CN102827916A - Method for quantifying microalgae using inorganic carbon approach - Google Patents
Method for quantifying microalgae using inorganic carbon approach Download PDFInfo
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- CN102827916A CN102827916A CN2012103484479A CN201210348447A CN102827916A CN 102827916 A CN102827916 A CN 102827916A CN 2012103484479 A CN2012103484479 A CN 2012103484479A CN 201210348447 A CN201210348447 A CN 201210348447A CN 102827916 A CN102827916 A CN 102827916A
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 84
- 229910052799 carbon Inorganic materials 0.000 title claims abstract description 84
- 238000013459 approach Methods 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title abstract description 7
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims abstract description 117
- 235000017557 sodium bicarbonate Nutrition 0.000 claims abstract description 64
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims abstract description 53
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims abstract description 38
- 239000001569 carbon dioxide Substances 0.000 claims abstract description 13
- 229910002092 carbon dioxide Inorganic materials 0.000 claims abstract description 13
- 235000015097 nutrients Nutrition 0.000 claims abstract description 8
- 241000195493 Cryptophyta Species 0.000 claims description 92
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 claims description 39
- 230000000155 isotopic effect Effects 0.000 claims description 31
- 238000002372 labelling Methods 0.000 claims description 31
- 239000000203 mixture Substances 0.000 claims description 11
- 235000011089 carbon dioxide Nutrition 0.000 claims description 9
- 238000006467 substitution reaction Methods 0.000 claims description 3
- 238000002474 experimental method Methods 0.000 abstract description 5
- 238000012360 testing method Methods 0.000 abstract 2
- 102000003846 Carbonic anhydrases Human genes 0.000 description 19
- 108090000209 Carbonic anhydrases Proteins 0.000 description 19
- 210000004027 cell Anatomy 0.000 description 11
- 239000000126 substance Substances 0.000 description 10
- 230000037361 pathway Effects 0.000 description 9
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 8
- 241000195585 Chlamydomonas Species 0.000 description 7
- 238000005194 fractionation Methods 0.000 description 6
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- 108090000790 Enzymes Proteins 0.000 description 5
- YSCNMFDFYJUPEF-OWOJBTEDSA-N 4,4'-diisothiocyano-trans-stilbene-2,2'-disulfonic acid Chemical compound OS(=O)(=O)C1=CC(N=C=S)=CC=C1\C=C\C1=CC=C(N=C=S)C=C1S(O)(=O)=O YSCNMFDFYJUPEF-OWOJBTEDSA-N 0.000 description 4
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000002512 suppressor factor Substances 0.000 description 3
- 101000755440 Buchnera aphidicola subsp. Acyrthosiphon pisum (strain APS) Uncharacterized protein BU181 Proteins 0.000 description 2
- 101000824312 Buchnera aphidicola subsp. Acyrthosiphon pisum (strain APS) Uncharacterized protein BU584 Proteins 0.000 description 2
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- 101000737401 Buchnera aphidicola subsp. Baizongia pistaciae (strain Bp) Uncharacterized protein bbp_529 Proteins 0.000 description 2
- 101000755397 Buchnera aphidicola subsp. Schizaphis graminum (strain Sg) Uncharacterized protein BUsg_175 Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- BZKPWHYZMXOIDC-UHFFFAOYSA-N acetazolamide Chemical compound CC(=O)NC1=NN=C(S(N)(=O)=O)S1 BZKPWHYZMXOIDC-UHFFFAOYSA-N 0.000 description 2
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for quantifying a microalgae using an inorganic carbon approach. The prior art can not quantify microalgae using an inorganic carbon approach. The solving method comprises the following steps of: respectively adding two sodium bicarbonate of which the delta 13C value difference values are larger than 0.8% as an isotope label 1 and an isotope label 2 to cultivate microalgae to be tested in a nutrient solution; and respectively cultivating the microalgae to be tested for 4 days under the above condition and the cultivating condition of 16mM of sodium bicarbonate and 10mM of MAZ, testing the delta 13C values, computing the portion fB of the used and added inorganic carbon source under various microalgae cultivating conditions and the delta 13C value delta a of a microalgae body when a carbon dioxide approach in the atmosphere is completely used, and obtaining the portions of the two approaches in which the microalgae uses the inorganic carbon according to the obtained data. The method can quickly quantify the microalgae using inorganic carbon approach, and can develop a cultivating test under the complete same experiment condition to obtain the reliable data of the portion of the microalgae using inorganic carbon approach, which can not be achieved in the prior art.
