CN103395775B - Graphene oxide reduced by microbial fuel cell anode microorganisms and preparation method thereof - Google Patents

Graphene oxide reduced by microbial fuel cell anode microorganisms and preparation method thereof Download PDF

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CN103395775B
CN103395775B CN201310323819.7A CN201310323819A CN103395775B CN 103395775 B CN103395775 B CN 103395775B CN 201310323819 A CN201310323819 A CN 201310323819A CN 103395775 B CN103395775 B CN 103395775B
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graphene oxide
fuel cell
anode
microbial fuel
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CN103395775A (en
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孙东平
范孟孟
孙瑞宁
朱春林
杨加志
黄洋
陈春涛
聂英
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Nanjing University of Science and Technology
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Abstract

The invention discloses graphene oxide reduced by microbial fuel cell anode microorganisms and a preparation method thereof. The preparation method comprises the following steps of: dispersing graphite oxide prepared by utilizing an improved Hummers method into water, and ultrasonically forming a graphene oxide suspension liquid with the relatively good dispersity; then adding a microbial fuel cell anode bacteria liquid into the graphene oxide suspension liquid, standing and culturing for several days under anaerobic conditions, and removing oxygen-containing functional groups of the dispersed graphene oxide under the action of bacteria so as to form graphene; and after finishing culturing, removing thalli, metabolites and other ions generated in a reduction process by utilizing hydrochloric acid, ethanol and deionized water, finally washing until the pH becomes neutral by utilizing the deionized water, and drying to obtain the pure graphene. According to the preparation method, the biological reduction is carried out on the graphene oxide by utilizing microbial fuel cell anode mixed microorganisms which can generate electricity and decompose organic matters, and relative to a method for reducing the graphene oxide by utilizing a single strain, the preparation method has the advantages of simple and convenient operation steps, few defects and number of layers of the prepared grapheme and the like.

Description

Anode of microbial fuel cell bacterium biological reducing graphene oxide and preparation thereof
Technical field
The present invention relates to the green bio controlled synthesis technology being prepared Graphene by graphene oxide, belong to technical field of biological material, particularly a kind of anode of microbial fuel cell bacterium living beings redox graphene.
Background technology
Because Graphene has the character of high specific surface area, outstanding heat conductivility and a series of excellence such as mechanical property and outstanding electron transmission performance thereof, so the Graphene preparing high quality and high yield is the focus that people pay close attention to always.Wherein chemical oxidation-reduction has become the effective way that may realize mass-producing and prepare Graphene, and namely graphite oxidation becomes graphite oxide, then obtains the graphite oxide solution of individual layer under ultrasound condition, then obtains Graphene by chemical reduction.In redox graphene process, in order to reduce environmental pollution, improve Graphene performance, micro-reduction starts the concern causing people, there is Shewanella (Gongming Wang at present, Fang Qian, Chad W. Saltikov, Yongqin Jiao.Microbial Reduction of Graphene Oxide by shewanella.Nano Res. 2011,4 (6): 563 – 570), intestinal bacteria (O. Akhavan, E.Ghaderi. Escherichia coli bacteria reduce graphene oxide to bactericidal graphene in a self-limiting manner. CARBON 50 (2012) 1853 – 1860) etc.
Anode bacterial reduction graphene oxide has obvious advantage relative to single reducing bacteria: first, anode bacterial reduction speed is fast, reaction conditions is gentle, both can carry out under room temperature, meet the theory of low-carbon (LC), medium component is cheap, reduces the preparation cost of Graphene, simple in technique; Eliminate repeatedly the complex steps of strain separating; Secondly, the Graphene that anode bacterial reduction obtains has lower textural defect relative to the Graphene of single culture reduction preparation, remains the performance of Graphene to greatest extent.Meanwhile, the process of anode bacterial reduction graphene oxide is easy to amplify, can amplification test further.
Microbiological fuel cell, except having the efficient feature of general fuel cell, also has the advantages such as raw material sources are extensive, operational condition is gentle concurrently.Microbiological fuel cell belongs to a kind of brand-new biomass utilization form.Existing research all shows, and the electricity generation ability of mixed bacterium MFC is far longer than the MFC of pure bacterium, and which illustrating mixed bacterium MFC has electron transfer capacity faster.
Summary of the invention
The object of the invention is, based on the transfer ability of the under anaerobic excellent electronics of anode bacterium, to reduce to graphene oxide with it, prepare the Graphene of low defect.
Another object of the present invention is to the preparation method of the anode bacterium living beings redox graphene that the low defect of a kind of green is provided.
