CN103074411B - Method for detecting and quantifying utilization of carbon source in calcium carbonate by microalgae - Google Patents
Method for detecting and quantifying utilization of carbon source in calcium carbonate by microalgae Download PDFInfo
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Abstract
The invention discloses a method for detecting and quantifying utilization of carbon source in calcium carbonate by microalgae. The method comprises the following steps: adding two types of calcium carbonate powder with great difference in Epsilon 13 C value but same crystal form respectively to cultivate the to-be-detected microalgae, measuring the protein content of the microalage at different incubation time and Epsilon 13 C value of the stable carbon isotope composition of the microalage after a harvest; then, remedying the measured Epsilon 13 C value of the microalage at the time of the harvest by using the protein content of the initial microalage and Epsilon 13 C value, and obtaining the share of carbon source in the added calcium carbonate utilized by the microalgae by using the isotope mixed model; through the data about the protein content of the microalage at different incubation time and Redfield value, fitting an exponential growth equation about the biomass and time; finally, calculating the amount of carbon source in calcium carbonate obtained by the detected microalgae of biomass per unit in unit time according to the share of carbon source in the added calcium carbonate utilized by the microalgae and the equation about the biomass and time.
Description
Technical field
The present invention relates to detection and quantivative approach that a kind of micro-algae utilizes calcium carbonate carbon source, belong to reply climate change and marine biotechnology field.
Background technology
Before the Industrial Revolution, the concentration of Carbon Dioxide in Air is only 280 ppmv, and the concentration of present airborne carbonic acid gas reaches 391 ppmv, has increased by 40%.Increase to leading whole world change by the concentration of carbonic acid gas in atmosphere, not only bring ecology and economic problems to the whole world, also brought political issue.Carbon content in Global Carbon Carbonate Rocks is 5 × 10
21mol, is considered to maximum carbon storehouse.Calcium carbonate is again the topmost composition of carbonatite, and its ratio shared in carbonatite is greater than 50%.In addition,, in modern abyssal sediment, tosca thing accounts for 32.2% (mean value).The generation place of carbonatite is not only in ocean and lake, is also main storage vault.Ocean and lake have covered 70% of earth surface, contain a large amount of plant planktons, and it contributes approximately 50% tellurian net primary productivity.Therefore, Marine ecosystems are that most important carbon converges and carbon source.
Micro-algae (microalgae) comprises the swim microphyte of mode of life of all Shui Zhongying of living in, and conventionally just refers to planktonic algae.Micro-algal structure is simple, and its physiological process is also relatively simple, and some kind is the model plant of scientific research, as: Chlamydomonas reinhardtii, chlorella, all right artificial culture of many types, this research that is us is provided convenience.
The flux that the data that the estimation that marine carbon converges at present utilizes are the carbonic acid gas in atmosphere, ignore the utilization of hydrobiont to this huge carbon storehouse of calcium carbonate in Marine ecosystems, the precision that this has a strong impact on carbon absorption estimation, causes some reply policies of climate change and the validity of measure to reduce.Quantitative micro-algae will contribute to science estimation carbon to converge to the utilization of the inorganic carbon source of calcium carbonate, and efficiently and effectively is formulated policy and the measure of reply climate change, meanwhile, and also for the development of Microalgae biotechnology and the improvement of wawter bloom red tide provide scientific basis.
Summary of the invention
The technical problem to be solved in the present invention is that the detection and the quantivative approach that provide a kind of micro-algae to utilize calcium carbonate carbon source, filled up the blank in carbon absorption estimation.
