 min; 94 DEG C of sex change 1 min, 55 DEG C of annealing 30 s, 72 DEG C extend 1.5 min, 29 circulations; 72 DEG C stop extension 10 min.Pcr amplification product, after agarose gel electrophoresis, cuts gel band; After carrying out purifying according to the operation steps of PCR primer purification kit, send Beijing three to win the order-checking of polygala root biotechnology company limited, record the 16S rDNA sequence of this bacterial strain as shown in SEQ ID NO: 1; Submit the sequence obtained to NCBI(www.ncbi.nlm.gov) carry out Blast comparison, transfer the correlated series that homology is higher, compare with clustalX software, adopt Mega software to carry out Phylogenetic Analysis, adopt adjacent method constructing system evolutionary tree, see Fig. 4.As can be seen from Figure 4, this bacterial strain 16SrDNA gene is carried out to PCR amplification and checks order, in its sequence and Gen-bank, known sequence compares, and result shows this bacterial strain 16SrRNA gene and citrobacter freundii (Citr obacter freuudii)it is KM272633 that 16SrDNA genetic homology is greater than 99%, GenBank reception number.According to the morphological structure of bacterial strain, physiological and biochemical property and bacterial strain 16S rDNA the sequencing results, determine that this bacterial strain belongs to citric acid Pseudomonas.
The application of embodiment 3 electrogenesis bacterium in biofuel cell
Build microbiological fuel cell (MFC) system by existing method, this system comprises cathode compartment, anolyte compartment, resistance box and proton exchange membrane, and its electrode materials is carbon fiber felt; Two interpolars are connected (ZX21,0-100000 Ω) with a precision resister case by copper conductor, connect a slice proton exchange membrane (Ultrex CMI7000, MI Mem. Int. Inc.) between two Room by short side pipe.
(1) the electroactive detection of citrobacter freundii HBUZL-1
By MFC reactor and anolyte after 121 DEG C of sterilizing 20min, at anolyte compartment's inoculation citrobacter freundii HBUZL-1, bring into operation and adopt data collecting system (DAM-3058R voltage acquisition module, Beijing Altay Science and Technology Ltd.), 1min records primary voltage, when output voltage reduces to below 0.3V, change anode fuel, temperature is room temperature (about 25 DEG C).Investigate its electrogenesis characteristic after steady running, peak power is obtained by polarization curve, when voltage stable output, by changing the resistance (0-90000 Ω) of external circuit, and stabilized voltage when being recorded in different external resistance.
Anolyte: glucose (2g/L), NaHCO 3(3.13g/L), NH 4cl(0.31g/L), Na 2hPO 4(2.75g/L), (NH 4) 2sO 4(0.56g/L), MgSO 4(0.2g/L), KCl(0.13g/L), CaCl 2(15mg/L), MnSO 4(20mg/L), FeCl 3(1mg/L); Anolyte also can be: sodium acetate (3g/L), NaHCO 3(3.13g/L), NH 4cl(0.31g/L), Na 2hPO 4(2.75g/L), (NH 4) 2sO 4(0.56g/L), MgSO 4(0.2g/L), KCl(0.13g/L), CaCl 2(15mg/L), MnSO 4(20mg/L), FeCl 3(1mg/L).
The K of catholyte: 25mmol/L 3[Fe (CN) 6], volume ratio is the NaH of 1:1 2pO 4and Na 2hPO 4each 50mmol/L.
Battery operation, to voltage plateau, is to electrode with anode carbon felt electrode, and cathode electrode is working electrode, Ag/AgCl is reference electrode, adjustment sweep limit, scanning speed etc., the cyclic voltammetry curve (CV) of electrode is measured with electrochemical workstation (Shanghai occasion China, CHI660C).Regulate initial potential E 0(V): 0.5, peak potential E 1(V): 0.5, peak potential E 2(V) :-0.7, sampling interval (V): 0.001, scanning speed (V/s) 0.002.
Experimental result is shown in Fig. 5, and Fig. 5 take glucose as the cyclic voltammetry curve that substrate measures, and it is that substrate acquired results is similar with it with sodium acetate, and cyclic voltammetry curve shows obvious redox peak as seen from the figure.
(2) with citrobacter freundii HBUZL-1 be electrogenesis bacterium, Cellulose,ether with glycolic acid for substrate electricity generation performance measure
Experimental procedure is:
A, aseptically, citrobacter freundii HBUZL-1 is inoculated in cellulosic pulp substratum (carboxymethyl cellulose 20 g, Na 2hPO 41 .5 g, peptone 2.5 g, yeast extract paste 0.5g, distilled water 1000ml, pH value is 7.0-7.2) in, cultivate 6-8 h, make cell be in logarithmic phase, must bacterium be inoculated, save backup for 30 DEG C;
B, anolyte compartment load anolyte, cathode compartment load catholyte, anolyte consists of: carboxymethyl cellulose 276 mg/L, NaHCO 33.13g/L, NH 4cl 0.31g/L, Na 2hPO 42.75g/L, (NH 4) 2sO 40.56g/L, MgSO 40.2g/L, KCl 0.13g/L, CaCl 215mg/L, MnSO 420mg/L, FeCl 31mg/L; Catholyte is identical with the catholyte in the step (1) of this example;
C, get inoculation bacterium 5mL, aseptic lower collected by centrifugation, adds electrogenesis in the anolyte compartment being inoculated into microbiological fuel cell;
D, resistance box is connected external circuit, adopt described data collecting system to carry out data gathering.
The experimental result of gained is shown in Fig. 6, and Fig. 6 shows, take carboxymethyl cellulose as substrate, citrobacter freundii HBUZL-1 has good electricity generation performance, and through domestication after a while, maximum voltage can reach 600mV.