Realizing technical solution of the present invention is: a kind of anode of microbial fuel cell bacterium biological reducing graphene oxide, comprises following preparation process:
The first step, by the strain inoculation of Staphylococcus in the anolyte of microbiological fuel cell, joins in anode of microbial fuel cell room by anode bacterium liquid, quiescent culture in constant incubator;
Second step, mixed by the anode bacterium liquid that graphene oxide suspension and cultivation terminate, quiescent culture in thermostat container, carries out biological reducing;
3rd step, the graphene oxide after the biological reducing of second step is standing, centrifugal, adopt dilute hydrochloric acid, ethanol, water repeatedly repeatedly to clean, precipitate, to pH is neutrality, finally drying is carried out to pure Graphene.
The inoculum size of the Staphylococcus species seed liquor described in the first step is 5%-20%, quiescent culture 2-5 days in constant incubator, culture temperature is 25-35 DEG C, stop when microbiological fuel cell open circuit voltage reaches 200mV-500mV, the composition of anolyte comprises: glucose 2.0g/L, NaHCO 33.13g/L, NH 4cl 0.31g/L, NaH 2pO 44.97g/L, (NH 4) 2sO 40.56g/L, KCl 0.13g/L, CaCl 215mg/L, MnSO 420mg/L, FeCl 31mg/L, Na 2hPO 412H 2o 6.93g/L, MgSO 40.2g/L; Catholyte composition comprises: K 3fe (CN) 616.5g/L, NaH 2pO 42H 2o 0.4g/L, Na 2hPO 412H 2o 6.0g/L.
Graphene oxide suspension concentration described in second step is 0.22%; Described anode bacterium liquid and the volume ratio of graphene oxide suspension are 1:1-1:10, and the described mixed culture time is 0.5-3d, and culture temperature is 25-35 DEG C.
Dilute hydrochloric acid mass concentration described in 3rd step is 0.1-5mol/L, the volume fraction of ethanol is 70%-80%, centrifugal rotational speed is 8000-12000r/min, centrifugation time 15-80min, the drying of dry employing conventional vacuum or vacuum lyophilization, conventional vacuum drying temperature is 50-100 DEG C, and vacuum lyophilization temperature is-20-(-80) DEG C, vacuum tightness≤160Pa, time of drying is 24-48h.
The present invention compared with prior art, there is significant advantage (1) and utilize stable anode of microbial fuel cell bacterium, graphene oxide is reduced, electric transmission faster can be carried out by the effect of biological reducing enzyme and electron carrier, realize the reduction of graphene oxide faster; (2) graphene oxide of biological reducing reduces the textural defect of Graphene, maintains original excellent specific property of Graphene to greatest extent; (3) innovative point of the present invention is to use mixed strains instead of single culture to carry out biological reducing graphene oxide, this not only eliminates the step being repeatedly separated bacterial classification and medium sterilization, and substantially increase the speed of biological reducing graphene oxide, in order to further illustrate the superiority of anode bacterium, we select single Shewanella bacterial classification to prepare Graphene process to contrast.
Accompanying drawing explanation
Accompanying drawing 1 is the schematic flow sheet of anode of microbial fuel cell bacterium biological reducing graphene oxide of the present invention.
Accompanying drawing 2 is effect contrast figures (the blank bacterium liquid of a, the blank graphene oxide c of b, 2 days d anode bacterium redox graphene recovery times, 3 days) of anode of microbial fuel cell bacterium biological reducing graphene oxide of the present invention.
Accompanying drawing 3 is Raman spectrums of the embodiment of the present invention 1 anode of microbial fuel cell bacterium biological reducing graphene oxide.
Accompanying drawing 4 is field emission scanning electron microscope figure of the embodiment of the present invention 1 anode of microbial fuel cell bacterium biological reducing graphene oxide.
Accompanying drawing 5 is cyclic voltammograms (sweep velocity 5mV/s) of the embodiment of the present invention 1 anode of microbial fuel cell bacterium biological reducing graphene oxide.
Embodiment
By reference to the accompanying drawings 1, anode of microbial fuel cell bacterium biological reducing graphene oxide of the present invention, is obtained by following preparation method:
The first step, disperse improving the standby graphite oxide of Hummers legal system all in water, ultrasonic disperse, forms uniform graphene oxide suspension;
Second step, is inoculated into Staphylococcus species in the anolyte of microbiological fuel cell, is joined by anode bacterium liquid and assembles in microbiological fuel cell, quiescent culture in constant incubator, to obtain eugonic bacterium;
3rd step, mixes graphene oxide suspension with the anode bacterium liquid grown, cultivates, carry out biological reducing in thermostat container;
4th step, graphene oxide after the biological reducing of the 3rd step is standing, centrifugal, adopt repeatedly cleaning, the centrifugation repeatedly of dilute hydrochloric acid, ethanol, water, dissolve the thalline and other ion removing and produce in oxidising process, to pH is neutrality, finally drying is carried out to pure Graphene.
