CN103509735A - Tolumonas osonensis bacterial strain with electrogenesis characteristic, and applications thereof in microbial fuel cells - Google Patents
Tolumonas osonensis bacterial strain with electrogenesis characteristic, and applications thereof in microbial fuel cells Download PDFInfo
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- CN103509735A CN103509735A CN201310244780.XA CN201310244780A CN103509735A CN 103509735 A CN103509735 A CN 103509735A CN 201310244780 A CN201310244780 A CN 201310244780A CN 103509735 A CN103509735 A CN 103509735A
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Abstract
The invention relates to Tolumonas osonensis P2-A-1 bacterial strain with electrogenesis characteristic. The bacterial strain is obtained by steps of enriching, separating, purifying and screening from sludge of sewage treatment plant; belongs to Tolumonas osonensis species, Tolumonas genus; and is named as Tolumonas osonensis P2-A-1. The bacterial strain is preserved in China General Microbiological Culture Collection Center, on May, 2013, and the preservation number is CGMCC No.7562. The bacterial strain possesses following characteristics: short rod-liked mycelial morphology; 2.4 to 3.6 [mu]m in length, and 0.9 [mu]m in width; the bacterial strain is distributed separately or in pairs, possesses no flagellum, is capable of growing with or without oxygen; no bacterial spore is produced; gram negative; at a temperature of 22 DEG C, after 48h of culturing on tryptone soybean broth plating medium, white bacterial colonies are formed, are round in shape, are easy to be stirred up, and are nontransparent, the edge is smooth, and diameter is 2 to 4mm; the bacterial strain is capable of using a plurality of substrates such as glucose and sodium acetate. The bacterial is used for electrogenesis in microbial fuel cells, electrogenesis capacity is relatively high, and power density reaches 509mW/m<2>. The electrogenesis capacity of T.osonensisa, and applications of T.osonensisa in microbial fuel cells are disclosed for the first time.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of electrogenesis characteristic of Tolumonas osonensis bacterial strain and the applied research in microbiological fuel cell thereof.
Background technology
Along with the development of society and economy, energy consumption will increase year by year.A large amount of consumption of the Nonrenewable energy resources sustainable growths such as coal, oil, Sweet natural gas, not only make the pressure of facing mankind resource exhaustion, have also brought the problems such as global warming, acid rain increase simultaneously.Therefore, for stablizing of mankind's energy, supply lastingly, realize economic sustainable growth, change the production and consumption mode of the energy, developing renewable energy source supply mode new, environmental sound, non-petroleum-type is the main thought of future source of energy development.
Microbiological fuel cell (Microbial Fuel Cells, MFCs) is a kind ofly utilize microorganism to do catalyst oxidation organism and directly chemical energy changed into the new device of electric energy.It is in conjunction with fuel cell and biology techniques means, realized biological chemistry can with the direct conversion of electric energy, be a kind of clean sustainable energy, become gradually the focus of new energy development.
The basic structure of MFCs and other types fuel cell similar ，You anolyte compartment and cathode compartment form.Organism in anolyte compartment is under anaerobic oxidized to meta-bolites and is discharged electronics and proton by bacteria metabolism effect; Electronics is delivered to anode surface by microorganism, then through external circuit, is transmitted to negative electrode by anode, and proton arrives cathode compartment by proton membrane simultaneously; In cathode compartment, there is reduction reaction in electronics, proton and electron acceptor(EA) (being generally air or dissolved oxygen), and product is H
2o.The electronics producing in anolyte compartment so is constantly transmitted, is consumed, thereby form loop line, produces sustained current, has so just realized the generation of organic removal and electric current simultaneously.
