CN102827916A - Method for quantifying microalgae using inorganic carbon approach - Google Patents

Abstract

The invention discloses a quantitative method for microalgae to utilize inorganic carbon pathways. Existing techniques fail to quantify inorganic carbon pathways utilized by microalgae. The solution is: add two kinds of sodium bicarbonate whose δ ¹³ C value difference is greater than 8‰ as isotope label 1 and isotope label 2 to the culture medium to cultivate the microalgae to be tested; After culturing for 4 days under the culture conditions of 16mM sodium bicarbonate and 10mMAZ, the δ ¹³ C value was measured, and the fraction f _B of the added inorganic carbon source used by the microalgae under each culture condition and the time when the microalgae fully utilized the carbon dioxide in the atmosphere were calculated. The δ ¹³ C value δ _a of the microalgae was calculated, and then the shares of the two pathways for microalgae to utilize inorganic carbon were calculated based on the obtained data. The invention can quickly quantify the use of inorganic carbon pathways by microalgae; carry out culture experiments under exactly the same experimental conditions, and obtain reliable data on the proportion of inorganic carbon pathways used by microalgae, which cannot be achieved by the prior art.

Description

Quantification of Inorganic Carbon Utilization Pathways by Microalgae

technical field

The invention relates to a quantitative method for utilizing inorganic carbon pathways by microalgae, and belongs to the fields of ecological environment management and marine biological engineering.

Background technique

Microalgae (microalgae) include all tiny plants that live in water and engage in a planktonic lifestyle, usually referring to planktonic algae. The structure of microalgae is simple, and its physiological process is also relatively simple. Some species are model plants for scientific research, such as Chlamydomonas reinhardtii and Chlorella, and many species can also be cultivated artificially, which provides convenience for our research. There are two ways for microalgae to utilize inorganic carbon in water bodies. (1) Utilize carbon dioxide in the atmosphere. As a linear non-polar molecule, CO ₂ is electrically neutral. It can freely diffuse into the cell bilayer lipid membrane, and the CO ₂ entering the cell is used by the photosynthesis of microalgae cells; (2) using bicarbonate in the solution ion. Bicarbonate ions can be transported both directly and indirectly into cells for use by microalgal cells. The direct transport of bicarbonate ions refers to the direct transport of bicarbonate ions into the cell through the carrier protein or anion exchange protein on the surface of the plasma membrane, and the conversion into CO ₂ by carbonic anhydrase in the cell or the direct transfer of bicarbonate ions The form is actively transported into the chloroplast by chloroplast membrane proteins, converted to CO by carbonic anhydrase for fixation by _ribulose -1,5-bisphosphate carboxylation/oxygenase (Rubisco); the indirect transport of bicarbonate ions refers to the dependence of Indirect transport of bicarbonate ions by extracellular carbonic anhydrase. Carbonic anhydrase (EC 4.2.1.1) is a zinc-containing metalloenzyme that catalyzes the reversible conversion of CO ₂ to HCO ₃ ^- : CO ₂ +H ₂ O↔H ⁺ +HCO ₃ ^- . There are two types of carbonic anhydrase: intraplasmic membrane and extraplasmic membrane. Carbonic anhydrase outside the plasma membrane (also known as extracellular carbonic anhydrase) connects with the inner surface of the cell wall through metal ions, and catalyzes the rapid hydrolysis of bicarbonate ions in the cell diffusion layer into free CO ₂ , thus ensuring the rapid supply of CO ₂ to the cells .

Some algae only use CO ₂ ; some algae can not only use CO ₂ , but also use bicarbonate ions directly, or indirectly through extracellular carbonic anhydrase, or both. However, existing techniques cannot quantify the use of inorganic carbon by microalgae. Knowing the utilization mode and share of microalgae will help the development of microalgae biotechnology, and at the same time, provide a scientific basis for the control of algal blooms and red tides.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for identifying the way of using inorganic carbon by microalgae, so as to overcome the deficiency that the prior art cannot quantify the way of using inorganic carbon by microalgae.

