CN103275887A - Shewanella haliotis strain and its application in bioelectricity generation - Google Patents
Shewanella haliotis strain and its application in bioelectricity generation Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
- Y02E60/30—Hydrogen technology
- Y02E60/50—Fuel cells
Abstract
The invention discloses a Shewanella haliotis strain and its application in bioelectricity generation. The Shewanella haliotis strain is Shewanella haliotis Z4, is preserved by China Center for Type Culture Collection called CCTCC for short, has a preservation number of CCTCC NO:M2012444, and is preserved on Nov., 6, 2012. The strain can transmit electrons to an extracellular electron acceptor under an anaerobic condition, and can degrade organic matters and generate electric energy when the strain is inoculated to a microbial fuel cell. The Shewanella haliotis Z4 has a very strong electrochemical activity, and can generate electricity by utilizing a plurality of types of organic matters as a sole carbon source. The Shewanella haliotis Z4 has a very good application prospect in polluted environment restoration and biological energy recovery based on the above characteristics.
Description
Technical field
The invention belongs to environmental pollution biological treatment and bioenergy technical field, be specifically related to a strain Bao Xiwa Salmonella and the application in producing bioelectricity thereof.
Background technology
Enter 21 century, the environmental pollution that causes in energy shortage and the energy exploitation and application is the two big main challenges that the Sustainable development of human modern society faces.Solve this two key points that problem will be Sustainable development.And sewage recycling is just like one of model who reaches resource utilization in decontamination.
Microbiological fuel cell (MFC) is to utilize microorganism as catalyzer, with oxidation operation, chemical energy is produced the device that is converted into electric energy by its metabolism.From the energy and point of view of environment protection; microbiological fuel cell is applied to field of waste water treatment; the MFC technology is used in the wastewater treatment; when handling organic waste water, obtain electric energy; being the effective way of alleviating current energy dilemma and solving environmental problem, also is one of the hot research problem in environmental energy field.
Bacterial classification is this quality factor that influences electricity generation performance of microbial fuel cell.In recent years, increase gradually about the report that can in microbiological fuel cell, produce the microorganism of electric current.Though have been found that more new electrogenesis bacterium, its electrogenesis power is still comparatively low.And in the electricity generation performance that improves these electrogenesis microorganisms, also to investigate many substrate utilizations of bacterial classification, in order to adapt to the environment of complicated waste water, repair polytype environmental pollution.Therefore still need screen electrogenesis microorganism more efficiently.
Shiva Salmonella (Shewanella sp.) is that typical marine microorganism belongs to, be a kind of Gram-negative, amphimicrobian heterotrophic bacterium, normally separates to obtain from marine alga, fish, oceanic sediment and shellfish.Shewanella sp. is the new genus that was stood in addition again in 1985 by Macdonell, also is to breathe the most significant biology of diversity so far, and this also is the most tangible characteristic of this Pseudomonas.Someone think the Shewanella bacterium can be in MFC electrogenesis, but also only have only several Shewanella kinds to carry out electrogenesis test and checking, and a lot of new kind of separating does not also obtain the report of more checkings.
Summary of the invention
The present invention has overcome above-mentioned defective, and a kind of electrochemical activity bacterium is provided, and the mainly application in environmental pollution reparation and bioenergy recovery.
A kind of electrochemical activity bacterium provided by the present invention; it derives from mangrove forest protection center, Shenzhen mangrove forest bed mud; obtain through the electrochemistry enrichment culture of microbiological fuel cell, artificial separation and purification; this bacterium is Bao Xiwa Salmonella (Shewanella haliotis) Z4; by China's typical culture collection center preservation, be called for short CCTCC, preserving number is: CCTCC NO:M 2012444; preservation date is on November 6th, 2012, and the address is China. Wuhan. and Wuhan University.This bacterium is at marine microorganism substratum 2216(DSMZ Medium604) bacterium colony be circular, orange-yellow, the edge is smooth, and is glossy, the about 2mm of diameter.The form of observing this bacterium under the transmission electron microscope is shaft-like, is about 1.3 μ m, wide about 0.6 μ m, and peritrichous and pod membrane, the growth logarithmic phase is 5～22h.The accession number of advancing total length 16S rDNA sequence of bacterial strain Z4 is JX286502.
