CN110320262A - A method of characterization bioenergetic metabolism - Google Patents
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Abstract
The invention belongs to field of biotechnology, are related to a kind of method using microbial metabolism electric current characterization external environment factor pair organism ATP metabolic effect.Its foundation is: 1) electric current of the electronics that biological cell generates in the metabolic process through being formed in electron transport chain transmittance process, i.e. metabolism electric current, is used to form a kind of transmembrane proton gradient, and then drive the synthesis of ATP；2) ATP is most direct energy source in organism, for cell activities carry out energy is provided；3) metabolism electric current is sensitiveer to the response ratio ATP of environment.Response of the organism to extraneous environmental factor in terms of energetic supersession can be presented in the present invention in real time, response especially in ATP level, compared with ATP content in detection organism, with real-time, quick, sensitive advantage, thus can be promoted and applied in fields such as medicine detection, biology, ecotoxicology.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of to characterize the external environment factor using microbial metabolism electric current
To the method for organism ATP metabolic effect.
Background technique
Energetic supersession is the essential characteristic of organism vital movement, in the metabolic process, along with energy release, transfer and
It utilizes.Adenosine triphosphate (ATP) is a kind of high energy phosphate compound, and in cell, it mutually converts realization energy storage with ADP's
And exoergic, to guarantee the energy supply of cell items vital movement.ATP can be generated by various kinds of cell approach, most typical
Approach is to be synthesized by oxidative phosphorylation by atp synthase.For eukaryocyte, oxidative phosphorylation occurs in mitochondria,
The system of participation oxidative phosphorylation on the inner membrance of mitochondria, constitutes respiratory chain with the formal distribution of complex, and also referred to as electronics passes
Pass chain.It is metabolized the formation of flow driving mitochondrial inner membrane two sides potential difference and proton concentration difference of the electronics on electron transport chain,
And then drive the synthesis of ATP.For prokaryotes, electron transport chain is distributed across on cell membrane.Existing research shows energy
It measures metabolic disorder and cancer and (Havas, 2017), neurodegenerative disease (Reale et al., 2014), dysgenesia occurs
(Bekhite et al., 2016;Solek et al., 2017) correlations such as.
In addition, the discharge of industrial waste gas, waste water, the use of agricultural pesticide, Insecticides (tech) & Herbicides (tech), to atmosphere, soil, water
Source causes serious pollution.The organism being exposed in pollutant can activate a series of stress reaction, to cope with these pollutions
Object is influenced caused by organism.The starting of stress response system, such as the table of superoxide dismutase, catalase antioxidase
Up to etc., the consumption of energy is necessarily increased, and then the abnormal energy metabolism of organism can be induced.Therefore, detection pollutant exposure item
The ATP level of organism can reflect pollutant to the health effect of organism, especially to organism to a certain extent under part
The influence of energetic supersession.
The method of detection ATP at present, such as fluorescence method, need to carry out clasmatosis, just can be carried out ATP dyeing, even
Visitain, that coloration result is mainly presented is also the intracorporal ATP of specific time biology horizontal, and cannot be presented in biology in real time
ATP variation.In addition, the content of ATP in vivo is generally relative constant, it is horizontal only by the intracorporal ATP of measurement biology
It is often difficult to really reflect the influence situation of pollutant ATP metabolism intracorporal to biology.Water flow as flowing through the river of reservoir
Amount variation can more reflect that a regional rainfall variation is the same than the water variation in reservoir, the change of electric current on electron transport chain
The changes in energy metabolism of organism can more be reflected by changing the variation than ATP in organism.Based on this, the present invention is passed by detection electronics
The variation that electric current is metabolized on chain is passed to indicate response of the organism to environment in terms of energetic supersession, especially in ATP level
Response.
Summary of the invention
(1) the technical issues of solving
The total amount of ATP in organism under characterization specific time is only limitted to for existing ATP detection technique, the present invention provides one kind
Sound of the organism to environmental factor in ATP level is presented in real time by being metabolized the variation of electric current on detection electron transport chain
It answers.