The present invention adopts the following technical scheme: it includes the following steps: first, select two kinds of sodium bicarbonate whose δ ¹³ C value difference is greater than 8 ‰ as isotope label 1 and isotope label 2 and add them to the culture solution to cultivate the microbes to be tested. algae; the δ ¹³ C value of isotope-labeled sodium bicarbonate 1 is δ _C1 , and the δ ¹³ C value of isotope-labeled sodium bicarbonate 2 is δ _C2 ; while measuring Inorganic carbon δ ¹³ C value is δ _C0 ;

Second, after the microalgae to be tested were cultured under the culture conditions under investigation and under the culture conditions of 16 mM sodium bicarbonate and 10 mM AZ for 4 days, the corresponding culture conditions of the two isotope-labeled culture solutions were respectively determined. δ _T1 , δ _T2 , δ _T1-AZ and δ _T2-AZ of the stable carbon isotope composition δ ¹³ C of microalgae under investigation;

，计算出微藻各培养条件下利用添加的无机碳源的份额f_B； Third, through the equation

, calculate the fraction f _B of the added inorganic carbon source utilized by the microalgae under each culture condition;

Fourth, based on the stable carbon isotope value δ _T1-AZ or δ _T2-AZ of the microalgae to be tested under the culture conditions of 16 mM sodium bicarbonate and 10 mM AZ, according to the equation: δ _a = δ _Ti - f _B D _i , to calculate the δ ¹³ _C value of the algal body when the microalgae fully utilizes the carbon dioxide in the atmosphere;

Fifth, based on the stable carbon isotope values δ _T1 and δ _T2 of the microalgae to be tested under each culture condition; the δ ¹³ C value δ _a of the algal body when the microalgae fully utilizes the carbon dioxide in the atmosphere; the culture conditions of the microalgae Using the fraction of added inorganic carbon source f _B and the corresponding δ ¹³ C value δ _Ci of isotope-labeled sodium bicarbonate and the value of inorganic carbon δ ¹³ C δ _C0 in the original culture solution without adding sodium bicarbonate and completely from air Substituting the information of the difference D _i into the equation: f _b = 1000 (δ _Ti -δ _a - f _B D _i )/9 to calculate the share of bicarbonate ion pathway used by microalgae f _b , 1-f _b is microalgae Share of algal carbon dioxide utilization pathways;

The advantages of the present invention are as follows :

There are two stable isotopes of carbon in nature: ¹² C and ¹³ C, and their natural average abundances are 98.89% and 1.11%, respectively. The stable carbon isotope composition is usually represented by δ ¹³ C (‰), and the variation of δ ¹³ C in nature is -90‰~+20‰. The strong fractionation signature of stable carbon isotopes is the basis for identifying the source of inorganic carbon in microalgae. Mass balance principles, together with isotope mixing models and chemometric methods, are the basis for the quantitative identification of inorganic carbon sources in microalgae.

The differences in isotopic fractionation caused by the two inorganic carbon utilization pathways in microalgae are significantly different. The maximum isotopic fractionation (δ _a ) can be caused by using the carbon dioxide pathway in the atmosphere. The direct transport of bicarbonate ions and the indirect transport of bicarbonate ions dependent on extracellular carbonic anhydrase resulted in 9‰ less isotopic fractionation than the isotopic fractionation using atmospheric carbon dioxide (PDB).

Acetazolamide (AZ) is a heterocyclic sulfonamide carbonic anhydrase extracellular enzyme inhibitor containing 1, 3, 4-thiadiazole ring. Using carbonic anhydrase ectoenzyme inhibitors to specifically inhibit the characteristics of carbonic anhydrase ectoenzyme, to study the isotope changes of microalgae under the inhibition of carbonic anhydrase ectoenzyme activity, can reversely identify the dependence on extracellular carbonic anhydride Contribution and share of the indirect transport pathway of the enzymatic bicarbonate ion to inorganic carbon utilization. While the indirect bicarbonate ion transport pathway, which depends on extracellular carbonic anhydrase, is inhibited, the direct bicarbonate ion transport pathway is also inhibited. High concentrations of sodium bicarbonate also have an inhibitory effect on the extracellular enzyme of carbonic anhydrase. Under the action of high concentration of sodium bicarbonate and 10mM AZ, the indirect transport pathway of bicarbonate ion and bicarbonate ion depend on extracellular carbonic anhydrase The direct transport pathway of ions will be completely inhibited at the same time.