Electrogenesis microorganism (Electricigens) refers to that those under anaerobic can become CO by complete oxidation organism
2, then the electronics producing in oxidising process is delivered to the microorganism of generation current on electrode by electron transport chain.The microorganism that can move MFCs electrogenesis under without amboceptor condition of having reported at present comprises bacterium class and Mycophyta.Wherein, bacterium class electrogenesis microorganism mainly comprises genus Shewanella (Shewanella), ground Bacillaceae (Geobacter), pseudomonas Pseudomonas (Pseudomonas), arc Pseudomonas (Arcobacter), the electrogenesis bacterium of hydrogen-producing bacteria family is (as clostridium butylicum (Clostridium butyricum), enteroaerogen (Enterobacter aerogenes), iron 3b vat red 3b spirillum (Rhodoferax ferrireducens), human pallid bacillus (Ochrobactrum anthropi), cryophilic bacteria (Geopsychrobacter electrodiphilus), Cray Bai Shi pulmonitis strain (Klebsiella pneumoniae L17) etc.The electrogenesis microorganism of Mycophyta mainly comprises Hansenula anomala (Hansenula anomala), swamp Rhodopseudomonas (Rhodopseudomonas palustris), chlorella (Chlorellavulgaris) etc.Electrogenesis microorganism is biological catalyst important in microbiological fuel cell, directly affects efficiency of fuel cell generation.Therefore excavate the microorganisms with this function for the diversity of enriching electrogenesis microorganism more, improve efficiency of fuel cell generation significant.
Tolumonas osonensis is a novel species of the toluene zygosaccharomyces (Tolumonas) of discovery in 2011.Cell dyeing is Gram-negative.Amphimicrobian, optimum growth temperature and pH are respectively 22 ℃ and 7.5.At present, in Tolumonas osonensis kind, electrogenesis characteristic and being applied in aspect microbiological fuel cell thereof of microorganism not yet found relevant report both at home and abroad.
Summary of the invention
The object of the present invention is to provide a kind of new bacterial strain with electricity generation ability---Tolumonas osonensis P2-A-1 and the application in microbiological fuel cell thereof.
The present invention, by the mud of sewage work is carried out to enrichment, separation, purifying and screening, obtains a strain Tolumonas osonensis P2-A-1.This bacterial strain can be used as the anode catalyst of microbiological fuel cell, carries out catalytic substrate electrogenesis, thereby has realized object of the present invention.
The bacterial strain called after Tolumonas osonensis P2-A-1 that the present invention obtains, in May, 2013, be kept at (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences), China Committee for Culture Collection of Microorganisms's common micro-organisms center, it is referred to as CGMCC, and deposit number is CGMCC7562.
Tolumonas osonensis P2-A-1 of the present invention, through microscopic examination, staining reaction, the experiment of conventional physiological and biochemical property, cell fatty acid chemical composition analysis and the 16S rDNA sequence alignment of bacterium colony and form, is accredited as Tolumonas osonensis.There is following characteristics: (1) thalli morphology is rod-short, 2.4-3.6 μ m (length) * 0.9 μ m (wide), single or existence in pairs, whole body atrichia, without gemma; (2) Gram-negative; (3) can under anaerobism and aerobic conditions, grow; (4) on 22 ℃, pancreas peptone soybean broth plate culture medium, cultivate 48h, form circular, easy picking, opaque, the white colony of neat in edge, diameter 2-4mm; (5) catalase, gelatine liquefication experiment, urease experimental result are all positive, and hydrogen sulfide experiment and indoles experimental result are all negative; (6) can utilize glucose, lactose, maltose, Sodium Propionate, sodium acetate, Sodium.alpha.-hydroxypropionate, sodium succinate, ethanol, glycerine, fumarate, pyruvate salt as substrate; (7) the 16S rDNA gene of this bacterial strain has the nucleotide sequence as shown in sequence table, and sequence length is 1425bp, and the 16S rDNA gene order input GenBank by obtaining, compares analysis by Blast program to all sequences in database.Found that the bacterial strain Tolumonas osonensis OCF7 of the 16S rDNA gene order of P2-A-1 bacterial strain of the present invention and toluene zygosaccharomyces (Tolumonas)
thave higher similarity (accession number on Genbank is GU370947), homology reaches 99%.
According to above-mentioned form, physio-biochemical characteristics and the result for retrieval of 16S rDNA gene order in GenBank thereof, identify that this bacterial strain belongs to Tolumonas osonensis.
Bacterial strain Tolumonas osonensis P2-A-1 of the present invention can utilize the multiple substrates such as glucose and sodium acetate to be directly used in microbiological fuel cell to carry out electrogenesis, and this is the bacterial strain that in this kind, reported first has electrogenesis characteristic.This bacterial strain has very high electrogenesis activity in microbiological fuel cell simultaneously, has important prospects for commercial application and theoretical investigation and is worth.