(2) technical solution
To achieve the above object, the invention provides the following technical scheme:
A method of characterization bioenergetic metabolism, comprising the following steps:
(1) strain culturing of extracellular breathing bacterium: by thallus streak inoculation in solid LB media, 30 DEG C of 20 ~ 30 h of culture are chosen
It takes single bacterium to drop down onto LB liquid medium, 30 DEG C, 180 rpm, cultivates 10 ~ 16 h, thalline were collected by centrifugation, with 0.5 mL final concentration
The PBS buffer solution of 0.2 M, pH 7.0 is resuspended, and is made bioelectrochemical system (Bioelectrochemical system, BES)
Inoculation liquid；
(2) bioelectrochemical system constructs: 100 mL of single chamber BES volume, working electrode and be carbon plate, reference electrode to electrode
For Ag/AgCl, after assembling, addition sterilized, 80 mL of electrode solution of deoxygenation, and 20 spaces mL reserve on top, and access BES is inoculated with
Liquid；
(3) current acquisition: being control with reference electrode, applies potential to carbon anode plate by electrochemical workstation, when BES is produced
When electric current rises to 0.2 ~ 0.5 mA, apply environmental factor, and record current intensity in real time；
(4) current strength-time (i- t) curve plotting: it is drawn using origin 8.0i- t curve compares environmental factor addition
Whether produced electricity to BES intensity of flow influence.
Preferably, extracellular breathing bacterium is any to have electricity production activity and can be using electrode as electron acceptor in step (1)
Microorganism.
Preferably, in BES building process, electrode solution is consisted of the following compositions: NaHCO30.1 g/L of 2.5 g/L, KCl,
NH4Cl 0.25 g/L, NaH2PO40.6 g/L, 10 mL vitamin stock solutions, 10 mL microelement mother liquors, adjusting pH is 7.2
Afterwards, the sodium acetate of final concentration of 15 mM is added.
Preferably, vitamin stock solution consists of the following compositions: 2.0 mg/L of biotin, 2.0 mg/L of folic acid, hydrochloric acid pyrrole
Tremble 10.0 mg/L of alcohol, 5.0 mg/L of riboflavin, 5.0 mg/L of thiamine, 5.0 mg/L of niacin, 5.0 mg/L of pantothenic acid, cobalt
Amine element 0.1 mg/L, 5.0 mg/L of p-aminobenzoic acid, 5.0 mg/L of lipoic acid.
Preferably, microelement mother liquor consists of the following compositions: nitrilotriacetic acid trisodium salt 1.5 g/L, MgSO4·7H2O 3.0
G/L, MnSO4·H2O 0.5 g/L, NaCl 1.0 g/L, FeSO4·7H2O 0.1 g/L, CaCl20.1 g/L, CoCl2·
6H2O 0.1 g/L, ZnSO4·7H2O 0.1 g/L, Cu2SO4·5H2O 0.01 g/L, AlK (SO4)2·12H2O 0.01 g/
L, H3BO30.01 g/L, Na2MoO4 0.025 g/L, NiCl2·6H2O 0.024 g/L, Na2WO4·2H2O 0.025 g/
L。
Preferably, the environmental factor in step (3) includes all factors that can be impacted to the energetic supersession of organism,
Such as electromagnetic field intensity, environment temperature, intensity of illumination, oxygen and gas concentration lwevel.
The characterizing method is using the electric current generated in biological cell metabolic process as evaluation index, according to metabolism electric current
Variation as response of the characterization organism to specific environment in terms of energetic supersession, being metabolized the variation of electric current, to pass through electric current strong
Degree-time plot slope indicates.