The present invention adopts the following ideas: respectively add two kinds of sodium bicarbonate with very different δ ¹³ C values to simultaneously cultivate the microalgae to be tested, measure the δ ¹³ C value of the algae body, and use the isotope mixing model of the two ends to obtain the microalgae. The inorganic carbon source of the air and the proportion of the added inorganic carbon source are used, and then two kinds of high-concentration sodium bicarbonate with different δ ¹³ C values are added respectively, and the microalgae to be tested are cultivated under the condition of 10mM AZ, and the algae body is measured. The δ ¹³ C value is used to obtain the isotope fractionation value (δ _a ) caused by the microalgae’s complete use of carbon dioxide in the atmosphere. Finally, according to the above information, use the isotope mixing model of the two end members to calculate the shares of the two pathways.

The principle of obtaining the share of inorganic carbon sources:

There are four forms of total dissolved inorganic carbon (DIC) in water: CO ₂ , HCO ₃ ^- , H ₂ CO ₃ and CO ₃ ^2- , and they have the following chemical equilibrium: CO ₂ +H ₂ O→H ₂ CO ₃ →HCO ₃ ^- +H ⁺ →CO ₃ ^2- +2H ⁺ . There are two types of inorganic carbon— _CO2 and _HCO3 ^— that are available to microalgae, whether derived from air or added. Therefore, the isotope mixing model of two-terminal elements can be used to obtain the share of microalgae using inorganic carbon sources from air and utilizing added inorganic carbon sources.

The isotope mixing model of both ends can be expressed as:

δ _Ti = δ _Ai - f _Bi δ _Ai + f _Bi δ _Bi (i=1, 2, 3, ------) (1)

Here δ _Ti is the δ ¹³ C value of the microalgae, δ _Ai is the δ ¹³ C value of the algal body when it is assumed that the microalgae completely utilizes the inorganic carbon source of the air, and δ _Bi is the value of the algal body when it is assumed that the microalgae completely utilizes the added inorganic carbon source The δ ¹³ C value of the algal body, f _Bi is the proportion of the added inorganic carbon source used by the investigated microalgae.

Obviously, it is difficult to obtain f _Bi only by knowing δ _Ti . Therefore, the present invention uses sodium bicarbonate with a relatively large difference in δ ¹³ C values to simultaneously culture microalgae, and to identify microalgae with stable carbon isotope double labeling. Share of inorganic carbon sources.

For isotope label 1 (i=1), equation (1) is expressed as follows:

δ _T1 = δ _A1 - f _B1 δ _A1 + f _B1 δ _B1 (2)

Here δ _T1 is the δ ¹³ C value of the microalgae cultured with sodium bicarbonate with the first known δ ¹³ C value, and δ _A1 is the δ ¹³ value of the algal body when it is assumed that the microalgae completely utilizes air as an inorganic carbon source C value, δ _B1 is the δ ¹³ C value of the algal body when the microalgae fully utilizes the added inorganic carbon source, and f _B1 is the proportion of the microalgae under investigation using the added inorganic carbon source.

For isotope label 2 (i=2), equation (1) is expressed as follows:

δ _T2 = δ _A2 - f _B2 δ _A2 + f _B2 δ _B2 (3)

Here δ _T2 is the δ ¹³ C value of the microalgae cultured with sodium bicarbonate with the first known δ ¹³ C value, and δ _A2 is the δ ¹³ value of the algal body when it is assumed that the microalgae completely utilizes the air as an inorganic carbon source C value, δ _B2 is the δ ¹³ C value of the algal body when it is assumed that the microalgae fully utilizes the added inorganic carbon source, f _B2 is the share of the microalgae under investigation using the added inorganic carbon source.