Accompanying drawing explanation
Fig. 1 is the colonial morphology figure of bacterial strain Tolumonas osonensis P2-A-1 of the present invention;
Fig. 2 is the gramstaining result of bacterial strain Tolumonas osonensis P2-A-1 of the present invention;
Fig. 3 is the thalline scanning electron microscope (SEM) photograph of bacterial strain Tolumonas osonensis P2-A-1 of the present invention;
Fig. 4 is the power density schematic diagram of bacterial strain Tolumonas osonensis P2-A-1 of the present invention in microbiological fuel cell.
Embodiment
The separation and purification of microorganism on embodiment 1:MFCs anode microbial film
Get the mud of TEDA in Tianjin's sludge of sewage treatment plant between concentrated, after simple elutriation and precipitation process, remove the throw out of inorganicization of bottom and the clear water on top layer, in the middle of getting water ratio at the mud of 95% left and right as inoculum, (mud 10mL, mineral solution 625 μ L, vitamin solution 250 μ L, 100mM PBS buffered soln 5mL, glucose 0.05g mend distilled water to 50mL with mud nutritive medium.) add together in the reactor of single chamber MFCs.Reactor body is a long 2.0cm of being, the synthetic glass cylinder that cross-sectional diameter is 3.0cm, and useful volume is 14mL, the useful area of anode and cathode is 7cm
2.Anode and negative electrode are placed in respectively two surveys of MFCs main body, and fix with seal with elastometic washer.Anode one end covers with plexiglass cover, and the lid of negative electrode one end is opening, and negative electrode carbon cloth carries platinum, and negative electrode is all connected with titanium silk electronics is derived or imported with anode, and last whole device is tightened fixing with screw and leading screw.Reactor successfully constructs rear airtight anolyte compartment, and system is moved continuously 14d left and right at 30 ℃, and the voltage at pull-up resistor two ends is stable gradually, forms macroscopic thick microbial film on anode.By the whole thalline inoculating needle scraping on this microbial film, and be suspended in and in sterilized water, be prepared into uniform bacteria suspension, adopt 10 times of stepwise dilution methods using suitable dilution bacterium earnestly liquid coat and add ironic citrate as electron donor (final concentration is 5.000g/L), in the PBBM solid medium of glucose as substrate (final concentration is 1.000g/L), under 30 ℃ of anaerobic conditions, cultivate 3d, the variform bacterium colony growing on flat board is separated with the line of carrying out repeatedly under culture condition at above-mentioned identical substratum, finally obtain a plurality of purebred strain isolateds, be numbered and named.
The electricity generation performance analysis of purebred microorganism on embodiment 2:MFCs anode microbial film
Each strain isolated that picking embodiment 1 obtains, be inoculated into respectively ironic citrate as in electron donor (final concentration is 5.000g/L), the PBBM liquid nutrient medium of glucose as substrate (final concentration is 1.000g/L), 22 ℃, standing cultivation 48h under aerobic conditions.The nutrient solution of each strain isolated, under 4 ℃ of conditions, is collected to thalline after the centrifugal 20min of 6000r/min.
Respectively the thalline of the equal in quality of different isolates being inoculated in to anolyte 1 (adds ironic citrate as electron donor (final concentration is 5.000g/L) in PBBM liquid nutrient medium, glucose is as substrate (final concentration is 1.000g/L), pH=7.2, specific conductivity is 7.8mS/cm.), the inoculation liquid obtaining is injected MFCs reactor, and reactor connects with external resistance 1000 Ω and data collector respectively, under room temperature, moves.In operational process, need to regularly replace inoculation liquid (having inoculated the anolyte of corresponding strain isolated thalline), general 24h changes once, during each replacing, first the former inoculation liquid in MFCs is taken out, then add fresh inoculation liquid, question response device occurs that the stabilized voltage of continuous three can be thought and starts successfully.After starting successfully, enter the commencement of commercial operation phase, when the output voltage of monitoring is during lower than 50mV, only need to change anolyte, when output voltage again reaches the highest and tends towards stability, carry out the mensuration of polarization curve and power density curve, stop afterwards MFCs operation.In whole process, utilize PISO-813 type data collecting system to carry out real-time online data monitoring and record to voltage, get the mean value in 30min during analytical data, sampling precision is 0.001V.The electricity generation performance of each strain isolated in MFCs the results are shown in Table 1.