Description
Technical field
The present invention relates to the quantivative approach that a kind of little algae utilizes the inorganic carbon approach, belong to ecological environment treatment and marine biotechnology field.
Background technology
Little algae (microalgae) comprises that all live in the swim microphyte of mode of life of Shui Zhongying, just refers to planktonic algae usually.Little algal structure is simple, and its physiological process is also simple relatively, and some kind is the model plant of scientific research, as: Chlamydomonas reinhardtii, chlorella, all right artificial culture of many types, this research for us is provided convenience.Little algae has dual mode to the utilization of water body inorganic carbon, and (1) utilizes Atmospheric Carbon Dioxide.CO
2As linear non-polar molecule, be electroneutral, it can freely diffuse into the cell bilayer lipid membrane, gets into the CO in the cell
2By the photosynthesis of microalgae cell is utilized; (2) utilize bicarbonate ion in the solution.Bicarbonate ion both can directly have been transported also and utilized by microalgae cell in the transporte to cells indirectly.The direct transhipment of bicarbonate ion refers to through cytoplasmic membrane surface carrier proteins or anion-exchange protein, directly in the bicarbonate ion transporte to cells, in born of the same parents, is converted into CO through carbonic anhydrase
2Or directly with the form of bicarbonate ion by chloroplast membranes albumen active transport in chloroplast(id), change into CO through carbonic anhydrase
2Supply ribulose-1,5-bisphosphate, 5 di-phosphate carboxylation/oxygenases (Rubisco) are fixing; The indirect transhipment of bicarbonate ion is meant the indirect transhipment of the bicarbonate ion that depends on the outer carbonic anhydrase of born of the same parents.Carbonic anhydrase (EC 4.2.1.1) is a kind of zinciferous metalloenzyme, catalysis CO
2With HCO
3 -Reversible conversion: CO
2+ H
2O H
++ HCO
3 -Carbonic anhydrase is divided in the plasma membrane and outer two kinds of plasma membrane.The outer carbonic anhydrase of plasma membrane (claiming born of the same parents outer carbonic anhydrase again) is connected with the cell walls internal surface through metals ion, and bicarbonate ion is hydrolyzed into free CO rapidly in the catalysis cellular invasion layer
2Thereby, guaranteed the CO of cell
2Fast supply.
Some algae only utilizes CO
2Some algae can not only utilize CO
2, can also or directly utilize bicarbonate ion, or through the outer carbonic anhydrase indirect utilization bicarbonate ion of born of the same parents, perhaps both have both at the same time.But prior art can't be utilized the inorganic carbon approach by quantitative little algae.Understand little algae and utilize mode and share will help little algae development of biology, simultaneously, for the improvement of wawter bloom red tide provides scientific basis.
Summary of the invention
The technical problem that the present invention will solve is, provides a kind of little algae to utilize the recognition methods of inorganic carbon approach, can't quantitative little algae utilizes the deficiency of inorganic carbon approach to overcome prior art.