In the two equations (2) and (3), δ _A1 = δ _A2 , f _B = f _Bi = f _B1 = f _B2 , solve simultaneously

(4)

(4) In the formula, δ _B1 - δ _B2 can be converted into the difference between the δ ¹³ C value δ _C1 of isotope-labeled sodium bicarbonate 1 and the δ ¹³ C value δ _C2 of isotope-labeled sodium bicarbonate 2, then:

（5）

(5)

Therefore, by measuring the δ ¹³ C value δ _C1 of isotope-labeled sodium bicarbonate 1 and the δ ¹³ C value δ _C2 of isotope-labeled sodium bicarbonate 2, the microalgae cultured with the corresponding labeled sodium bicarbonate can be measured simultaneously. The δ ¹³ C value, that is, the measured δ _T1 and δ _T2 values, was used to calculate the proportion of the added inorganic carbon source used by the investigated microalgae according to formula (5).

The principle of obtaining the share of microalgal inorganic carbon utilization pathways:

There are two types of inorganic carbon— _CO2 and _HCO3 ^— that are available to microalgae, whether derived from air or added. Therefore, the isotope mixing model of the terminal element can be used to obtain the share information of the two pathways of inorganic carbon utilization in microalgae.

The isotope mixing model of both ends 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 _Ti is the δ ¹³ C value of the microalgae, δ _a is the δ ¹³ C value of the algal body when the microalgae completely utilizes the carbon dioxide pathway in the atmosphere, and δ _bi is assumed to be the microalgae using the added inorganic carbon source to complete the carbon dioxide pathway is the δ ¹³ C value of the algal body, and f _b is the proportion of the bicarbonate ion pathway utilized by the investigated microalgae.

Here, δ _bi - δ _a can be converted into the δ ¹³ C value δ _Ci of isotope-labeled sodium bicarbonate (or δ ¹³ C value δ C1 of isotope-labeled sodium bicarbonate 1 or δ _C1 of isotope-labeled sodium bicarbonate 2 The difference D _i between ^{the 13} C value δ _C2 ) and the inorganic carbon δ ¹³ C value δ _C0 in the original culture solution without adding sodium bicarbonate and completely from air.

Therefore, formula (6) can be simplified as:

f _b = 1000 (δ _Ti -δ _a - f _B D _i )/9 (i=1, 2) (7)

The δ ¹³ C value of the algal body when the microalgae fully utilizes the carbon dioxide in the atmosphere, that is, the δ _a assignment principle:

Under the action of high concentration sodium bicarbonate and 10mM AZ, (7) formula f _b =0

That is, δ _Ti -δ _a - f _B D _i =0,

δ _a = δ _Ti - f _B D _i (i=1, 2) (8)

Comprehensive utilization of the above principles can quickly quantify the use of inorganic carbon pathways by microalgae; carry out culture experiments under exactly the same experimental conditions to obtain reliable data on the share of inorganic carbon pathways used by microalgae, which cannot be achieved by existing technologies.

Detailed ways

Embodiments of the present invention: the first step is to measure sodium bicarbonate produced by different manufacturers, and select two kinds of sodium bicarbonate whose δ ¹³ C value difference is greater than 8 ‰ as isotope label 1 and isotope label 2 and add them to the culture solution respectively. The δ ¹³ C value of isotope-labeled sodium bicarbonate is denoted as δ _Ci , wherein the δ ¹³ C value of isotope-labeled sodium bicarbonate 1 is δ _C1 , and the δ ¹³ C value of isotope-labeled 2 sodium bicarbonate is δ _C2 . The difference between the δ ¹³ C value δ _C of isotope-labeled sodium bicarbonate and the δ ¹³ C value δ _C0 of inorganic carbon in the original culture solution without sodium bicarbonate and completely from air is D _i , where D ₁ is the isotope-labeled 1 The difference between the δ ¹³ C value δ _C1 of sodium bicarbonate and the δ ¹³ C value δ _C0 of inorganic carbon in the original culture solution without adding sodium bicarbonate and completely from air, D ₂ is the δ ¹³ C of sodium bicarbonate labeled with 2 The difference between the value δ _C2 and the value δ _C0 of inorganic carbon δ ¹³ C in the original culture solution without adding sodium bicarbonate and completely from air.