The present invention takes following technical scheme: it comprises the following steps: the first, select two kinds of δ
13c value difference value be greater than 8 ‰ and the identical calcium carbonate powders of crystal formation add to respectively in nutrient solution and cultivate and treat micrometer algae as isotopic labeling 1 and isotopic labeling 2, and measure the initial stable carbon isotope composition δ that treats micrometer algae
13c value and protein content N
0; The δ of the calcium carbonate of carbon isotope labelling 1
13c value is δ
c1, the δ of the calcium carbonate of carbon isotope labelling 2
13c value is δ
c2; The initial stable carbon isotope composition δ that treats micrometer algae
13c value is δ
0;
Second, in culturing process, measure the protein content of the micro-algae of different incubation times under each culture condition, after cultivating 7 to 9 days, results frond, measures respectively stable carbon isotope under corresponding each culture condition that two kinds of isotope-labeled nutrient solutions cultivate, that investigated micro-algae and forms δ
13the value δ of C
h t1, δ
h t2with protein content N
1, N
2;
The 3rd, utilize the protein content N of initial frond
0with stable carbon isotope composition δ
13c value δ
0the protein content N of micro-algae during with results
i, pass through updating formula: δ
h ti=(N
0/ N
i) δ
0+ (1-N
0/ N
i) δ
tithe frond δ measuring during to results
13c value δ
h tiproofread and correct, calculate the frond δ after correction
13c value δ
ti;
The 4th, pass through equation
, calculate the share f that utilizes the calcium carbonate carbon source of adding under the each culture condition of micro-algae
b;
The 5th, calculate the mean value of micro-algae protein content of two kinds of isotope-labeled nutrient solution cultivations under the different culture condition of different incubation times, the biomass temporal evolution of the protein metering of the micro-algae of utilization index growth equation matching, draw under each culture condition, investigated micro algae biomass Q
tthe equation that T changes in time, Q
t=q+ae
bT, wherein Q
t: the micro algae biomass that representative is measured with protein in the time of time T, q, a and b: represent fitting parameter, e:2.7183; The biological significance of b is the biomass being increased within the unit time by the micro-algae of investigation of unit biomass.
The 6th, the share f of the calcium carbonate carbon source that during according to results, under each culture condition, micro-algae utilization is added
b, unit biomass investigated micro-algae the biomass b and the conversion factor F that within the unit time, increase, utilize formula d=Fbf
b, calculate the amount d that is investigated the calcium carbonate carbon source obtained of micro-algae within the unit time of unit mass biomass.
advantage of the present invention is as follows:
Micro-algae has two kinds of modes to the utilization of water body inorganic carbon, and (1) utilizes the carbonic acid gas in atmosphere.CO
2as linear non-polar molecule, be electric neutrality, it can enter cell bilayer lipid membrane by free diffusing, enters the CO in cell
2by the photosynthesis of microalgae cell is utilized; (2) utilize bicarbonate ion in solution.And the reversible hydration katalysis of carbonic acid gas (carbonic anhydride) of the outer carbonic anhydrase of born of the same parents
play important promoter action in micro-algae in to the utilization of inorganic carbon.
In biological presence, there is following reaction in calcium carbonate in ocean or lake:
Therefore, micro-algae possesses the condition of utilizing the carbon in calcium carbonate completely.Forefathers generally believe the weathering of carbonatite and corrosion neither carbon converge neither carbon source, the calcium carbonate of dissolving can precipitate again very soon, the few notice that micro-algae can utilize the carbon in calcium carbonate, more nobody utilizes calcium carbonate carbon source to carry out quantitatively to micro-algae.
Occurring in nature carbon has two kinds of stable isotopes:
12c and
13c, their natural average abundance is respectively 98.89% and 1.11%.Stable carbon isotope composition is used δ conventionally
13c(‰) represent occurring in nature δ
13c is changed to-90 ‰ ~+20 ‰.The strong fractionation feature of stable carbon isotope is the basis in the micro-algae inorganic carbon of identification source.Mass balance principle and isotropic substance mixture model and chemometrics method are the bases in the micro-algae inorganic carbon of quantitatively identification source.
The present invention takes following thinking: add respectively two kinds of δ
13the calcium carbonate powders that the different great disparity of C value difference and crystal formation are identical is cultivated simultaneously and is treated micrometer algae, measures protein content and the rear frond stable carbon isotope composition of the results δ of different incubation time fronds
13c value.Then, utilize protein content and the δ of initial frond
13the frond δ that C value was measured whens results
13c value is proofreaied and correct, and the isotropic substance mixture model of recycling two end members obtains the share that micro-algae utilization comes from the calcium carbonate carbon source of interpolation.Then, utilize the protein content data of different incubation time fronds, with exponential growth equation model biomass temporal evolution.Finally, utilize share and the time dependent equation of biomass of the calcium carbonate carbon source of adding according to micro-algae, the amount of the calcium carbonate carbon source of being obtained by the micro-algae of investigation of the unit's of calculating biomass within the unit time.