Staphylococcus species of the present invention is derived from the anaerobic sludge of city east, Nanjing sewage work, through this seminar domestication culture of isolated, through bio-chemical characteristics and comparison uncle outstanding Bacteria Identification handbook, determine this anode bacterium mainly belong to Staphylococcus ( staphylococcus), amphimicrobian, this bacterium has electricity generation ability, can as the main bacteria seed of redox graphene.
Embodiment 1: anode of microbial fuel cell bacterium biological reducing graphene oxide of the present invention and preparation thereof, comprise the following steps:
The first step, graphene oxide pre-treatment.In 1L triangular flask, add the 2wt% graphene oxide of 50mL, add deionized water to 400mL in triangular flask, in the Ultrasonic Cleaners of 250W, supersound process 30min under room temperature, obtains the good graphene oxide brown suspension of dispersing property;
Second step, preparing microorganism fuel battery negative pole liquid, each 200mL of anolyte, natural ph.10% seed liquor is inoculated in anolyte, in anolyte, passes into the N of 20min 2quiescent culture 2 days in 30 DEG C of constant incubators, namely stop when microbiological fuel cell open circuit voltage reaches 200mV, wherein seed liquor is passed through the strain inoculation of the Staphylococcus on inclined-plane in LB substratum, cultivate in the constant-temperature table of 30 DEG C and obtain for three days, wherein anode of fuel cell liquid composition comprises: glucose 2.0g/L, NaHCO 33.13g/L, NH 4cl 0.31g/L, NaH 2pO 44.97g/L, (NH 4) 2sO 40.56g/L, KCl 0.13g/L, CaCl 215mg/L, MnSO 420mg/L, FeCl 31mg/L, Na 2hPO 412H 2o 6.93g/L, MgSO 40.2g/L; Catholyte composition comprises: K 3fe (CN) 616.5g/L, NaH 2pO 42H 2o 0.4g/L, Na 2hPO 412H 2o 6.0g/L;
3rd step, divide the triangular flask being filled to 4 250mL, the loading amount 50mL of every bottle by the anode of microbial fuel cell bacterium liquid that growth terminates, the loading amount of every bottle of graphene oxide suspension is 50mL, every bottle of N passing into 20min 2, to remove dissolved oxygen.Bottleneck is sealed, prevents oxygen from entering, in thermostat container, leave standstill Anaerobic culturel 2d, 30 DEG C, carry out biological reducing.
4th step, leaves standstill fermented liquid complete for the 3rd step biological reducing, the centrifugal 30min of 10000r/min rotating speed; In the mixture precipitation obtained, add 1mol/L dilute hydrochloric acid 100mL, stir, left at room temperature 1h; With the centrifugal 30min of 10000r/min rotating speed, incline supernatant liquor, by 80% ethanolic soln washing and precipitating, stir, the centrifugal 30min of left at room temperature 1h, 10000r/min rotating speed inclines supernatant liquor, finally precipitate by washed with de-ionized water, as above repeat 3 times, till finally washing neutrality with deionized water, precipitation is placed in vacuum freezing case,-50 DEG C, vacuum tightness≤160Pa, time of drying is 48h, obtains pure Graphene.
As shown in Figure 2, the basic all blackening of graphene oxide suspension, at the bottom of being deposited in bottle, illustrate that anode bacterium has very strong reductibility to graphene oxide.
Carry out Raman spectrum test to the anode bacterium living beings redox graphene obtained, Graphene prepared by biological reducing graphene oxide is at 1350cm -1, 1580cm -1there is D peak and two, G peak characteristic peak respectively in left and right, Graphene D/G prepared by biological reducing graphene oxide be less than Shewanella reduction preparation Graphene (see reference document Gongming Wang, Fang Qian, Chad W. Saltikov, Yongqin Jiao.Microbial Reduction of Graphene Oxide by shewanella.Nano Res. 2011,4 (6): 563 – 570), show that anode bacterium living beings redox graphene has lower defect and randomness;
Field emission microscopy observation is carried out to the graphene oxide of the biological reducing obtained, as shown in Figure 4.That can see Graphene has which floor (general 1-6 layer) of obvious minority, and this also can be able to be verified by the red shift at G peak in Fig. 3.