In the second step, the culture solution of two kinds of isotope-labeled sodium bicarbonate is simultaneously used under the culture condition of being investigated and under the culture condition of 16 mM sodium bicarbonate and 10mM AZ respectively simultaneously, and the microalgae being investigated are cultivated, after 4 days, Simultaneously measure the stable carbon isotope composition δ ¹³ C values δ _T1 , δ _T2 , δ _T1-AZ and δ _T2-AZ of the investigated microalgae under the corresponding culture conditions of two isotope-labeled culture solutions ;.

，计算出各个培养条件下被考察微藻利用添加的无机碳源的份额f_B，微藻利用空气的无机碳源份额为1-f_B。 The third step is to use the δ ¹³ C value of sodium bicarbonate with isotope label 1 as δ _C1 , the δ ¹³ C value of microalgae cultured by isotope label 1 under various culture conditions as δ _T1 , and the value of isotope label 2 The δ ¹³ C value of sodium bicarbonate was taken as δ _C2 , and the δ ¹³ C value of the investigated microalgae cultured with isotope label 2 under the corresponding culture conditions was taken as δ _T2 , which was brought into

, calculate the proportion f _B of the added inorganic carbon source used by the investigated microalgae under each culture condition, and the proportion of the inorganic carbon source used by the microalgae in the air is 1-f _B .

The fourth step is based on the stable carbon isotope value δ _Ti (δ _T1-AZ or δ _T2-AZ ) of the investigated microalgae cultured under the culture conditions of 16 mM sodium bicarbonate and 10 mM AZ, according to the equation: δ _a = δ _Ti -f _B D _i , calculate the δ ¹³ C value δ _a of the algal body when the microalgae fully utilizes the carbon dioxide in the atmosphere.

The fifth step is based on the stable carbon isotope value δ _Ti of the microalgae to be tested under each culture condition, wherein the stable carbon isotope value δ T1 of the microalgae to be tested under isotope label 1 sodium bicarbonate culture is δ _T1 under isotope label 2 The stable carbon isotope value δ _T2 of the tested microalgae under sodium bicarbonate culture; the δ ¹³ C value δ _a of the algal body when the microalgae fully utilizes the carbon dioxide in the atmosphere; The fraction f _B and the corresponding δ ¹³ C value δ _Ci of isotope-labeled sodium bicarbonate (δ ¹³ C value δ C1 of isotope-labeled sodium bicarbonate δ _C1 or δ ¹³ C value δ C2 of isotope-labeled sodium bicarbonate ₂ ) and the information of the difference D _i of the inorganic carbon δ ¹³ C value δ _C0 in the original culture solution without adding sodium bicarbonate and completely from the air, substituting into the equation: f _b = 1000 (δ _Ti -δ _a - f _B D _i ) In /9, calculate the share f _b of the bicarbonate ion pathway used by the microalgae, and 1-f _b is the share of the carbon dioxide pathway used by the microalgae.

Implementation effect of the present invention is as follows:

The culture materials are: Chlamydomonas and Chlorella. The basic culture medium uses SE medium, and the basic culture conditions are: photoperiod L/D: 12h/12h; temperature 25°C; light intensity 100µmolm ^-2 s ^-1 , pH value 8.0 or 8.2 (adjusted with hydrochloric acid and sodium hydroxide ). Other culture conditions are shown in Table 1, where the δ ¹³ C of the added sodium bicarbonate is -26.3‰ (PDB) (δ _C1 ) and -16.5‰ (PDB) (δ _C2 ), respectively, 16 mM sodium bicarbonate and 10 mM AZ Culture conditions are required. The value of inorganic carbon δ ¹³ C δ _C0 in the original culture solution without adding sodium bicarbonate and completely from air was -11.8‰ (PDB). After 4 days of culture, the δ ¹³ C values of microalgae were measured for each culture condition and each experiment. Using the method of the present invention, the proportion f _B of the added inorganic carbon source and the proportion 1-f _B of the inorganic carbon source utilizing air under each culture condition of Chlamydomonas and Chlorella are obtained, as shown in Table 2. Subsequently, using the present invention, the shares of Chlamydomonas and Chlorella using bicarbonate ion pathways and carbon dioxide pathways under each culture condition were obtained, as shown in Table 3.