Obtain the principle of calcium carbonate carbon source share:
Under different culture condition, cultivate frond, the δ of frond
13c value is different.So, process the δ that cultivates the rear frond of measuring
13c value is the δ of frond while inoculating at first
13the δ of the frond of growing in C value and culturing process
13the mixed result of C value, and the δ of the frond of growing in culturing process
13c value more can reflect the utilize situation of frond to different carbon sources.The δ of the frond of measuring when therefore, we can use isotropic substance mixture model to results
13c proofreaies and correct.
Its calculation formula can be expressed as:
δ
h Ti=(N
0/N
i)δ
0+ (1-N
0/N
i)δ
Ti （1）
Here δ
h tithe δ of micro-algae of measuring during for results
13c value, δ
0the δ of micro-algae of measuring during for inoculation
13c value, δ
ti: the δ of the micro-algae after correction
13c value, N
0: initial frond biomass, N
i: the biomass of frond when different markers are cultivated lower results.
The inorganic carbon source that micro-algae utilizes is the carbon in the calcium carbonate adding and the inorganic carbon except the calcium carbonate carbon source of adding.Therefore, can utilize the isotropic substance mixture model of two end members to obtain the share that micro-algae utilizes the share of the calcium carbonate carbon source of adding and comes from the inorganic carbon source beyond the calcium carbonate carbon source of 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，------）（2）
Here δ
tifor the δ of the micro-algae after proofreading and correct
13c value, δ
aithe δ of frond while utilizing the inorganic carbon source beyond the calcium carbonate carbon source that comes from interpolation completely for being assumed to micro-algae
13c value, δ
bithe δ of frond while utilizing the calcium carbonate carbon source of interpolation completely for being assumed to micro-algae
13c value, f
bifor the micro-algae of this investigation utilizes the shared share of calcium carbonate carbon source of adding.
Obviously, only know δ
tibe difficult to obtain f
bi, therefore, the present invention adopts the δ with larger difference
13c value calcium carbonate powders is cultivated respectively micro-algae simultaneously, identifies the share of the calcium carbonate carbon source of micro-algae utilization interpolation with stable carbon isotope two-way mark.
For isotopic labeling 1(i=1), equation (2) is expressed as follows formula:
δ
T1=δ
A1- f
B1δ
A1 +f
B1δ
B1 （3）
Here δ
t1for using the known δ of the first
13δ after micro-algae frond that the calcium carbonate powders of C value is cultivated is proofreaied and correct
13c value, δ
a1the δ of frond while utilizing the inorganic carbon source beyond the calcium carbonate carbon source that comes from interpolation completely for being assumed to micro-algae
13c value, δ
b1the δ of frond while utilizing the calcium carbonate carbon source of interpolation completely for being assumed to micro-algae
13c value, f
b1for the micro-algae of this investigation utilizes the shared share of calcium carbonate carbon source of adding.
For isotopic labeling 2(i=2), equation (2) is expressed as follows formula:
δ
T2=δ
A2 - f
B2δ
A2 +f
B2δ
B2 （4）
Here δ
t2for using the known δ of the second
13δ after micro-algae frond that the calcium carbonate of C value is cultivated is proofreaied and correct
13c value, δ
a2the δ of frond while utilizing the inorganic carbon source beyond the calcium carbonate carbon source that comes from interpolation completely for being assumed to micro-algae
13c value, δ
b2the δ of frond while utilizing the calcium carbonate carbon source of interpolation completely for being assumed to micro-algae
13c value, f
b2for the micro-algae of this investigation utilizes the shared share of calcium carbonate carbon source of adding.
(3) δ and in (4) two equations
a1=δ
a2, f
b=f
bi=f
b1=f
b2, simultaneous solution
(5) δ in formula
b1-δ
b2can be converted into the δ of the calcium carbonate of isotopic labeling 1
13c value δ
c1δ with the calcium carbonate of isotopic labeling 2
13c value δ
c2poor:
Therefore, can be by the δ of the calcium carbonate of mensuration isotopic labeling 1
13c value δ
c1δ with the calcium carbonate of isotopic labeling 2
13c value δ
c2, measure the micro-algae δ cultivating with the calcium carbonate of corresponding mark simultaneously
13c value, determines δ
t1and δ
t2value, calculates the micro-algae of this investigation according to (6) formula and utilizes the shared share of calcium carbonate carbon source of adding.