As shown in Figure 5, (see reference the Graphene prepared than Shewanella of Graphene prepared by anode bacterium document Gongming Wang, Fang Qian, Chad W. Saltikov, Yongqin Jiao.Microbial Reduction of Graphene Oxide by shewanella.Nano Res. 2011,4 (6): 563 – 570) there is stronger redox peak and better symmetry, illustrate that Graphene prepared by anode bacterium has better electrochemical properties and deposits less defect.
Embodiment 2: anode of microbial fuel cell bacterium biological reducing graphene oxide of the present invention and preparation thereof, comprise the following steps:
The first step, graphene oxide pre-treatment.In 1L triangular flask, add 2% graphene oxide of 50mL, add deionized water to 400mL in triangular flask, in the Ultrasonic Cleaners of 250W, supersound process 30min under room temperature, obtains the good graphene oxide brown suspension of dispersing property;
Second step, the molten each 200mL of preparing microorganism fuel battery negative pole liquid, anode, natural ph.By culture medium inoculated 10% anode bacterium seed liquor, quiescent culture 3d in 30 DEG C of constant incubators, namely stops when microbiological fuel cell open circuit voltage reaches 300mV, and wherein anode of fuel cell liquid composition comprises: glucose 2.0g/L, NaHCO 33.13g/L, NH 4cl 0.31g/L, NaH 2pO 44.97g/L, (NH 4) 2sO 40.56g/L, KCl 0.13g/L, CaCl 215mg/L, MnSO 420mg/L, FeCl 31mg/L, Na 2hPO 412H 2o 6.93g/L, MgSO 40.2g/L; Catholyte composition comprises: K 3fe (CN) 616.5g/L, NaH 2pO 42H 2o 0.4g/L, Na 2hPO 412H 2o 6.0g/L;
3rd step, divide the triangular flask being filled to 4 250mL, the loading amount 10mL of every bottle by the anode of microbial fuel cell bacterium liquid that growth terminates, the loading amount of every bottle of graphene oxide is 100mL, every bottle of N passing into 20min 2, to remove dissolved oxygen.Bottleneck is sealed, prevents oxygen from entering, in thermostat container, leave standstill Anaerobic culturel 0.5d, 30 DEG C, carry out biological reducing.
4th step, leaves standstill fermented liquid complete for the 3rd step biological reducing, the centrifugal 20min of 12000r/min rotating speed; In the mixture precipitation obtained, add 1mol/L dilute hydrochloric acid 100mL, stir, left at room temperature 1h; With the centrifugal 30min of 12000r/min rotating speed, incline supernatant liquor, by 80% ethanolic soln washing and precipitating, stir, the centrifugal 30min of left at room temperature 1h, 12000r/min rotating speed inclines supernatant liquor, finally precipitate by washed with de-ionized water, as above repeat 3 times, till finally washing neutrality with deionized water, precipitation is placed in conventional vacuum loft drier, 50 DEG C, vacuum tightness≤160Pa, time of drying is 48h, obtains pure Graphene.

Claims (6)

1. an anode of microbial fuel cell bacterium biological reducing graphene oxide, is characterized in that comprising following preparation process:
The first step, by the strain inoculation of Staphylococcus in the anolyte of microbiological fuel cell, joins in anode of microbial fuel cell room by anode bacterium liquid, quiescent culture in constant incubator;
The inoculum size of described Staphylococcus species seed liquor is 5%-20%, quiescent culture 2-5 days in constant incubator, and culture temperature is 25-35 DEG C;
The composition of the anolyte of described microbiological fuel cell comprises: glucose 2.0g/L, NaHCO 33.13g/L, NH 4cl0.31g/L, NaH 2pO 44.97g/L, (NH 4) 2sO 40.56g/L, KCl0.13g/L, CaCl 215mg/L, MnSO 420mg/L, FeCl 31mg/L, Na 2hPO 412H 2o6.93g/L, MgSO 40.2g/L; Catholyte composition comprises: K 3fe (CN) 616.5g/L, NaH 2pO 42H 2o0.4g/L, Na 2hPO 412H 2o6.0g/L;
Second step, mixed by the anode bacterium liquid that graphene oxide suspension and cultivation terminate, quiescent culture in thermostat container, carries out biological reducing;
3rd step, the graphene oxide after the biological reducing of second step is standing, centrifugal, cleaning, precipitation, dry.