Electric current in step (3) is that the electronics by generating in cellular process is raw during transmitting on electron transport chain
At the electric current on electron transport chain can be detected by electro-chemical systems；Electric current on electron transport chain, based on it all thin
Function in born of the same parents' biology is mainly to drive the synthesis of ATP, for eukaryotic cells, if without suitable detection technique, it can
It is detected indirectly with the microorganism that can be carried out extracellular electron transmission by detection；Can be carried out the microorganism of extracellular electron transmission, can
Using electrode as extracellular electron acceptor, it can for constructing the microorganism of bioelectrochemical system.
(3) beneficial effect
The present invention provides a kind of methods of characterization bioenergetic metabolism, according to being: 1) biological cell is in the metabolic process
Electric current of the electronics of generation through being formed in electron transport chain transmittance process, i.e. metabolism electric current are used to form a kind of cross-film proton ladder
Degree, and then drive the synthesis of ATP；2) ATP is most direct energy source in organism, provide for cell activities
Energy；3) metabolism electric current is sensitiveer to the response ratio ATP of environment.Compared with prior art, the beneficial effects of the present invention are: 1)
The dynamic effects of plus environmental factor pair bioenergetic metabolism, the especially influence in ATP level can be presented in real time；2) it is not required to
Break process is carried out to histocyte, thus can more accurately reflect the influence of plus environmental factor pair ATP metabolism；3) it and examines
It surveys ATP content in organism to compare, there is real-time, quick, sensitive advantage, thus can be in medicine detection, biology, environment
The fields such as toxicology promote and apply.
Detailed description of the invention
Fig. 1 is the relationship for being metabolized electric current and ATP level intracellular；
Fig. 2 is the influence that the metabolism of 100 Gs electromagnetic field bioenergetics is indicated using metabolism electric current；
Fig. 3 is the variation that metabolism electric current is indicated using current strength-time graph slope.
Specific embodiment
The embodiments described below is exemplary, it is intended to is used to explain the present invention, and be should not be understood as to of the invention
Limitation.
Embodiment 1: the relationship of ATP level in metabolism electric current and biological cell.
(1) strain culturing: by electricity-producing microorganism Oneida Shewanella (Shewanella oneidensisMR-1,
ATCC700550) streak inoculation in solid LB media (10 g/L of tryptone, yeast extract 10 g/L of 5 g/L, NaCl,
7.0) agar powder 15 g, pH are adjusted to, 30 DEG C of 24 h of culture, picking single bacterium drops down onto LB liquid medium, and 30 DEG C, 180 rpm, training
Support 12 h；
(2) prepared by BES inoculation liquid: by 4 DEG C of 6000 r/min of bacterium solution in (1), being centrifuged 2 min, removes supernatant under anaerobic condition
Liquid obtains somatic cells, and with PBS buffer solution, ingredient is (/L): Na2HPO4·12H2O 22.2 g；NaH2PO4·2H2O 5.93
g；0.13 g of KCl, three times, 0.5 mL suspends again for cleaning, and BES inoculation liquid is made；
(3) prepared by BES electrode solution: electrode solution is basic culture medium (NaHCO32.5 g/L；KCl 0.1 g/L；NH4Cl 0.25
g/L； NaH2PO40.6 g/L；10 mL vitamin stock solutions；10 mL microelement mother liquors；7.2) and electron donor pH is adjusted to
The mixed liquor of sodium acetate, final concentration of 15 mM of sodium acetate.Electrode solution high-temperature sterilization (121 DEG C, 20 min), then fills gaseous mixture
(N2:CO2=80:20 v/v) 30 min, exclude the oxygen in liquid, sealing；The above reagent is that analysis is pure.
(4) BES is constructed: 100 mL of single chamber three-electrode electro Chemical reactor volume, working electrode and be carbon plate to electrode
(3 × 2.5 × 0.5 cm), reference electrode Ag/AgCl；After assembling, addition sterilized, 80 mL of electrode solution of deoxygenation, top
Reserved 20 spaces mL, access BES inoculation liquid.