The application of described Bao Shiva Salmonella in producing bioelectricity specifically comprises the steps:
(1) the described Bao Shiva of claim 1 Salmonella is inoculated in the paramount salt LB substratum, 30 ± 5 ℃ of anaerobism are cultivated; The phase growth bacterium liquid in mid-term of taking the logarithm, centrifugal, abandon supernatant liquor, add the PBS damping fluid and shake up,, clean so repeatedly 2 times gained bacteria suspension recentrifuge by above-mentioned steps, add the PBS damping fluid and make bacteria suspension;
(2) start the single chamber air cathode microbial fuel cell, will (1) in acquisition bacteria suspension be inoculated in microbiological fuel cell after nutritive medium mixes, under 30 ± 5 ℃ of constant temperatures, move and get final product.When the output voltage of MFC reaches more than the 100mV, be considered as starting successfully.After this, only change the nutritive medium that contains electron donor.
Preferably, described high salt LB nutrient media components is as follows: Tryptones (Tryptone) 10gL
-1, yeast extract (Yeast extract) 5gL
-1, sodium-chlor (NaCl) 20gL
-1
Preferably, described nutrient composition is 20mmolL
-1Electron donor, 50mmolL
-1PBS damping fluid and a small amount of VITAMIN and trace element.
Preferably, described electron donor is one or more in lactic acid salt, glucose, pyruvic acid and the formate; The prescription of described PBS damping fluid is NH
4Cl 0.31gL
-1, NaH
2PO
4H
2O 2.452gL
-1, Na
2HPO
44.576gL
-1, KCl 0.13gL
-1, pH 7.0.
Preferably, the OD of described bacteria suspension
600Be 0.6.
Preferably, the pH of described substratum and nutritive medium is 7.0～7.4.
Preferably, the described culture temperature of step (1) is 30 ± 1 ℃, and incubation time is 16-18h; Described centrifugal condition is centrifugal 10 minutes of 5000r/min.
Preferably, the volume ratio of described bacteria suspension and nutritive medium is 1:5.
Preferably, microbiological fuel cell described in the step (2) is that cylindrical (diameter of section is 2cm, useful volume 6.28cm
3), material is polycarbonate. air cathode is for carrying platinum carbon paper (0.5mgcm
-2), anode is Nitric Acid Modified carbon felt, and the negative electrode inboard is one deck cationic exchange membrane, and interelectrode distance is 2cm, does the electronics collector with the titanium silk.The concrete method of modifying of Nitric Acid Modified carbon felt is: pretreated carbon felt is soaked 5h with concentrated nitric acid (65%), washes repeatedly to neutrality with deionized water, put into 120 ℃ of baking ovens oven dry 2h after, place in the moisture eliminator standby.Carbon felt pretreatment process is: after soaking 3h with acetone, take out through vacuum pump and to wash, to remove the oil-soluble substance on surface; With deionized water rinsing and soak, boil, change water 1 time every 0.5h again, boil 3h altogether, electrode materials is put into 120 ℃ of baking ovens oven dry 2h after, place in the moisture eliminator standby.
Preferably, the output voltage (U) of MFC described in the step (2) adopts Keithley 2700 to gather.
Preferably, cationic exchange membrane soaks 24h with 5%NaCl in advance described in the step (2).
The present invention compared with prior art advantage is: this bacterial strain CCTCC M 2012444 shows stronger electrochemical activity, compare with other bacterial strains, can produce comparatively significant electric energy, when being electron donor with the lactic acid salt, the single chamber air cathode MFC of this strain construction can produce maximum output voltage 0.279V, maximum power density 274mWm
-2, except lactic acid salt, this bacterium can also utilize glucose, pyruvic acid and formate electrogenesis.This explanation bacterial strain Shewanella haliotis Z4 not only has stronger electrochemical activity, and can utilize polytype organism electrogenesis, this means this bacterium will various industrial sewage handle and the environmental pollution reparation in play a significant role.Simultaneously, this bacterial strain is the amphimicrobian type, do not need strict anaerobic environment in the electricity generation process, has reduced in the practical application requirement to waste water and environmental pollution treatment device.