Nitrogen in organism is almost all present in the middle of protein, and nitrogen element content in protein is about 16%.Meanwhile, can know the O of marine phytoplankton according to classical Redfield value
2: C:N:P generally approaches 138:106:16:1.And organic content has accounted for the more than 90% of total mass in plant plankton.Like this, as long as know that the content of protein just can calculate organic carbon content and the biomass of frond.
According to the biomass data of the micro-algae of different time, with exponential growth equation model biomass Q
tt changes (Q in time
t=q+ae
bT), q, a and b are equation parameter here, to this exponential growth equation differentiate, draw the rate of rise equation of micro algae biomass, that is: V
t=abe
bT, V here
tfor the rate of rise of micro algae biomass.The rate of rise V of micro algae biomass
twith biomass Q
tlinear, its slope is b, and the biological significance of b is the biomass being increased within the unit time by the micro-algae of investigation of unit biomass.The amount d that is investigated the calcium carbonate carbon source obtained of micro-algae within the unit time of unit mass biomass is the share f from interpolation calcium carbonate carbon source in biomass b that micro-algae increases within the unit time and the micro-algae of this investigation that investigated of unit biomass
bwith the product of conversion factor F, that is: d=Fbf
b.Can know according to classical Redfield value, the biomass of the every increase unit mass of plant plankton will increase the carbon of corresponding mass.Therefore,
from biological significance, F represents the organic carbon content that the biomass of every increase unit mass increases, N represents protein content, 16% represents the per-cent of nitrogen element in protein, 5.7 represent the carbon-nitrogen ratio of plant plankton, 29 representatives quality biomass corresponding to unit nitrogen element in plant plankton, and the 1000th, milligram biomass is converted into a gram biomass.And the biological significance of above formula Fb is the quality of the organic carbon being increased within the unit time by the micro-algae of investigation of unit mass biomass.
Advantage of the present invention is as follows:
1) present method can detect and quantitative micro-algae utilizes the amount of calcium carbonate carbon source, has filled up the blank in carbon sink estimation;
2) present method does not need to obtain the isotropic substance δ of two end members
13the absolute value of C, only need are measured the δ of two isotope-labeled calcium carbonate
13c value, therefore needs step few, calculates simple;
3) present method needs location parameter less, and experiment is simple, and cost is low;
4) present method is carried out two groups of culture experiment under identical experiment condition, and notice the problem of calcium carbonate crystal formation on experimental result impact by great many of experiments, therefore, obtain micro-algae and utilize the data of amount of calcium carbonate carbon source more reliable, under different algal species and different treatment, micro-algae utilizes the amount of adding calcium carbonate carbon source also to have comparability.
Embodiment
Embodiments of the invention: first step, measure the calcium carbonate stable carbon isotope composition δ that different manufacturers is produced
13c value and crystal formation, select two kinds of δ
13c value difference value be greater than 8 ‰ and the identical calcium carbonate powders of crystal formation be added to respectively in nutrient solution and cultivate and treat micrometer algae as isotopic labeling 1 and isotopic labeling 2, and measure the initial stable carbon isotope composition δ that treats micrometer algae
13c value δ
0with protein content N
0.The δ of isotope-labeled calcium carbonate
13c value is designated as δ
ci, the wherein δ of the calcium carbonate of isotopic labeling 1
13c value is δ
c1, the δ of the calcium carbonate of isotopic labeling 2
13c value is δ
c2.
Second step, in culturing process, measure the protein content of the micro-algae of different incubation times under each culture condition, after cultivation finishes, results frond, measures respectively stable carbon isotope under corresponding each culture condition that two kinds of isotope-labeled nutrient solutions cultivate, that investigated micro-algae and forms δ
13c value δ
h tiwith protein content N
i, by δ under isotopic labeling 1 culture condition that cultivate, each, that investigated micro-algae
13c value and protein content are respectively as δ
h t1and N
1, by δ under that cultivate, the corresponding culture condition of isotopic labeling 2, that investigated micro-algae
13c value and protein content are respectively as δ
h t2and N
2.