The present invention takes following technical scheme: it may further comprise the steps: the first, select two kinds of δ
13C value difference value is added to respectively to cultivate in the nutrient solution as isotopic labeling 1 and isotopic labeling 2 greater than 8 ‰ sodium hydrogencarbonate and is treated the micrometer algae; The δ of the sodium hydrogencarbonate of isotopic labeling 1
13The C value is δ
C1, the δ of the sodium hydrogencarbonate of isotopic labeling 2
13The C value is δ
C2Measure simultaneously and do not add sodium hydrogencarbonate, complete from inorganic carbon δ in the original culture of air
13The C value is δ
C0
Second; Treat the micrometer algae simultaneously by the culture condition of investigating down with in the cultivation culture condition of 16 mM sodium hydrogencarbonates and 10mM AZ under after 4 days, measure stable carbon isotope composition δ under corresponding each culture condition of two kinds of isotope-labeled nutrient solutions cultivations, that investigated little algae respectively
13The value δ of C
T1, δ
T2, δ
T1-AZAnd δ
T2-AZ
The 3rd, through equation
, calculate the share f that utilizes the inorganic carbon source of adding under each culture condition of little algae
B
The 4th, according to the stable carbon isotope value δ that treats the micrometer algae under the culture condition of 16 mM sodium hydrogencarbonates and 10mM AZ
T1-AZOr δ
T2-AZ, according to equation: δ
a=δ
Ti-f
BD
i, the δ of frond when calculating little algae and utilizing the Atmospheric Carbon Dioxide approach fully
13The C value is δ
a
The 5th, according to the stable carbon isotope value δ that treats the micrometer algae under each culture condition
T1And δ
T2The δ of frond when little algae utilizes the Atmospheric Carbon Dioxide approach fully
13C value δ
aEach culture condition of little algae utilizes the share f of the inorganic carbon source of adding down
BAnd the δ of corresponding isotope-labeled sodium hydrogencarbonate
13C value δ
CiWith do not add sodium hydrogencarbonate, fully from inorganic carbon δ in the original culture of air
13C value δ
C0Poor D
iInformation, substitution equation: f
b=1000 (δ
Ti-δ
a-f
BD
iIn)/9, calculate the share f that little algae utilizes the bicarbonate ion approach
b, 1-f
bUtilize the share of carbonic acid gas approach for little algae;
Advantage of the present invention is following:
The occurring in nature carbon has two kinds of stable isotopes:
12C with
13C, their natural average abundance is respectively 98.89% and 1.11%.The stable carbon isotope composition is used δ usually
13C (‰) expression, occurring in nature δ
13C is changed to-90 ‰ ~+20 ‰.The strong fractionation characteristic of stable carbon isotope is the basis in the little algae inorganic carbon of identification source.Mass balance principle and isotropic substance mixture model and chemometrics method are the bases in the little algae inorganic carbon of quantitatively identification source.
The isotope fractionation difference that two kinds of inorganic carbons of little algae utilize approach to cause has obvious difference.Utilize the Atmospheric Carbon Dioxide approach can cause maximum isotope fractionation (δ
a).The direct transhipment of bicarbonate ion and depend on the isotope fractionation Billy that the indirect transporting pathway of the bicarbonate ion of the outer carbonic anhydrase of born of the same parents causes and cause isotope fractionation little by 9 ‰ (PDB) with the Atmospheric Carbon Dioxide approach.
(acetazolamide is to contain 1,3 AZ) to acetazolamide, the heterocyclic sulfonamide carbonic anhydrase extracellular enzyme suppressor factor of 4-thiadiazoles ring.Utilize carbonic anhydrase extracellular enzyme suppressor factor can suppress the characteristics of carbonic anhydrase extracellular enzyme single-mindedly; Little algae isotropic substance under the research carbonic anhydrase extracellular enzyme activity inhibited changes, and can reversal of identification depend on contribution and the share of the indirect transporting pathway of the bicarbonate ion of carbonic anhydrase outside the born of the same parents to the inorganic carbon utilization.The indirect transporting pathway of the bicarbonate ion of carbonic anhydrase is suppressed simultaneously outside depending on born of the same parents, and the direct transporting pathway of bicarbonate ion also is suppressed simultaneously.High density carbonic acid hydrogen sodium also has restraining effect to the carbonic anhydrase extracellular enzyme; Under the effect of high density carbonic acid hydrogen sodium and 10mM AZ, depending on the indirect transporting pathway of the bicarbonate ion of carbonic anhydrase outside the born of the same parents and the direct transporting pathway of bicarbonate ion will be suppressed simultaneously fully.