Description of drawings
Fig. 1 is the MFC trigger voltage figure of inoculum with the mangrove forest bed mud for embodiment 1;
Fig. 2 is the bacterial strain cyclic voltammetry curve figure of embodiment 1;
Fig. 3 is MFC voltage, power density and the enclosed pasture efficiency diagram of matrix with multiple organism for the bacterial strain of embodiment 2;
Fig. 4 is MFC voltage (a), polarization curve (b) and the electrode potential figure (c) of matrix with the lactic acid salt for the bacterial strain of embodiment 3;
Fig. 5 is the cyclic voltammetry curve figure of MFC anode bacteriological filtration liquid among the embodiment 3.
Embodiment
Below in conjunction with specific embodiment the present invention is done further concrete detailed description the in detail, but embodiments of the present invention are not limited thereto, the processing parameter for not indicating especially can carry out with reference to routine techniques.Used mangrove forest bed mud is from Shenzhen mangrove forest protection center in the embodiment of the invention.
Embodiment 1
One, the screening of bacterial strain CCTCC M 2012444 and separation
Get mangrove forest bed mud 300mL and transfer in the 500mL beaker, add glucose nutritive medium 200mL, stir, use NaHCO
3Solution is adjusted about pH value to 7.0 (6.7-7.2), places 30 ℃ of incubators to cultivate 1 day.Measure the pH value, and use NaHCO in good time
3Solution is adjusted the pH value to 6.7-7.2, stirs, and treats its post precipitation, abandoning supernatant.So repeat to cultivate 3 days standby.
Start the single chamber air cathode microbial fuel cell, described MFC is rectangular parallelepiped (length * wide * height=8.5 * 7 * 6.5cm
3, useful volume 200mL), material is the synthetic glass of 0.5cm, and an end seals with blind plate, and the other end is placed negative electrode with clamping plate.Air cathode is for carrying platinum carbon paper (0.5mg/m
2), anode is Nitric Acid Modified carbon felt (BET 1200m
2/ g, 2mm), interelectrode distance is 3cm, does the electronics collector with the titanium silk.During startup, get the mud after the pre-cultivation, and inoculate MFC after the mixed of nutritive medium by 1:1, extrernal resistance is 1000 Ω, moves under (30 ± 1) ℃ constant temperature.Nutritive medium is for containing 1gL
-1The PBS(pH 7.0 of glucose) and a small amount of VITAMIN and trace element, use behind 121 ℃ of sterilization 20min.When the output voltage of MFC reaches more than the 100mV, be considered as starting successfully.After this, only change the nutritive medium that contains electron donor, gather the output voltage (U) of MFC by Keithley 2700.The result as shown in Figure 1, start battery 170 as a child output voltage reaches stable substantially, maximum output voltage is 0.152V.Can observe one deck microbial film at the MFC anode surface.
Treat this MFC steady running after 1 month, take out the long biomembranous anode that has, it is some to scrape the anode microbial film with blade, and inoculation marine microorganism liquid nutrient medium places under (30 ± 1) ℃ condition anaerobism to cultivate 1d..Then, get culture coating marine microorganism solid medium.Cultivate after one day, according to features such as colonial morphology, color, the transparencys, the bacterium colony of picking surface characteristic obvious difference is seeded to respectively and carries out the anaerobism cultivation in the liquid nutrient medium of ocean. and go down to posterity 7-8 time so repeatedly, obtain many strains pure culture bacterial strain.Pure bacterium is carried out its electrochemical activity of cyclic voltammetric analysis verification, obtain a strain electrochemical activity bacterium Z4 at last.The bacterium colony of this bacterium on marine microorganism 2216 substratum is circular, and orange-yellow, the edge is smooth, and is glossy, the about 2mm of diameter.The form of observing this bacterium under the transmission electron microscope is shaft-like, is about 1.3 μ m, wide about 0.6 μ m, and peritrichous and pod membrane, the growth logarithmic phase is 5～22h.The homology of the 16S rDNA sequence of bacterial strain Z4 and known bacterial strain Shewanella haliotis DW01 reaches 100%, therefore, in conjunction with Physiology and biochemistry and Molecular Identification result, bacterial strain Z4 is accredited as Bao Xiwa Salmonella (Shewanella haliotis).Described liquid culture based formulas is: NaCl 19.45g, MgCl
28.8g, peptone (Peptone) 5.0g, Na
2SO
33.24g, CaCl
21.8g, yeast extract 1.0g, KCl 0.55g, NaHCO
30.16g, (ironic citrate) Ferric citrate0.1g, KBr 0.08g, SrCl
20.03g, H
3BO
30.02g, Na
2HPO
48.0mg, Na
2SiO
34.0mg, NaF 2.4mg, NH
4NO
31.6mg, if be configured to solid medium then add 15gL
-1Agar powder adopts 1molL
-1NaOH be adjusted to pH7.6 ± 0.2.