Third step, utilizes the protein content N of initial frond
0with stable carbon isotope composition δ
13c value δ
0the protein content N of micro-algae during with results
i, pass through updating formula: δ
h ti=(N
0/ N
i) δ
0+ (1-N
0/ N
i) δ
tithe frond δ measuring during to results
13c value δ
h tiproofread and correct, calculate the frond δ after correction
13c value δ
ti, by the δ after correction under isotopic labeling 1 culture condition that cultivate, each, that investigated micro-algae
13c value is δ
t1, by the δ after correction under that cultivate, the corresponding culture condition of isotopic labeling 2, that investigated micro-algae
13c value is as δ
t2.
The 4th step, by δ
c1, δ
c2, δ
t1and δ
t2bring into
, calculate the share f that is investigated the calcium carbonate carbon source of micro-algae utilization interpolation under each culture condition
b.
The 5th step, calculates under different culture condition under different incubation times the mean value of micro-algae protein content that two kinds of isotope-labeled nutrient solutions cultivate the biomass Q of the protein of the micro-algae of utilization index growth equation matching metering
tt changes in time, draw under corresponding each culture condition, investigated the time dependent equation of micro algae biomass, Q
t=q+ae
bT;
The 6th step, the share f of the calcium carbonate carbon source that during according to results, under each culture condition, micro-algae utilization is added
b, unit biomass investigated micro-algae the biomass b and the conversion factor F that within the unit time, increase, utilize formula d=Fbf
b, calculate the amount d that is investigated the calcium carbonate carbon source obtained of micro-algae within the unit time of unit mass biomass.
different concns acetazolamide is processed lower chlamydomonas and is utilized the mensuration of adding calcium carbonate carbon source amount:
Cultivated material is: chlamydomonas.Basic culture solution adopts SE substratum, and basic culture condition is: photoperiod L/D:12h/12h; 25 DEG C of temperature; Intensity of illumination is
, pH value 8.0(regulates with hydrochloric acid and sodium hydroxide).Add respectively 1g δ
13c value difference value be greater than 8 ‰ and the identical calcium carbonate powders of crystal formation to basic SE nutrient solution, contain 0.1mmol/L born of the same parents outside the SE nutrient solution of carbonic anhydrase inhibitor acetazolamide (AZ) outside the SE nutrient solution of carbonic anhydrase inhibitor acetazolamide (AZ) and the born of the same parents of containing 10mmol/L, the δ of the calcium carbonate of interpolation
13c value is respectively 0.34 ‰ (PDB) (δ
c1) and-15.80 ‰ (PDB) (δ
c2).After inoculation algae, measure the δ of initial chlamydomonas frond
13c value δ
0with protein biomass N
0.In the common culturing bottle that is stamped air-permeable envelope, cultivate chlamydomonas, and measure the protein content of different incubation time chlamydomonas under each processing.Results are cultivated the chlamydomonas after 7 days, measure under different treatment the δ of the chlamydomonas frond that two kinds of calcium carbonate markers cultivate respectively
13c value δ
h t1, δ
h t2with protein biomass N
1, N
2(table 1).Utilize updating formula: δ
h ti=(N
0/ N
i) δ
0+ (1-N
0/ N
i) δ
tithe chlamydomonas frond δ measuring during to results
13c value δ
h tiproofread and correct, calculate the chlamydomonas frond δ after correction under different treatment
13c value δ
ti(table 1).Use again equation
, show that chlamydomonas under different treatment utilizes the share f of the calcium carbonate carbon source of adding
b(table 1).
Calculate the mean value of the chlamydomonas protein content of two kinds of isotope-labeled nutrient solution cultivations under the different incubation times of different treatment, and the biomass Q of the protein of utilization index growth equation matching chlamydomonas metering
tt changes in time, draw under corresponding each culture condition, investigated the time dependent equation of chlamydomonas biomass, Q
t=q+ae
bT(table 2).