Table 1 Culture conditions of each culture experiment of Chlamydomonas and Chlorella

Table 2 The share of added inorganic carbon sources utilized by Chlamydomonas and Chlorella under various culture conditions

Table 3 The share of bicarbonate ion pathway and carbon dioxide utilization pathway in Chlamydomonas and Chlorella under various culture conditions Training number f _b 1-f _b Training 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

It can be seen from Table 2 that the proportion of the added inorganic carbon source was significantly different under different culture conditions, showing a trend that the more sodium bicarbonate was added, the greater the share of the added inorganic carbon source (except YB6 and XB6) . YBA2, YBA3 and XBA3 with fractions of added inorganic carbon source less than 0 are due to measurement errors, but are also very close to 0. These are consistent with the actual situation, that is, within a certain concentration range, the more sodium bicarbonate is added, the greater the proportion of the added inorganic carbon source is used. When the concentration of sodium bicarbonate exceeds a certain range, certain physiological processes are inhibited. Inhibition, resulting in a lower share of the utilization of the added inorganic carbon source. It shows that the proportion of the added inorganic carbon source used by the microalgae obtained by the present invention is reliable.

It can be seen from Table 3 that the share of bicarbonate ion pathway utilized by microalgae was significantly different under different culture conditions, showing a trend that the greater the concentration of AZ was added, the smaller the share of microalgae utilized bicarbonate ion pathway. This is because AZ is an inhibitor of extracellular carbonic anhydrase. Overall, Chlamydomonas utilized bicarbonate ion pathways more than Chlorella. DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulfonate) can inhibit bicarbonate ions from entering cells. Using DIDS to specifically inhibit bicarbonate ions from entering cells, research on bicarbonate ions entering cells The microalgae isotopic changes under blocking can reversely identify the contribution and share of the direct transport pathway of bicarbonate ions to inorganic carbon utilization. While the direct bicarbonate ion transport pathway was completely inhibited, the indirect bicarbonate ion transport pathway dependent on extracellular carbonic anhydrase was also completely inhibited. Low concentrations of DIDS had little effect on bicarbonate ion utilization pathways. A high concentration of DIDS (2mM) has a greater effect on the pathway utilizing bicarbonate ions and almost completely inhibits the pathway utilizing bicarbonate ions. This is consistent with the actual situation. Due to the measurement error, there are some situations greater than 1 or less than 0, which can be corrected to 1 or 0. For example, the shares of YB4 and XB4 using the bicarbonate ion pathway are 1.03 and 1.04, which can be corrected to 1. Using carbon dioxide The share of the pathways can be corrected to 0, indicating that when adding 4mM sodium bicarbonate, the two microalgae did not use the carbon dioxide pathway, but only used the bicarbonate ion pathway, which is also consistent with the actual situation. Some large deviations appeared for XD5, YBAT1, XBAT1, and XBAT2 because, compared with the microalgae cultured under 16 mM NaHCO3 and 10 mMAZ, the growth rate of the microalgae was small, the cell volume was smaller, and the microalgae The measured δ ¹³ C value of algae is minus 1 to 2‰ (PDB) than the theoretical value. Therefore, apparently, the share of microalgae using carbon dioxide pathways increases, but this situation (very little supply of inorganic carbon) occurs in nature Rarely occurs. YB5 and XB5 also have large deviations. This is because, compared with the microalgae cultured under 16 mM sodium bicarbonate and 10 mMAZ, the growth rate of the microalgae at this time is large, the cell volume is large, and the measured δ ¹³ C value of the microalgae It is 0.5 to 1‰ PDB higher than the theoretical value, but this situation (sodium bicarbonate up to 8mM) also rarely occurs in nature. For situations that often occur in nature, the present invention can well estimate the share of bicarbonate ion pathways used by microalgae.

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.