Two, the electrochemical activity of bacterial strain CCTCC M 2012444 is measured
Z4 inoculates (Tryptones 10gL in the paramount salt LB substratum with Bao Xiwa Salmonella (Shewanella haliotis)
-1, yeast extract 5gL
-1, sodium-chlor 20gL
-1, pH7.4), (30 ± 1) ℃ anaerobism is cultivated 18h.The phase growth bacterium liquid in mid-term of taking the logarithm, centrifugal 10 minutes of 5000r/min abandons supernatant liquor, adds the PBS damping fluid and shakes up, and, cleans so repeatedly 2 times gained bacteria suspension recentrifuge by above-mentioned steps, adds the PBS damping fluid and makes bacteria suspension, OD
600Be about 0.6.With electrochemical workstation the gained bacteria suspension is done the cyclic voltammetric test, three-electrode system is adopted in test, is working electrode with the glass-carbon electrode, and the Ag/AgCl electrode is reference electrode, and platinized platinum is counter electrode, sweeps speed and is 100mVs
-1, sweep limit is-0.7V～0.7V.Experimental result is seen Fig. 2, bacterial strain Shewanella haliotis Z4 cultivates 1d in high salt LB liquid nutrient medium after, obtain cyclic voltammetry curve a pair of tangible redox peak is arranged, wherein oxidation peak-130mV～-180mV between, reduction peak is between 140mV～180mV.
The bacterial strain CCTCC M 2012444 of present embodiment explanation resulting separation can carry out the transmission of born of the same parents' exoelectron, has stronger electrochemical activity.
With the paramount salt LB substratum of described bacterial strain CCTCC M 2012444 inoculations, (30 ± 1) ℃ anaerobism is cultivated 18h.The phase growth bacterium liquid in mid-term of taking the logarithm, centrifugal 10 minutes of 5000r/min abandons supernatant liquor, adds the PBS damping fluid and shakes up, and, cleans so repeatedly 2 times gained bacteria suspension recentrifuge by above-mentioned steps, adds the PBS damping fluid and makes bacteria suspension, OD
600Be about 0.6.
Start the single chamber air cathode microbial fuel cell, described microbiological fuel cell is that cylindrical (diameter of section is 2cm, useful volume 6.28cm
3), material is polycarbonate.Air cathode is for carrying platinum carbon paper (0.5mgcm
-2), anode is Nitric Acid Modified carbon felt, and the negative electrode inboard is one deck cationic exchange membrane, and interelectrode distance is 2cm, does the electronics collector with the titanium silk.During startup, the phase growth bacterium liquid in mid-term of taking the logarithm and is inoculated MFC after the mixed of nutritive medium by 1:5, and extrernal resistance is 1000 Ω, moves under (30 ± 1) ℃ constant temperature.Described nutrient composition is: 20mmolL
-1Electron donor (Sodium.alpha.-hydroxypropionate, glucose, pyruvic acid or sodium formiate), 50mmolL
-1PBS damping fluid and a small amount of VITAMIN and trace element use behind 121 ℃ of sterilization 20min.When the output voltage of MFC reaches more than the 100mV, be considered as starting successfully.After this, only change the nutritive medium that contains electron donor.The output voltage of MFC (U) adopts Keithley 2700 to gather;
Calculate.M is the molecular weight of oxygen, 32; F is the uncommon constant in not bright Delhi; B is every mole of available amount of electrons of oxygen, 4; υ
AnBe anolyte compartment's volume; Δ COD is t
bThe amount of the COD of time internal consumption.