The share f of the calcium carbonate carbon source that during according to results, under each culture condition, chlamydomonas utilization is added
b, unit biomass investigated chlamydomonas the biomass b and the conversion factor F that within the unit time, increase, utilize formula d=Fbf
b, calculate the amount d (table 3) that is investigated the calcium carbonate carbon source obtained of chlamydomonas within the unit time of unit mass biomass.
low CO
2
concentration is coerced the mensuration of processing lower chlamydomonas utilization interpolation calcium carbonate carbon source amount with different concns acetazolamide:
Cultivated material is: chlamydomonas.Basic culture solution adopts SE substratum, and basic culture condition is: photoperiod L/D:12h/12h; 25 DEG C of temperature; Intensity of illumination is
, pH value 8.0(regulates with hydrochloric acid and sodium hydroxide).Add respectively 1g δ
13c value difference value be greater than 8 ‰ and the identical calcium carbonate powders of crystal formation to basic SE nutrient solution, contain 0.1mmol/L born of the same parents outside the SE nutrient solution of carbonic anhydrase inhibitor acetazolamide (AZ) outside the SE nutrient solution of carbonic anhydrase inhibitor acetazolamide (AZ) and the born of the same parents of containing 10mmol/L, the δ of the calcium carbonate of interpolation
13c value is respectively 0.34 ‰ (PDB) (δ
c1) and-15.80 ‰ (PDB) (δ
c2).After inoculation algae, measure the δ of initial chlamydomonas frond
13c value δ
0with protein biomass N
0.Be furnished with except CO
2in the culturing bottle of device, cultivate chlamydomonas to be measured, and measure the protein content of different incubation time chlamydomonas under each processing.
Results are cultivated the chlamydomonas after 7 days, measure under different treatment the δ of the chlamydomonas frond that two kinds of calcium carbonate markers cultivate respectively
13c value δ
h t1, δ
h t2with protein biomass N
1, N
2(table 4).Utilize updating formula: δ
h ti=(N
0/ N
i) δ
0+ (1-N
0/ N
i) δ
tithe chlamydomonas frond δ measuring during to results
13c value δ
h tiproofread and correct, calculate the chlamydomonas frond δ after correction under different treatment
13c value δ
ti(table 4).Use again equation
, show that chlamydomonas under different treatment utilizes the share f of the calcium carbonate carbon source of adding
b(table 4).
Calculate the mean value of the chlamydomonas protein content of two kinds of isotope-labeled nutrient solution cultivations under the different incubation times of different treatment, and the biomass Q of the protein of utilization index growth equation matching chlamydomonas metering
tt changes in time, draw under corresponding each culture condition, investigated the time dependent equation of chlamydomonas biomass, Q
t=q+ae
bT(table 5).
The share f of the calcium carbonate carbon source that during according to results, under each culture condition, chlamydomonas utilization is added
b, unit biomass investigated micro-chlamydomonas the biomass b and the conversion factor F that within the unit time, increase, utilize formula d=Fbf
b, calculate the amount d (table 6) that is investigated the calcium carbonate carbon source obtained of chlamydomonas within the unit time of unit mass biomass.
different concns acetazolamide is processed lower chlorella and is utilized the mensuration of adding calcium carbonate carbon source amount:
Cultivated material is: chlorella.Basic culture solution adopts SE substratum, and basic culture condition is: photoperiod L/D:12h/12h; 25 DEG C of temperature; Intensity of illumination is
, pH value 8.0(regulates with hydrochloric acid and sodium hydroxide).Add respectively 1g δ
13c value difference value be greater than 8 ‰ and the identical calcium carbonate powders of crystal formation to basic SE nutrient solution, contain 0.1mmol/L born of the same parents outside the SE nutrient solution of carbonic anhydrase inhibitor acetazolamide (AZ) outside the SE nutrient solution of carbonic anhydrase inhibitor acetazolamide (AZ) and the born of the same parents of containing 10mmol/L, the δ of the calcium carbonate of interpolation
13c value is respectively 0.34 ‰ (PDB) (δ
c1) and-15.80 ‰ (PDB) (δ
c2).After inoculation algae, measure the δ of initial chlorella frond
13c value δ
0with protein biomass N
0.In the common culturing bottle that is stamped air-permeable envelope, cultivate chlorella, and measure the protein content of different incubation time chlorellas under each processing.Results are cultivated the chlorella after 7 days, measure under different treatment the δ of the chlorella frond that two kinds of calcium carbonate markers cultivate respectively
13c value δ
h t1, δ
h t2with protein biomass N
1, N
2(table 7).Utilize updating formula: δ
h ti=(N
0/ N
i) δ
0+ (1-N
0/ N
i) δ
tithe bead frond δ measuring during to results
13c value δ
h tiproofread and correct, calculate the bead frond δ after correction under different treatment
13c value δ
ti(table 7).Use again equation
, show that chlorella under different treatment utilizes the share f of the calcium carbonate carbon source of adding
b(table 7).