Table 1
Embodiment 3: the 16S rDNA sequencing analysis of bacterial strain P2-A-1
Adopt bacterial genomes DNA extraction test kit, the genomic dna of extracting bacterial strain P2-A-1 is as masterplate, and the universal primer (27F, 1492R) of bacterial 16 S r DNA is as primer, the 16S rDNA fragment of amplification bacterial strain P2-A-1.The 16S rDNA gene of this bacterial strain has the nucleotide sequence as shown in sequence table, sequence length is 1425bp, by the 16S rDNA gene order input GenBank obtaining, by Blast program, all sequences in database is compared to analysis, found that the bacterial strain Tolumonas osonensisOCF7 of the 16S rDNA gene order of P2-A-1 bacterial strain of the present invention and toluene zygosaccharomyces (Tolumonas)
thave higher similarity (accession number on Genbank is GU370947), homology reaches 99%.
Embodiment 4: the biological assay of bacterial strain P2-A-1
1. the morphological specificity of bacterial strain
(1) colony morphology characteristic: bacterial strain P2-A-1 at ironic citrate as electron donor (final concentration is 5.000g/L), on the PBBM solid medium of glucose as substrate (final concentration is 1.000g/L), 30 ℃ of anaerobism are cultivated after 3d, bacterium colony is rounded, pink projection, smooth surface, grease-like, diameter is 2mm.
According to the result of embodiment 3, bacterial strain P2-A-1 inoculation, to being applicable to, on the pancreas peptone soybean broth plate culture medium of Tolumonas osonensis, cultivating 48h under 22 ℃ of aerobic conditions, is formed to circular, easy picking, opaque, the white colony of neat in edge, diameter 2-4mm.As shown in Figure 1.
(2) morphological features: the Gram-stained result of bacterial strain P2-A-1 shows that this bacterial strain is Gram-negative bacteria (seeing Fig. 2).Utilize sem observation bacterial strain, thalli morphology is rod-short, 2.4-3.6 μ m (length) * 0.9 μ m (wide), and single or existence in pairs, whole body atrichia, without gemma (being shown in Fig. 3).
2. the Physiology and biochemistry of bacterial strain experiment
Consult handbooks such as " Preface to the First Edition ofBergey ' s Manual of Systematic Bacteriology ", and in conjunction with the bacterial strain T.osonensis OCF7 of bibliographical information
tphysiological and biochemical property, select following experimental project to operate, comprise the experiment of gelatine liquefication experiment, hydrogen sulfide, indoles experiment, catalase experiment and substrate utilization experiment etc.
Table 2
Remarks: "+" represents positive; "-" represents negative
Table 3
Remarks: "+" representative can utilize; "-" representative can not utilize
From table 2 and table 3, the catalase of bacterial strain P2-A-1, gelatine liquefication experiment, urease experimental result are all positive, and hydrogen sulfide experiment and indoles experimental result are all negative.This bacterial strain can utilize glucose, lactose, maltose, Sodium Propionate, sodium acetate, Sodium.alpha.-hydroxypropionate, sodium succinate, glycerine, ethanol, fumarate, pyruvate salt as substrate.The bacterial strain T.osonensis OCF7 reporting in this result and document
tphysiological and biochemical property consistent.
The 16S rDNA sequence alignment of the morphologic observation of process bacterial strain, physiological and biochemical property experiment and embodiment 3, bacterial strain P2-A-1 is accredited as Tolumonas osonensis.Its judgement reference is: Matthew E.Caldwell, Toby D.Allen, Paul A.Lawson and Ralph S.Tanner.Tolumonas osonensis sp.nov., isolated from anoxic freshwater sediment, and emended description of the genus Tolumonas.International Journal of Systematic and Evolutionary Microbiology (2011), 61,2659-2663.
Embodiment 5: bacterial strain P2-A-1 is at the electrogenesis of MFCs
By the optimization of the conditions such as buffered soln in thalline starting point concentration, anolyte substrate kind and concentration, anolyte pH, external resistance and anolyte in inoculation liquid, and utilize chemical agent to carry out permeabilized processing to improve the output rating of MFCs to cell.Result shows, bacterial strain P2-A-1 is inoculated in the 100mL triangular flask that 50mL pancreas peptone soybean broth substratum is housed, standing cultivation 48h (OD under 22 ℃ of aerobic conditions
600=2.714), the thalline of centrifugal collection, after 1mM EDTA processes 20min, is inoculated in anolyte II, in PBBM liquid nutrient medium, adds sodium acetate as substrate (final concentration is 2.000g/L), ironic citrate is as electron donor (final concentration is 5.000g/L), pH7.2.The inoculation liquid that the stem cell concentration preparing is 3.394g/L, injects MFCs reactor, and external 1000 Ω resistance move 14d under room temperature, and generation power density is 509mW/m
2, than the 194mW/m before optimizing
2, improve 162.4%.