The present invention takes following thinking: add two kinds of δ respectively
13The sodium hydrogencarbonate of the different great disparity of C value difference is cultivated simultaneously and is treated the micrometer algae, measures frond δ
13The C value utilizes the isotropic substance mixture model of two end members to obtain the share that little algae utilizes inorganic carbon source that comes from air and the inorganic carbon source of utilizing interpolation, adds two kinds of δ subsequently respectively
13C value difference sodium hydrogencarbonate different great disparity, high density is cultivated under the condition of 10mM AZ and is treated the micrometer algae, measures frond δ
13The C value is obtained the isotope fractionation value (δ that little algae utilizes the Atmospheric Carbon Dioxide approach to cause fully
a), last, according to above-mentioned information, utilize the isotropic substance mixture model of two end members, obtain the share of two kinds of approach respectively.
Obtain the principle of inorganic carbon source share:
There are four kinds of form: CO in total dissolved inorganic carbon (DIC) in the water body
2, HCO
3 -, H
2CO
3And CO
3 2-, and they exist following chemical equilibrium: CO
2+ H
2O → H
2CO
3→ HCO
3 -+ H
+→ CO
3 2-+ 2H
+Derive from the inorganic carbon of air or the inorganic carbon that adds all has two kinds of inorganic carbons---CO
2And HCO
3 -Supply little algae to utilize.Therefore, can utilize the isotropic substance mixture model of two end members to obtain the share that little algae utilizes inorganic carbon source that comes from air and the inorganic carbon source of utilizing interpolation.
The isotropic substance mixture model of two end members can be expressed as:
δ
Ti=δ
Ai- f
Biδ
Ai +f
Biδ
Bi （i=1，2，3，------）（1）
Here δ
Tiδ for little algae
13The C value, δ
AiThe δ of frond when utilizing the inorganic carbon source of air fully for being assumed to little algae
13The C value, δ
BiThe δ of frond when utilizing the inorganic carbon source of interpolation fully for being assumed to little algae
13The C value, f
BiInvestigate little algae for this and utilize the shared share of inorganic carbon source of adding.
Obviously, only know δ
TiBe difficult to obtain f
Bi, therefore, the present invention adopts has the δ than big-difference
13C value sodium hydrogencarbonate is the while culturing microalgae respectively, discerns the share that little algae utilizes the inorganic carbon source of adding with the stable carbon isotope double-tagging.
For isotopic labeling 1 (i=1), equation (1) expression as shown in the formula:
δ
T1=δ
A1- f
B1δ
A1 +f
B1δ
B1 （2）
Here δ
T1For with first kind of known δ
13The δ of the sodium hydrogencarbonate cultured microalgae frond of C value
13The C value, δ
A1The δ of frond when utilizing the inorganic carbon source of air fully for being assumed to little algae
13The C value, δ
B1The δ of frond when utilizing the inorganic carbon source of interpolation fully for being assumed to little algae
13The C value, f
B1Investigating the inorganic of little algae utilization interpolation for this is the shared share of carbon source.
For isotopic labeling 2 (i=2), equation (1) expression as shown in the formula:
δ
T2=δ
A2 - f
B2δ
A2 +f
B2δ
B2 （3）
Here δ
T2For with first kind of known δ
13The δ of the sodium hydrogencarbonate cultured microalgae frond of C value
13The C value, δ
A2The δ of frond when utilizing the inorganic carbon source of air fully for being assumed to little algae
13The C value, δ
B2The δ of frond when utilizing the inorganic carbon source of interpolation fully for being assumed to little algae
13The C value, f
B2Investigate little algae for this and utilize the shared share of inorganic carbon source of adding.
(2) δ and in (3) two equations
A1=δ
A2, f
B=f
Bi=f
B1=f
B2, simultaneous solution
（4）
(4) δ in the formula
B1-δ
B2Then can be converted into the δ of the sodium hydrogencarbonate of isotopic labeling 1
13C value δ
C1δ with the sodium hydrogencarbonate of isotopic labeling 2
13C value δ
C2Poor, then:
Therefore, can pass through the δ of the sodium hydrogencarbonate of mensuration isotopic labeling 1
13C value δ
C1δ with the sodium hydrogencarbonate of isotopic labeling 2
13C value δ
C2, measure δ simultaneously with the sodium hydrogencarbonate cultured microalgae of corresponding mark
13The C value promptly determines δ
T1And δ
T2Value calculates this according to (5) formula and investigates the shared share of inorganic carbon source that little algae utilizes interpolation.