Experimental result as shown in Figure 3, under 30 ℃, the condition of extrernal resistance 1000 Ω, this bacterium is respectively with 20mmolL
-1Sodium.alpha.-hydroxypropionate, glucose, Sodium.alpha.-ketopropionate, when sodium formiate is electron donor, the regulated output voltage of generation is respectively: 0.279V, 0.11603V, 0.243V, 0.247V.The maximum power density that obtains during for electron donor with the lactic acid salt and enclosed pasture efficient are respectively 274mWm
-2, 15.16%; When being electron donor with glucose, the maximum power density of generation and enclosed pasture efficient are respectively 43mWm
-2, 7.33%; When being electron donor with the Sodium.alpha.-ketopropionate, the maximum power density of generation and enclosed pasture efficient are respectively 188mWm
-2, 6.37%; When being electron donor with the sodium formiate, the maximum power density of generation and enclosed pasture efficient are respectively 194mWm
-2, 77.3%.
By present embodiment as can be known, bacterial strain CCTCC M 2012444 can utilize polytype organism electrogenesis, for the application of this bacterium in repairing the recovery of broad variety environmental pollution and bioenergy laid a good foundation.
With the paramount salt LB substratum of described bacterial strain CCTCC M 2012444 inoculations, (30 ± 1) ℃ anaerobism is cultivated 18h.The phase growth bacterium liquid in mid-term of taking the logarithm, centrifugal 10 minutes of 5000r/min abandons supernatant liquor, adds the PBS damping fluid and shakes up, and, cleans so repeatedly 2 times gained bacteria suspension recentrifuge by above-mentioned steps, adds the PBS damping fluid and makes bacteria suspension, OD
600Be about 0.6.
Start the single chamber air cathode microbial fuel cell, described MFC is that cylindrical (diameter of section is 2cm, useful volume 6.28cm
3), material is polycarbonate.Air cathode is for carrying platinum carbon paper (0.5mgcm
-2), anode is Nitric Acid Modified carbon felt, and the negative electrode inboard is one deck cationic exchange membrane, and interelectrode distance is 2cm, does the electronics collector with the titanium silk.During startup, bacteria suspension and nutritive medium are inoculated MFC after the mixed by 1:5, extrernal resistance is 1000 Ω, moves under (30 ± 1) ℃ constant temperature.Described nutrient composition is: 20mmolL
-1Sodium.alpha.-hydroxypropionate, 50mmolL
-1PBS and a small amount of VITAMIN and trace element use behind 121 ℃ of sterilization 20min.When the output voltage of MFC reaches more than the 100mV, be considered as starting successfully.After this, only change the nutritive medium that contains electron donor.
By the output voltage (U) of Keithley 2700 collection MFC, the reference electrode during the potential electrode electromotive force is the Ag/AgCl electrode.Electric current (I) is by Ohm's law I=U/R
ExtCalculate and obtain R
ExtBeing external loop resistance. the drafting of polarization curve and power density curve, is calculated and curve plotting behind the output voltage of measurement correspondence by regulating external loop resistance.Current density I
sUtilize formula I respectively with volumetric power density P
s=U/ (R
ExtA
An) and P=U
2/ (R
ExtV) calculate, wherein, A
AnBe respectively annode area and the useful volume of MFC with V.
When MFC obtain stable can repeat output voltage after, utilize electrochemical workstation (CHI700, Shanghai occasion China) that MFC anode filtrate is carried out the cyclic voltammetric test.Anolyte being poured in the electrolyzer after with 0.22 μ m filtering with microporous membrane degerming, utilized three-electrode system that it is carried out the cyclic voltammetric test, is working electrode with the glass-carbon electrode, and the Ag/AgCl electrode is reference electrode, and platinized platinum is counter electrode, sweeps speed and is 100mVs
-1, sweep limit is-1.0V～1.0V.