Calculate the mean value of the chlorella protein content of two kinds of isotope-labeled nutrient solution cultivations under the different incubation times of different treatment, and the biomass Q of the protein of utilization index growth equation matching chlorella metering
tt changes in time, draw under corresponding each culture condition, investigated the time dependent equation of chlorella biomass, Q
t=q+ae
bT(table 8).
The share f of the calcium carbonate carbon source that during according to results, under each culture condition, chlorella utilization is added
b, unit biomass investigated chlorella the biomass b and the conversion factor F that within the unit time, increase, utilize formula d=Fbf
b, calculate the amount d (table 9) that is investigated the calcium carbonate carbon source obtained of chlorella within the unit time of unit mass biomass.
low CO
2
concentration is coerced the mensuration of processing lower chlorella utilization interpolation calcium carbonate carbon source amount with different concns acetazolamide:
Cultivated material is: chlorella.Basic culture solution adopts SE substratum, and basic culture condition is: photoperiod L/D:12h/12h; 25 DEG C of temperature; Intensity of illumination is
, pH value 8.0(regulates with hydrochloric acid and sodium hydroxide).Add respectively 1g δ
13c value difference value be greater than 8 ‰ and the identical calcium carbonate powders of crystal formation to basic SE nutrient solution, contain 0.1mmol/L born of the same parents outside the SE nutrient solution of carbonic anhydrase inhibitor acetazolamide (AZ) outside the SE nutrient solution of carbonic anhydrase inhibitor acetazolamide (AZ) and the born of the same parents of containing 10mmol/L, the δ of the calcium carbonate of interpolation
13c value is respectively 0.34 ‰ (PDB) (δ
c1) and-15.80 ‰ (PDB) (δ
c2).After inoculation algae, measure the δ of initial chlorella frond
13c value δ
0with protein biomass N
0.Be furnished with except CO
2in the culturing bottle of device, cultivate chlorella to be measured, and measure the protein content of different incubation time chlorellas under each processing.Results are cultivated the chlorella after 7 days, measure under different treatment the δ of the chlorella frond that two kinds of calcium carbonate markers cultivate respectively
13c value δ
h t1, δ
h t2with protein biomass N
1, N
2(table 10).Utilize updating formula: δ
h ti=(N
0/ N) δ
0+ (1-N
0/ N) δ
tithe bead frond δ measuring during to results
13c value δ
h tiproofread and correct, calculate the bead frond δ after correction under different treatment
13c value δ
ti(table 10).Use again equation
, show that chlorella under different treatment utilizes the share f of the calcium carbonate carbon source of adding
b(table 10).
Calculate the mean value of the chlorella protein content of two kinds of isotope-labeled nutrient solution cultivations under the different incubation times of different treatment, and the biomass Q of the protein of utilization index growth equation matching chlorella metering
tt changes in time, draw under corresponding each culture condition, investigated the time dependent equation of chlorella biomass, Q
t=q+ae
bT(table 11).
The share f of the calcium carbonate carbon source that during according to results, under each culture condition, chlorella utilization is added
b, unit biomass investigated chlorella the biomass b and the conversion factor F that within the unit time, increase, utilize formula d=Fbf
b, calculate the amount d (table 12) that is investigated the calcium carbonate carbon source obtained of chlorella within the unit time of unit mass biomass.