Obtain the principle that little algae inorganic carbon utilizes the approach share:
Derive from the inorganic carbon of air or the inorganic carbon that adds all has two kinds of inorganic carbons---CO
2And HCO
3 -Supply little algae to utilize.Therefore, can utilize the isotropic substance mixture model of two end members to obtain the share information that two kinds in little algae utilizes the inorganic carbon approach.
The isotropic substance mixture model of two end members can be expressed as:
δ
Ti=(1-f
B)[(1-f
b)δ
a+f
b(δ
a +9‰)]+f
B [(1-f
b)δ
bi+f
b(δ
bi +9‰)]（i=1，2）（6）
Here δ
Tiδ for little algae
13The C value, δ
aThe δ of frond when utilizing the Atmospheric Carbon Dioxide approach fully for little algae
13The C value, δ
BiBe assumed to that little algae utilizes the inorganic carbon source of adding, the δ of frond when carrying out the carbonic acid gas approach fully
13The C value, f
bInvestigate little algae for this and utilize the shared share of bicarbonate ion approach.
Here, δ
Bi-δ
aCan be converted into the δ of isotope-labeled sodium hydrogencarbonate
13C value δ
Ci(or the δ of the sodium hydrogencarbonate of isotopic labeling 1
13C value δ
C1Or the δ of the sodium hydrogencarbonate of isotopic labeling 2
13C value δ
C2) and do not add sodium hydrogencarbonate, complete in inorganic carbon δ in the original culture of air
13C value δ
C0Poor D
i
Therefore, (6) formula can be simplified to:
f
b= 1000 (δ
Ti-δ
a- f
BD
i)/9 （i=1，2） （7）
The δ of frond when little algae utilizes the Atmospheric Carbon Dioxide approach fully
13The C value also is δ
aThe assignment principle:
Under the effect of high density carbonic acid hydrogen sodium and 10mM AZ, (7) formula f
b=0
Also be δ
Ti-δ
a-f
BD
i=0,
δ
a=δ
Ti- f
BD
i （i=1，2） （8）
Fully utilize above-mentioned principle, can utilize the inorganic carbon approach by the little algae of fast quantification; Under identical experiment condition, carry out culture experiment, obtain little algae and utilize the data of inorganic carbon approach share reliable, this is that prior art all can't be accomplished.
Embodiment
Embodiments of the invention: first step, measure the sodium hydrogencarbonate that different manufacturers is produced, select two kinds of δ
13C value difference value is added in the nutrient solution respectively as isotopic labeling 1 and isotopic labeling 2 greater than 8 ‰ sodium hydrogencarbonate.The δ of isotope-labeled sodium hydrogencarbonate
13The C value is designated as δ
Ci, the δ of the sodium hydrogencarbonate of isotopic labeling 1 wherein
13The C value is δ
C1, the δ of the sodium hydrogencarbonate of isotopic labeling 2
13The C value is δ
C2The δ of isotope-labeled sodium hydrogencarbonate
13C value δ
CWith do not add sodium hydrogencarbonate, fully from inorganic carbon δ in the original culture of air
13C value δ
C0Difference be D
i, D wherein
1δ for the sodium hydrogencarbonate of isotopic labeling 1
13C value δ
C1With do not add sodium hydrogencarbonate, fully from inorganic carbon δ in the original culture of air
13C value δ
C0Poor, D
2δ for the sodium hydrogencarbonate of isotopic labeling 2
13C value δ
C2With do not add sodium hydrogencarbonate, fully from inorganic carbon δ in the original culture of air
13C value δ
C0Poor.