Experimental result as shown in Figure 4, when being matrix with the Sodium.alpha.-hydroxypropionate, the single chamber air cathode MFC of this strain construction belongs to the fast rise period of voltage at postvaccinal 0-67h, and starts successfully after about 13 hours.Each matrix of changing, voltage is all accelerated significantly, moves 5 all after dates and stable repeatably output voltage occurs, and maximum output voltage reaches 0.279V, and obtaining maximum power density when extrernal resistance is 900 Ω is 274mWm
-2Along with the increase of current density, anode potential raises gradually, and cathode potential reduces gradually.The cyclic voltammetry curve of anode filtrate has at least two pairs of redox peaks to occur as shown in Figure 5, and illustrating has multiple redox material to exist in the anolyte.
Present embodiment shows, the running status of bacterial strain CCTCC M 2012444 in MFC is good, can under the condition of not adding external source electronics transmission intermediate, utilize lactic acid salt to produce higher electric energy, and can transmit electronics by the mode of secretion electronics transmission intermediate, has stronger electrochemical activity, can reduce the practical application cost of this bacterium in environmental pollution reparation and bioenergy recovery, for its application in environmental pollution reparation and bioenergy recovery provides assurance.
Claims (10)
1. a strain Bao Xiwa Salmonella is characterized in that, this bacterium is Bao Xiwa Salmonella (Shewanella haliotis) Z4, by China's typical culture collection center preservation, be called for short CCTCC, preserving number is: CCTCC NO:M 2012444, preservation date are on November 6th, 2012.
2. the application of the described Bao Shiva of claim 1 Salmonella in producing bioelectricity.
3. application according to claim 2 is characterized in that, comprises the steps:
(1) the described Bao Shiva of claim 1 Salmonella is inoculated in the paramount salt LB substratum, 30 ± 5 ℃ of anaerobism are cultivated; The phase growth bacterium liquid in mid-term of taking the logarithm, centrifugal, abandon supernatant liquor, add the PBS damping fluid and shake up,, clean so repeatedly 2 times gained bacteria suspension recentrifuge by above-mentioned steps, add the PBS damping fluid and make bacteria suspension;
(2) start the single chamber air cathode microbial fuel cell, will (1) in acquisition bacteria suspension be inoculated in microbiological fuel cell after nutritive medium mixes, temperature-constant operation gets final product under the culture temperature of step (1).
4. application according to claim 3 is characterized in that, described high salt LB nutrient media components is as follows: Tryptones 10gL
-1, yeast extract 5gL
-1, sodium-chlor 20gL
-1Described nutrient composition is 20mmolL
-1Electron donor, 50mmolL
-1PBS damping fluid and a small amount of VITAMIN and trace element.
5. application according to claim 4 is characterized in that, described electron donor is one or more in lactic acid salt, glucose, pyruvic acid and the formate; The prescription of described PBS damping fluid is NH
4Cl0.31gL
-1, NaH
2PO
4H
2O 2.452gL
-1, Na
2HPO
44.576gL
-1, KCl 0.13gL
-1, pH 7.0.
6. according to claim 3 or 4 or 5 described application, it is characterized in that microbiological fuel cell is cylindrical described in the step (2), material is polycarbonate; Air cathode is for carrying the platinum carbon paper, and anode is Nitric Acid Modified carbon felt, and the negative electrode inboard is one deck cationic exchange membrane, and interelectrode distance is 2cm, does the electronics collector with the titanium silk.
7. application according to claim 6 is characterized in that, described cationic exchange membrane soaks 24h with 5%NaCl in advance.
8. according to claim 3 or 4 or 5 described application, it is characterized in that the OD of described bacteria suspension
600Be 0.6; The pH of described substratum and nutritive medium is 7.0～7.4.
9. according to claim 3 or 4 or 5 described application, it is characterized in that the described culture temperature of step (1) is 30 ± 1 ℃, incubation time is 16-18h; Described centrifugal condition is centrifugal 10 minutes of 5000r/min.
10. according to claim 3 or 4 or 5 described application, it is characterized in that the volume ratio of described bacteria suspension and nutritive medium is 1:5.
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