(5) current acquisition: 30 ± 1 DEG C of cultivation temperature.It is control with reference electrode, passes through electrochemical workstation
(CHI1000C) 0.3 V potential is applied to carbon anode plate.Thallus incubation, the every primary electricity of 10 min record of data collection system
Intensity of flow；In the experimental stage, it is set as recording primary current intensity every 1 s.
(6) when the produced electricity stream of BES rises to 0.4 mA, the distilled water of the sterile deoxygenation of 0.5 mL is added in 2 BES for connecing bacterium；2
The Shanghai the external source ATP(Mike woods of the 0.5 sterile deoxygenation of mL10 M, CAS:987-65-5 is added in a BES for connecing bacterium).
(7) current strength-time (i- t) curve plotting: it is drawn using origin 8.0i- t curve compares external source ATP and adds
The influence of intensity of flow produced electricity to BES whether adding.
As a result as shown in Figure 1, after external source ATP is added, metabolism faradic yield declines rapidly, and water is added and produces to electric current
Amount will not impact, and show to be metabolized electric current and the intracellular horizontal correlation of ATP.
Embodiment 2: the influence that 100 Gs electromagnetic fields are metabolized bioenergetic is characterized using metabolism electric current.
(1) strain culturing: by electricity-producing microorganism Oneida Shewanella (Shewanella oneidensisMR-1,
ATCC700550) streak inoculation in solid LB media (10 g/L of tryptone, yeast extract 10 g/L of 5 g/L, NaCl,
7.0) agar powder 15 g, pH are adjusted to, 30 DEG C of 24 h of culture, picking single bacterium drops down onto LB liquid medium, and 30 DEG C, 180 rpm, training
Support 12 h；
(2) prepared by BES inoculation liquid: by 4 DEG C of 6000 r/min of bacterium solution in (1), being centrifuged 2 min, removes supernatant under anaerobic condition
Liquid obtains somatic cells, and with PBS buffer solution, ingredient is (/L): Na2HPO4·12H2O 22.2 g；NaH2PO4·2H2O 5.93
g；0.13 g of KCl, three times, 0.5 mL suspends again for cleaning, and BES inoculation liquid is made；
(3) prepared by BES electrode solution: electrode solution is basic culture medium (NaHCO32.5 g/L；KCl 0.1 g/L；NH4Cl 0.25
g/L； NaH2PO40.6 g/L；10 mL vitamin stock solutions；10 mL microelement mother liquors；7.2) and electron donor pH is adjusted to
The mixed liquor of sodium acetate, final concentration of 15 mM of sodium acetate.Electrode solution high-temperature sterilization (121 DEG C, 20 min), then fills gaseous mixture
(N2:CO2=80:20 v/v) 30 min, exclude the oxygen in liquid, sealing；The above reagent is that analysis is pure.
(4) BES is constructed: 100 mL of single chamber three-electrode electro Chemical reactor volume, working electrode and be carbon plate to electrode
(3 × 2.5 × 0.5 cm), reference electrode Ag/AgCl；After assembling, addition sterilized, 80 mL of electrode solution of deoxygenation, top
Reserved 20 spaces mL, access BES inoculation liquid.
(5) current acquisition: 30 ± 1 DEG C of cultivation temperature.It is control with reference electrode, passes through electrochemical workstation
(CHI1000C) 0.3 V potential is applied to carbon anode plate.Thallus incubation, the every primary electricity of 10 min record of data collection system
Intensity of flow；In the experimental stage, it is set as recording primary current intensity every 10 s.
(6) electromagnetic field exposure treatment: BES is placed in electromagnetic field experimental provision (Hunan is escaped forever, FE-3580AF),
When BES, which can stablize, generates electric current, electromagnetic field is opened, and be adjusted to 100 Gs, electromagnetic field persistently handles 10 hours.
(7) current strength-time (i- t) curve plotting: it is drawn using origin 8.0i- t curve compares electromagnetic field and applies
The influence of intensity of flow produced electricity to BES whether adding.