2. anode of microbial fuel cell bacterium biological reducing graphene oxide according to claim 1, is characterized in that the graphene oxide suspension concentration described in second step is 0.22%; Described anode bacterium liquid and the volume ratio of graphene oxide suspension are 1:1-1:10, and the described mixed culture time is 0.5-3d, and culture temperature is 25-35 DEG C.
3. anode of microbial fuel cell bacterium biological reducing graphene oxide according to claim 1, is characterized in that the cleaning described in the 3rd step adopts dilute hydrochloric acid, ethanol, water repeatedly to clean successively; Described dilute hydrochloric acid concentration is 0.1-5mol/L, and the volume fraction of ethanol is 70%-80%; Described centrifugal rotational speed is 8000-12000r/min, centrifugation time 15-80min, the drying of dry employing conventional vacuum or vacuum lyophilization, conventional vacuum drying temperature is 50-100 DEG C, vacuum lyophilization temperature is-20-(-80) DEG C, vacuum tightness≤160Pa, time of drying is 24-48h.
4. a preparation method for anode of microbial fuel cell bacterium biological reducing graphene oxide, is characterized in that comprising the following steps:
The first step, by the strain inoculation of Staphylococcus in the anolyte of microbiological fuel cell, joins in anode of microbial fuel cell room by anode bacterium liquid, quiescent culture in constant incubator;
The inoculum size of described Staphylococcus species seed liquor is 5%-20%, quiescent culture 2-5 days in constant incubator, and culture temperature is 25-35 DEG C;
The composition of the anolyte of described microbiological fuel cell comprises: glucose 2.0g/L, NaHCO 33.13g/L, NH 4cl0.31g/L, NaH 2pO 44.97g/L, (NH 4) 2sO 40.56g/L, KCl0.13g/L, CaCl 215mg/L, MnSO 420mg/L, FeCl 31mg/L, Na 2hPO 412H 2o6.93g/L, MgSO 40.2g/L; Catholyte composition comprises: K 3fe (CN) 616.5g/L, NaH 2pO 42H 2o0.4g/L, Na 2hPO 412H 2o6.0g/L;
Second step, mixed by the anode bacterium liquid that graphene oxide suspension and cultivation terminate, quiescent culture in thermostat container, carries out biological reducing;
3rd step, the graphene oxide after the biological reducing of second step is standing, centrifugal, cleaning, precipitation, dry.
5. the preparation method of anode of microbial fuel cell bacterium biological reducing graphene oxide according to claim 4, is characterized in that the graphene oxide suspension concentration described in second step is 0.22%; Described anode bacterium liquid and the volume ratio of graphene oxide suspension are 1:1-1:10, and the described mixed culture time is 0.5-3d, and culture temperature is 25-35 DEG C.
6. the preparation method of anode of microbial fuel cell bacterium biological reducing graphene oxide according to claim 4, is characterized in that the cleaning described in the 3rd step adopts dilute hydrochloric acid, ethanol, water repeatedly to clean; Described dilute hydrochloric acid concentration is 0.1-5mol/L, and the volume fraction of ethanol is 70%-80%; Described centrifugal rotational speed is 8000-12000r/min, centrifugation time 15-80min, the drying of dry employing conventional vacuum or vacuum lyophilization, conventional vacuum drying temperature is 50-100 DEG C, vacuum lyophilization temperature is-20-(-80) DEG C, vacuum tightness≤160Pa, time of drying is 24-48h.
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CN103803542B (en) * 2014-02-24 2016-01-20 南京理工大学 A kind of preparation method of microbiological fuel cell mixt bacteria nitrogen-doped graphene
CN104176732B (en) * 2014-08-20 2015-10-28 南京工业大学 Method for preparing graphene through biocatalysis
CN105217800A (en) * 2015-08-25 2016-01-06 南京工业大学 Graphene/polypyrrole bioelectrode and preparation method and application thereof
CA3003464A1 (en) * 2015-10-31 2017-05-04 Bego Technologies, Ltd Bioelectrochemical methods and systems for efficient production of graphene oxide and hydrogen
CN105733980B (en) * 2016-01-27 2019-04-09 大连理工大学 The double end bacterium of one plant of aerobic degradation graphene oxide and its application
CN105870464B (en) * 2016-05-16 2018-07-03 中国科学院过程工程研究所 A kind of microbiological fuel cell original position cathodic modification method
CN110203907B (en) * 2019-04-22 2022-09-16 武汉理工大学 Method for reducing graphene oxide by using extracellular polymer

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