Second step; Simultaneously with the nutrient solution of two kinds of isotopic labeling sodium hydrogencarbonates respectively simultaneously by under the culture condition of investigating and under the culture condition of 16 mM sodium hydrogencarbonates and 10mM AZ; Little algae that cultivation is investigated; After 4 days, measure corresponding each culture condition stable carbon isotope composition down, that investigated little algae δ that two kinds of isotope-labeled nutrient solutions are cultivated simultaneously respectively
13The value δ of C
T1, δ
T2, δ
T1-AZAnd δ
T2-AZ
Third step is with the δ of the sodium hydrogencarbonate of isotopic labeling 1
13The C value is as δ
C1, that cultivate by isotopic labeling 1, under each culture condition, as to be investigated little algae δ
13The C value is as δ
T1, the δ of the sodium hydrogencarbonate of isotopic labeling 2
13The C value is as δ
C2, that cultivate by isotopic labeling 2, under the corresponding culture condition, as to be investigated little algae δ
13The C value is as δ
T2, bring into
, calculate and investigated the share f that little algae utilizes the inorganic carbon source of adding under each culture condition
B, little algae utilizes the inorganic carbon source share of air to be 1-f
B
The 4th step is according to the stable carbon isotope value δ that is investigated little algae that under the culture condition of 16 mM sodium hydrogencarbonates and 10mM AZ, cultivates
Ti(δ
T1-AZOr δ
T2-AZ), according to equation: δ
a=δ
Ti-f
BD
i, the δ of frond when calculating little algae and utilizing the Atmospheric Carbon Dioxide approach fully
13C value δ
a
The 5th step is according to the stable carbon isotope value δ that treats the micrometer algae under each culture condition
Ti, the stable carbon isotope value δ that treats the micrometer algae under isotopic labeling 1 sodium hydrogencarbonate is cultivated wherein
T1, the stable carbon isotope value δ that treats the micrometer algae under isotopic labeling 2 sodium hydrogencarbonates are cultivated
T2The δ of frond when little algae utilizes the Atmospheric Carbon Dioxide approach fully
13C value δ
a, each culture condition of little algae utilizes down the share f of the inorganic carbon source of adding
BAnd the δ of corresponding isotope-labeled sodium hydrogencarbonate
13C value δ
Ci(the δ of the sodium hydrogencarbonate of isotopic labeling 1
13C value δ
C1Or the δ of the sodium hydrogencarbonate of isotopic labeling 2
13C value δ
C2) and do not add sodium hydrogencarbonate, complete in inorganic carbon δ in the original culture of air
13C value δ
C0Poor D
iInformation, substitution equation: f
b=1000 (δ
Ti-δ
a-f
BD
iIn)/9, calculate the share f that little algae utilizes the bicarbonate ion approach
b, 1-f
bUtilize the share of carbonic acid gas approach for little algae.
Implementation result of the present invention is following:
Cultivated material is: chlamydomonas and chlorella.Basic culture solution adopts the SE substratum, and basic culture condition is: photoperiod L/D:12h/12h; 25 ℃ of temperature; Intensity of illumination is 100 μ molm
-2s
-1, pH value 8.0 or 8.2 (regulating) with hydrochloric acid and sodium hydroxide.Other culture condition such as table 1, the δ of the sodium hydrogencarbonate that wherein adds
13C is respectively-26.3 ‰ (PDB) (δ
C1) and-16.5 ‰ (PDB) (δ
C2), the culture condition of 16 mM sodium hydrogencarbonates and 10mM AZ is essential.Do not add sodium hydrogencarbonate, complete from inorganic carbon δ in the original culture of air
13C value δ
C0Be-11.8 ‰ (PDB).After waiting to cultivate 4 days, measure little algae δ of each culture condition and each experiment respectively
13The C value.Use the inventive method, draw the share f that utilizes the inorganic carbon source of adding under each culture condition of chlamydomonas and chlorella
BAnd the inorganic carbon source share 1-f that utilizes air
B, like table 2.Subsequently, utilize the present invention, draw the share of utilizing the bicarbonate ion approach under each culture condition of chlamydomonas and chlorella and utilizing the carbonic acid gas approach, like table 3.