Claims (4)

1. an electrogenesis bacterium for degraded cellulose, is characterized in that, described bacterial strain preservation title be citrobacter freundii ( citrobacter freundii) HBUZL-1, depositary institution CGMCC, preserving number is CGMCC No.10335, preservation date on January 9th, 2015.
2. the electrogenesis bacterium of degraded cellulose according to claim 1, is characterized in that, the nucleotides sequence of the 16S rDNA of described bacterial strain is classified as SEQ ID NO: 1.
3. the application of electrogenesis bacterium in microbiological fuel cell of a degraded cellulose as claimed in claim 1.
4. the application of electrogenesis bacterium in microbiological fuel cell of degraded cellulose according to claim 3, is characterized in that, comprise the following steps:
A, aseptically, citrobacter freundii HBUZL-1 to be inoculated in cellulosic pulp substratum, to cultivate 6-8 h, be cultured to logarithmic phase, must bacterium be inoculated, save backup for 30 DEG C; Consisting of of described Mierocrystalline cellulose substratum: Mierocrystalline cellulose 20 parts, Na 2hPO 41.5 parts, peptone 2.5 parts, yeast extract paste 0.5 part and distilled water 1000 parts, the pH value of described Mierocrystalline cellulose substratum is 7.0-7.2;
B, structure microbial fuel cells system, this system comprises cathode compartment, anolyte compartment, proton exchange membrane and external resistance; In described anolyte compartment, anolyte is housed, cathode compartment is equipped with catholyte, described anolyte consists of: Mierocrystalline cellulose 276mg/L, NaHCO 33.13g/L, NH 4cl 0.31g/L, Na 2hPO 42.75g/L, (NH 4) 2sO 40.56g/L, MgSO 40.2g/L, KCl 0.13g/L, CaCl 215mg/L, MnSO 420mg/L, FeCl 31mg/L; Described catholyte consists of: the K of 25mmol/L 3[Fe (CN) 6], volume ratio is the NaH of 1:1 2pO 4and Na 2hPO 4each 50 mmol/L;
C, get described inoculation bacterium 5mL, collected by centrifugation under aseptic condition, is inoculated in anolyte compartment, carries out electrogenesis.
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CN105655598A (en) * 2015-12-30 2016-06-08 中山大学 Method for in-situ immobilization of microbiological fuel cell anode microbes
CN105655598B (en) * 2015-12-30 2018-02-16 中山大学 A kind of method of fixation in situ anode of microbial fuel cell microorganism

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