effect:
In summary it can be seen, under the culture condition without acetazolamide processing, the chlamydomonas of every increase unit mass of unit time is greater than the utilization of chlorella to calcium carbonate carbon source to the utilization of calcium carbonate carbon source.This conforms to the fact that the outer carbonic anhydrase activity of chlamydomonas born of the same parents is greater than the outer carbonic anhydrase activity of born of the same parents of chlorella.Meanwhile, for frond of the same race, at low CO
2micro-algae of every increase unit mass of lower unit time of condition that concentration is coerced is generally greater than at high CO the utilization of calcium carbonate carbon source
2the utilization of micro-algae to calcium carbonate carbon source under concentration conditions.This and low CO
2the Atmospheric CO that under concentration, micro-algae can be used
2less and be forced to utilize the fact of calcium carbonate carbon source to conform to.In addition, when without low CO
2when concentration is coerced, outside the born of the same parents of high density, carbonic anhydrase inhibitor AZ (10mmol/L) effect, micro-algae of every increase unit mass of unit time is greater than the outer carbonic anhydrase inhibitor AZ of born of the same parents of lower concentration to the utilization of calcium carbonate carbon source or without the utilization of micro-algae to calcium carbonate carbon source the outer carbonic anhydrase inhibitor AZ effect of born of the same parents.Under this acts on carbonic anhydrase inhibitor AZ (10mmol/L) outside the born of the same parents of high density, the outer almost total loss of carbonic anhydrase activity of born of the same parents of micro-algae, makes micro-algae not utilize the CO in atmosphere by the hydration katalysis of the outer carbonic anhydrase of born of the same parents
2thereby, reduced micro-algae to Atmospheric CO
2the fact of utilization conform to.In an embodiment of the present invention, there are two situations that calcium carbonate utilization of carbon source share is negative value in chlorella, this is because chlorella is now less to calcium carbonate utilization of carbon source amount, experimental error causes these values faintly partially negative, therefore in the calculating that it is utilized to calcium carbonate utilization of carbon source amount, be, 0 by its correction.In a word, above numerous conclusion can show that it is believable that micro-algae that the present invention invents utilizes the quantitative detecting method of calcium carbonate carbon source.
Claims (1)
1. micro-algae utilizes detection and a quantivative approach for calcium carbonate carbon source, it is characterized in that: it comprises the following steps:
The first, select two kinds of δ
13c value difference value be greater than 8 ‰ and the identical calcium carbonate powders of crystal formation add to respectively in nutrient solution and cultivate and treat micrometer algae as isotopic labeling 1 and isotopic labeling 2, and measure the initial stable carbon isotope composition δ that treats micrometer algae
13c value and protein content N
0; The δ of the calcium carbonate of carbon isotope labelling 1
13c value is δ
c1, the δ of the calcium carbonate of carbon isotope labelling 2
13c value is δ
c2; The initial stable carbon isotope composition δ that treats micrometer algae
13c value is δ
0;
Second, in culturing process, measure the protein content of the micro-algae of different incubation times under each culture condition, after cultivating 7 to 9 days, results frond, measures respectively stable carbon isotope under corresponding each culture condition that two kinds of isotope-labeled nutrient solutions cultivate, that investigated micro-algae and forms δ
13the value δ of C
h t1, δ
h t2with protein content N
1, N
2;
The 3rd, utilize the protein content N of initial frond
0with stable carbon isotope composition δ
13c value δ
0the protein content N of micro-algae during with results
i, pass through updating formula: δ
h ti=(N
0/ N
i) δ
0+ (1-N
0/ N
i) δ
tithe frond δ measuring during to results
13c value δ
h tiproofread and correct, calculate the frond δ after correction
13c value δ
ti;
The 4th, pass through equation
, calculate the share f that utilizes the calcium carbonate carbon source of adding under the each culture condition of micro-algae
b;
The 5th, calculate the mean value of micro-algae protein content of two kinds of isotope-labeled nutrient solution cultivations under the different culture condition of different incubation times, the biomass temporal evolution of the protein metering of the micro-algae of utilization index growth equation matching, draw under each culture condition, investigated micro algae biomass Q
tthe equation that T changes in time, Q
t=q+ae
bT, wherein Q
t: the micro algae biomass that representative is measured with protein in the time of time T, q, a and b: represent fitting parameter, e:2.7183; The biological significance of b is the biomass being increased within the unit time by the micro-algae of investigation of unit biomass;
The 6th, the share f of the calcium carbonate carbon source that during according to results, under each culture condition, micro-algae utilization is added
b, unit biomass investigated micro-algae the biomass b and the conversion factor F that within the unit time, increase, utilize formula d=Fbf
b, calculate the amount d that is investigated the calcium carbonate carbon source obtained of micro-algae within the unit time of unit mass biomass, wherein
from biological significance, F represents the organic carbon content that the biomass of every increase unit mass increases, N represents protein content, 16% represents the per-cent of nitrogen element in protein, 5.7 represent the carbon-nitrogen ratio of plant plankton, 29 representatives quality biomass corresponding to unit nitrogen element in plant plankton, the 1000th, milligram biomass is converted into a gram biomass.
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