The culture condition of table 1 chlamydomonas and each culture experiment of chlorella
Each culture condition of table 2 chlamydomonas and chlorella utilizes the share of the inorganic carbon source of adding down
Utilize bicarbonate ion approach and the share of utilizing the carbonic acid gas approach under each culture condition of table 3 chlamydomonas and chlorella
| Cultivate number | f b | 1-f b | Cultivate number | f b | 1-f b |
| YB1 | 0.97 | 0.03 | XB1 | 0.71 | 0.29 |
| YB2 | 0.99 | 0.01 | XB2 | 0.75 | 0.25 |
| YB3 | 1.07 | -0.07 | XB3 | 0.87 | 0.13 |
| YB4 | 1.03 | -0.03 | XB4 | 1.04 | -0.04 |
| YB5 | 1.11 | -0.11 | XB5 | 1.22 | -0.22 |
| YB6 | 0.46 | 0.54 | XB6 | 0.46 | 0.54 |
| YBA1 | 0.68 | 0.32 | XBA1 | 0.64 | 0.36 |
| YBA2 | 0.65 | 0.35 | XBA2 | 0.53 | 0.47 |
| YBA3 | 0.66 | 0.34 | XBA3 | 0.62 | 0.38 |
| YBA4 | 0.40 | 0.60 | XBA4 | 0.34 | 0.66 |
| YBA5 | 0.26 | 0.74 | XBA5 | 0.13 | 0.87 |
| YBA6 | 0.00 | 1.00 | XBA6 | 0.07 | 0.93 |
| YBAT1 | -0.27 | 1.27 | XBAT1 | -0.48 | 1.48 |
| YBAT2 | -0.03 | 1.03 | XBAT2 | -0.30 | 1.30 |
| YBAT3 | 0.12 | 0.88 | XBAT3 | -0.04 | 1.04 |
| YBAT4 | 0.25 | 0.75 | XBAT4 | -0.02 | 1.02 |
| YBAT5 | 0.18 | 0.82 | XBAT5 | 0.01 | 0.99 |
| YBAT6 | 0.00 | 1.00 | XBAT6 | 0.00 | 1.00 |
| YD1 | 1.08 | -0.08 | XD1 | 0.81 | 0.19 |
| YD2 | 1.07 | -0.07 | XD2 | 0.86 | 0.14 |
| YD3 | 1.09 | -0.09 | XD3 | 0.90 | 0.10 |
| YD4 | 1.14 | -0.14 | XD4 | 0.69 | 0.31 |
| YD5 | -0.04 | 1.04 | XD5 | -0.30 | 1.30 |
Can find out that from table 2 different culture condition utilize the share of the inorganic carbon source of adding obviously different down, it is many more to demonstrate the interpolation sodium hydrogencarbonate, utilizes the big more trend (except YB6 and XB6) of share of the inorganic carbon source of adding.The share of the inorganic carbon source that utilization is added is that error at measurment causes less than 0 YBA2, YBA3 and XBA3, but also very near 0.These are consistent with practical situation, and also promptly in certain concentration range, it is many more to add sodium hydrogencarbonate; Utilize the share of the inorganic carbon source of adding big more; When sodium hydrogencarbonate concentration surpassed certain limit, some physiological process was suppressed, and caused utilizing the share of the inorganic carbon source of interpolation to reduce.Show that little algae that the present invention draws utilizes the share of the inorganic carbon source of adding to have safety.