As a result as shown in Fig. 2, the produced electricity intensity of flow of BES improves nearly 150% under 100 Gs electromagnetic field exposure conditions, according to
The result of study of embodiment 1, i.e. metabolism electric current are mainly used for driving the generation of ATP, show that electromagnetic field exposure can promote Ao Nai
ATP up to Shewanella is synthesized, i.e. energetic supersession enhances, this may be since electromagnetic field processing can activate Oneida Shewanella
In stress response system so that ATP consumption increase caused by.
Embodiment 3: the variation of energetic supersession is indicated using current strength-time graph slope.
(1) strain culturing: by electricity-producing microorganism Oneida Shewanella (Shewanella oneidensisMR-1,
ATCC700550) streak inoculation in solid LB media (10 g/L of tryptone, yeast extract 10 g/L of 5 g/L, NaCl,
7.0) agar powder 15 g, pH are adjusted to, 30 DEG C of 24 h of culture, picking single bacterium drops down onto LB liquid medium, and 30 DEG C, 180 rpm, training
Support 12 h；
(2) prepared by BES inoculation liquid: by 4 DEG C of 6000 r/min of bacterium solution in (1), being centrifuged 2 min, removes supernatant under anaerobic condition
Liquid obtains somatic cells, and with PBS buffer solution, ingredient is (/L): Na2HPO4·12H2O 22.2 g；NaH2PO4·2H2O 5.93
g；0.13 g of KCl, three times, 0.5 mL suspends again for cleaning, and BES inoculation liquid is made；
(3) prepared by BES electrode solution: electrode solution is basic culture medium (NaHCO32.5 g/L；KCl 0.1 g/L；NH4Cl 0.25
g/L； NaH2PO40.6 g/L；10 mL vitamin stock solutions；10 mL microelement mother liquors；7.2) and electron donor pH is adjusted to
The mixed liquor of sodium acetate, final concentration of 15 mM of sodium acetate.Electrode solution high-temperature sterilization (121 DEG C, 20 min), then fills gaseous mixture
(N2:CO2=80:20 v/v) 30 min, exclude the oxygen in liquid, sealing；The above reagent is that analysis is pure.
(4) BES is constructed: 100 mL of single chamber three-electrode electro Chemical reactor volume, working electrode and be carbon plate to electrode
(3 × 2.5 × 0.5 cm), reference electrode Ag/AgCl；After assembling, addition sterilized, 80 mL of electrode solution of deoxygenation, top
Reserved 20 spaces mL, access BES inoculation liquid.
(5) current acquisition: 30 ± 1 DEG C of cultivation temperature.It is control with reference electrode, passes through electrochemical workstation
(CHI1000C) 0.3 V potential is applied to carbon anode plate.Thallus incubation, the every primary electricity of 10 min record of data collection system
Intensity of flow；In the experimental stage, it is set as recording primary current intensity every 1 s.
(6) electromagnetic field exposure treatment: BES is placed in electromagnetic field experimental provision (Hunan is escaped forever, FE-3580AF),
When the produced electricity stream of BES rises to 0.4 A or so, the electrochemical workstation data acquisition current generated to BES is set as 2 s/ time,
Electromagnetic field is opened after recording 20 min, and is adjusted to 2 Gs, electromagnetic field persistently handles 20 min, and after treatment continues record 20
min；Identical operation is carried out when handling under the conditions of 8,32,128 Gs electromagnetic field.
(7) current strength-time (i- t) curve plotting: it is drawn using origin 8.0i- t curve, and calculate the curve
The slope of Trendline the variation of electric current is metabolized when comparing processing using slope value before and after treatment as control.
Experimental result is as shown in figure 3, when electromagnetic field is handled, and the slope for being metabolized electric current is positive value, and the value is with electromagnetic field
The increase of intensity and increase, illustrate electromagnetic field processing can be improved Oneida Shewanella metabolism electric current yield, and the influence with
The increase of electromagnetic field intensity and enhance.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art are not departing from the principle of the present invention and objective
In the case where can make changes, modifications, alterations, and variations to the above described embodiments within the scope of the invention.It is all according to Shen of the present invention
Please the equivalent changes and modifications done of the scope of the patents, be all covered by the present invention.