Claims (5)
1. the bacterial strain Tolumonas osonensis P2-A-1 with electricity generation ability, it is characterized in that: the 16S rDNA gene of this bacterial strain has the nucleotide sequence as shown in sequence table, sequence length is 1425bp, accession number on Genbank is GU370947, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNo.7562.
2. bacterial strain Tolumonas osonensis P2-A-1 according to claim 1, is characterized in that: this bacterial strain obtains a strain gram negative bacterium from the mud of sewage work through enrichment, separation, purifying and screening.
3. bacterial strain Tolumonas osonensis P2-A-1 according to claim 1, is characterized in that: can under anaerobism and aerobic conditions, grow; On 22 ℃, pancreas peptone soybean broth plate culture medium, cultivate 48h, form circular, easy picking, opaque, the white colony of neat in edge, diameter 2-4mm; Thalli morphology is rod-short, 2.4-3.6 μ m (length) * 0.9 μ m (wide), and single or existence in pairs, whole body atrichia, without gemma.
4. bacterial strain Tolumonas osonensis P2-A-1 according to claim 1, it is characterized in that: catalase, gelatine liquefication experiment, urease experimental result are all positive, hydrogen sulfide experiment and indoles experimental result are all negative, can utilize glucose, lactose, maltose, Sodium Propionate, sodium acetate, Sodium.alpha.-hydroxypropionate, sodium succinate, ethanol, glycerine, fumarate, pyruvate salt as substrate.
5. the bacterial strain Tolumonas osonensis P2-A-1 as previously discussed application in microbiological fuel cell, is characterized in that: described bacterial strain can utilize the multiple substrates such as glucose and sodium acetate to be directly used in microbiological fuel cell, to carry out electrogenesis.
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Cited By (5)
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| CN104332645A (en) * | 2014-09-01 | 2015-02-04 | 山东科技大学 | Microbiological fuel cell for processing lead-containing sewage |
| CN104726369A (en) * | 2015-02-16 | 2015-06-24 | 天津科技大学 | Raoultella ornithinolytica and applications thereof |
| CN105524954A (en) * | 2016-02-01 | 2016-04-27 | 天津科技大学 | Method for producing 10-deacetyl-bacratin (10-DAB) III by utilizing microbial fermentation |
| CN106011016A (en) * | 2016-06-29 | 2016-10-12 | 哈尔滨工业大学 | Indole-degrading compound bacterial agent and reaction device thereof |
| CN109825459A (en) * | 2019-03-29 | 2019-05-31 | 天津科技大学 | One plant of coupling produces the dissimilatory iron reduction bacterium of hydrogen |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104332645A (en) * | 2014-09-01 | 2015-02-04 | 山东科技大学 | Microbiological fuel cell for processing lead-containing sewage |
| CN104332645B (en) * | 2014-09-01 | 2017-08-11 | 山东科技大学 | A kind of microbiological fuel cell for handling leaded sewage |
| CN104726369A (en) * | 2015-02-16 | 2015-06-24 | 天津科技大学 | Raoultella ornithinolytica and applications thereof |
| CN104726369B (en) * | 2015-02-16 | 2018-01-05 | 天津科技大学 | One plant of solution ornithine Raoul bacterium and its application |
| CN105524954A (en) * | 2016-02-01 | 2016-04-27 | 天津科技大学 | Method for producing 10-deacetyl-bacratin (10-DAB) III by utilizing microbial fermentation |
| CN105524954B (en) * | 2016-02-01 | 2018-09-28 | 天津科技大学 | A method of producing the deacetylated baccatin IIIs of 10- using microbial fermentation |
| CN106011016A (en) * | 2016-06-29 | 2016-10-12 | 哈尔滨工业大学 | Indole-degrading compound bacterial agent and reaction device thereof |
| CN106011016B (en) * | 2016-06-29 | 2020-03-13 | 哈尔滨工业大学 | Compound microbial inoculum for degrading indole and reaction device thereof |
| CN109825459A (en) * | 2019-03-29 | 2019-05-31 | 天津科技大学 | One plant of coupling produces the dissimilatory iron reduction bacterium of hydrogen |
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