Can find out that from table 3 little algae utilizes the share of bicarbonate ion approach obviously different under the different culture condition, it is big more to demonstrate interpolation AZ concentration, and little algae utilizes the more little trend of the share of bicarbonate ion approach.This is because AZ is the suppressor factor of the outer carbonic anhydrase of born of the same parents.See that on the whole chlamydomonas utilizes the share of bicarbonate ion approach to utilize the share of bicarbonate ion approach greater than chlorella.DIDS (4; 4'-diisothiocyano-stilbene-2; 2'-disulfonate) can suppress bicarbonate ion and get into cell; Utilize DIDS can suppress the characteristics that bicarbonate ion gets into cell, the research bicarbonate ion gets into the little algae isotropic substance that is blocked down to be changed single-mindedly, and direct transporting pathway that can the reversal of identification bicarbonate ion is to the contribution and the share of inorganic carbon utilization.When the direct transporting pathway of bicarbonate ion was suppressed fully, the indirect transporting pathway that depends on the bicarbonate ion of the outer carbonic anhydrase of born of the same parents also was suppressed simultaneously fully.The DIDS of lower concentration utilizes the influence of approach less to bicarbonate ion.The DIDS of high density (2mM) is bigger to the approach influence that utilizes bicarbonate ion, almost completely suppresses to utilize the approach of bicarbonate ion.This and practical situation are consistent.Because error at measurment, some appear greater than 1 or less than 0 situation, this all recoverable be 1 or 0; For example the share of utilizing the bicarbonate ion approach of YB4 and XB4 is respectively 1.03 and 1.04, and all recoverable is 1, and the share of utilizing the carbonic acid gas approach all recoverable is 0; When being illustrated in the sodium hydrogencarbonate that adds 4mM; Two kinds of little algaes all do not utilize the carbonic acid gas approach, only utilize the bicarbonate ion approach, and this also is consistent with practical situation.Some big deviations appear on XD5, YBAT1, XBAT1 and the XBAT2, this be because, compare with cultured microalgae under 16 mM sodium hydrogencarbonates and the 10mMAZ, this moment, micro algae growth speed was very little, cell volume is less, the δ that little algae is surveyed
13The C value is than negative partially 1 to 2 ‰ (PDB) of theoretical value, therefore, make little algae utilize the share of carbonic acid gas approach to increase on apparent, but this situation (inorganic carbon is supplied with few) seldom occurs at occurring in nature.Bigger deviation also appears in YB5 and XB5, this be because, compare with cultured microalgae under 16 mM sodium hydrogencarbonates and the 10mMAZ, this moment, micro algae growth speed was big, cell volume is big, the δ that little algae is surveyed
13The C value is than theoretical value polarization 0.5 to 1 ‰ PDB, but this situation (sodium hydrogencarbonate is up to 8mM) also seldom occurs at occurring in nature equally.The normal situation about occurring of nature, the present invention can estimate the share that little algae utilizes the bicarbonate ion approach well.
Claims (1)
1. a little algae utilizes the quantivative approach of inorganic carbon approach, and its characteristic comprises following steps:
The first, select two kinds of δ
13C value difference value is added to respectively to cultivate in the nutrient solution as isotopic labeling 1 and isotopic labeling 2 greater than 8 ‰ sodium hydrogencarbonate and is treated the micrometer algae; The δ of the sodium hydrogencarbonate of isotopic labeling 1
13The C value is δ
C1, the δ of the sodium hydrogencarbonate of isotopic labeling 2
13The C value is δ
C2Measure simultaneously and do not add sodium hydrogencarbonate, complete from inorganic carbon δ in the original culture of air
13The C value is δ
C0
Second; Treat the micrometer algae simultaneously by the culture condition of investigating down with in the cultivation culture condition of 16 mM sodium hydrogencarbonates and 10mM AZ under after 4 days, measure stable carbon isotope composition δ under corresponding each culture condition of two kinds of isotope-labeled nutrient solutions cultivations, that investigated little algae respectively
13The value δ of C
T1, δ
T2, δ
T1-AZAnd δ
T2-AZ
The 3rd, through equation
, calculate the share f that utilizes the inorganic carbon source of adding under each culture condition of little algae
B
The 4th, according to the stable carbon isotope value δ that treats the micrometer algae under the culture condition of 16 mM sodium hydrogencarbonates and 10mM AZ
T1-AZOr δ
T2-AZ, according to equation: δ
a=δ
Ti-f
BD
i, the δ of frond when calculating little algae and utilizing the Atmospheric Carbon Dioxide approach fully
13The C value is δ
a
The 5th, according to the stable carbon isotope value δ that treats the micrometer algae under each culture condition
T1And δ
T2The δ of frond when little algae utilizes the Atmospheric Carbon Dioxide approach fully
13C value δ
aEach culture condition of little algae utilizes the share f of the inorganic carbon source of adding down
BAnd the δ of corresponding isotope-labeled sodium hydrogencarbonate
13C value δ
CiWith do not add sodium hydrogencarbonate, fully from inorganic carbon δ in the original culture of air
13C value δ
C0Poor D
iInformation, substitution equation: f
b=1000 (δ
Ti-δ
a-f
BD
iIn)/9, calculate the share f that little algae utilizes the bicarbonate ion approach
b, 1-f
bUtilize the share of carbonic acid gas approach for little algae.
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