Claims (8)
1. a kind of method of characterization bioenergetic metabolism, which is characterized in that the foundation of the method the following steps are included:
(1) strain culturing of extracellular breathing bacterium: by thallus streak inoculation in solid LB media, 30 DEG C of 20 ~ 30 h of culture are chosen
It takes single bacterium to drop down onto LB liquid medium, 30 DEG C, 180 rpm, cultivates 10 ~ 16 h, thalline were collected by centrifugation, with 0.5 mL final concentration
The PBS buffer solution of 0.2 M, pH 7.0 is resuspended, and BES inoculation liquid is made；
(2) BES is constructed: 100 mL of single chamber BES volume, working electrode and be carbon plate to electrode, reference electrode Ag/AgCl,
After assembling, addition sterilized, 80 mL of electrode solution of deoxygenation, and 20 spaces mL reserve on top, access BES inoculation liquid；
(3) current acquisition: being control with reference electrode, applies potential to carbon anode plate by electrochemical workstation, when BES is produced
When electric current rises to 0.2 ~ 0.5 mA, apply environmental factor, and record current intensity in real time；
(4) current strength-time graph production: current strength-time graph is drawn using origin 8.0, compares environmental factor
The influence of intensity of flow produced electricity to BES whether addition.
2. the method according to claim 1, wherein extracellular breathing bacterium is any with electricity production work in step (1)
Property simultaneously can be using electrode as the microorganism of electron acceptor.
3. the method according to claim 1, wherein electrode solution consists of the following compositions in BES building process:
NaHCO32.5 g/L, KCl 0.1 g/L, NH4Cl 0.25 g/L, NaH2PO40.6 g/L, 10 mL vitamin stock solutions, 10
The sodium acetate of final concentration of 15 mM is added after adjusting pH is 7.2 in mL microelement mother liquor.
4. according to the method described in claim 3, it is characterized in that, vitamin stock solution consists of the following compositions: biotin 2.0
Mg/L, 2.0 mg/L of folic acid, 10.0 mg/L of puridoxine hydrochloride, 5.0 mg/L of riboflavin, 5.0 mg/L of thiamine, niacin
5.0 mg/L, 5.0 mg/L of pantothenic acid, 0.1 mg/L of cobalamin, 5.0 mg/L of p-aminobenzoic acid, 5.0 mg/L of lipoic acid.
5. according to the method described in claim 3, it is characterized in that, microelement mother liquor consists of the following compositions: nitrilotriacetic acid
Trisodium 1.5 g/L, MgSO4·7H2O 3.0 g/L, MnSO4·H2O 0.5 g/L, NaCl 1.0 g/L, FeSO4·7H2O
0.1 g/L, CaCl20.1 g/L, CoCl2·6H2O 0.1 g/L, ZnSO4·7H2O 0.1 g/L, Cu2SO4·5H2O 0.01
G/L, AlK (SO4)2·12H2O 0.01 g/L, H3BO30.01 g/L, Na2MoO4 0.025 g/L, NiCl2·6H2O
0.024 g/L, Na2WO4·2H2O 0.025 g/L。
6. the method according to claim 1, wherein the environmental factor in step (3) includes electromagnetic field intensity, ring
Border temperature, intensity of illumination, oxygen and gas concentration lwevel.
7. the method according to claim 1, wherein the characterizing method is to utilize to produce in biological cell metabolic process
As evaluation index, the variation for being metabolized electric current is indicated raw electric current by current strength-time plot slope.
8. -7 any methods are in characterization external environment factor pair bioenergetic metabolic effect according to claim 1
Using.
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| CN113376226A (en) * | 2021-06-08 | 2021-09-10 | 北京航空航天大学 | Method for enhancing water quality toxicity detection by reversing extracellular electron transfer direction of electrogenic bacteria |
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