CN112501237A - Method for predicting utilization capacity of microalgae bicarbonate - Google Patents

Method for predicting utilization capacity of microalgae bicarbonate Download PDF

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CN112501237A
CN112501237A CN202011265613.XA CN202011265613A CN112501237A CN 112501237 A CN112501237 A CN 112501237A CN 202011265613 A CN202011265613 A CN 202011265613A CN 112501237 A CN112501237 A CN 112501237A
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吴沿友
李海涛
吴沿胜
张承
苏跃
童成英
孙涛
方蕾
周英
刘丛强
王世杰
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Abstract

The invention discloses a method for predicting the utilization capacity of microalgae bicarbonate, and belongs to the field of coping with climate change and marine bioengineering. Separately adding two kinds of delta of different concentrations13Sodium bicarbonate with greatly different C values is used for simultaneously culturing the microalgae to be detected and measuring the algal body delta13C value and algal protein proliferation multiple, and the proportion of bicarbonate used by microalgae cultured by bicarbonate with different concentrations is obtained by using an isotope mixing model of two end members; and respectively fitting the equation of the microalgae protein increase times and the concentration of the bicarbonate and the equation of the proportion of the microalgae using the bicarbonate and the concentration of the bicarbonate by using a Gaussian equation to obtain various parameter values in the equation, and further obtaining a relation equation of the bicarbonate utilization capacity and the concentration of the bicarbonate. The bicarbonate concentration of the culture solution to be measured is substituted into the relational equation between the bicarbonate utilization capacity and the bicarbonate concentration, and the carbonate utilization capacity of the microalgae under the bicarbonate concentration to be measured can be calculated。

Description

Method for predicting utilization capacity of microalgae bicarbonate
Technical Field
The invention discloses a method for predicting the utilization capacity of microalgae bicarbonate, and belongs to the field of coping with climate change and marine bioengineering. The method not only can predict the bicarbonate utilization capacity of the microalgae under any bicarbonate concentration, but also can find the optimal bicarbonate concentration for the growth of the microalgae. The determination result can be quantified and compared, and scientific support is provided for accurate estimation of the carbon sink of the microalgae in the karst lake.
Background
Microalgae (microalgae) include all the tiny plants that live in a planktonic lifestyle in water, commonly referred to as planktonic algae. Microalgae are simple in structure and relatively simple in physiological process, and some species are model plants for scientific research, such as: chlamydomonas reinhardtii and chlorella can be artificially cultured, which provides convenience for the research of people. The microalgae can utilize inorganic carbon in water in two ways, (1) carbon dioxide in the atmosphere is utilized. CO 22Is a linear nonpolar molecule with neutral charge, can freely diffuse into cell double-layer lipid membrane, and enter CO in cells2Utilized for photosynthesis of microalgae cells; (2) utilizing bicarbonate ions in solution. The bicarbonate ions can be directly transported or indirectly transported into the cells to be utilized by the microalgae cells. The direct transport of bicarbonate ion refers to the direct transport of bicarbonate ion into cell via the plasma membrane surface carrier protein or anion exchange protein, and the intracellular CO is converted into CO via carbonic anhydrase2Or directly in the form of bicarbonate ion, is actively transported into chloroplast from chloroplast membrane proteinConversion of carbonic anhydrase to CO2For the fixation of ribulose-1, 5-bisphosphate carboxylation/oxygenase (Rubisco); indirect transport of bicarbonate ions refers to indirect transport of bicarbonate ions that is dependent on extracellular carbonic anhydrase.
There are 4 forms of inorganic carbon in water, which are respectively CO2,HCO3 -,H2CO3And CO3 2-These four forms exist in the following equilibrium:
Figure BDA0002774980880000011
thus, whether the microalgae utilize carbon dioxide or bicarbonate ions, they may have two sources, one from the inorganic carbon in the air and the other from the bicarbonate ions inherent in the solution. Whether the algae adopts a carbon dioxide utilization path or a bicarbonate ion utilization path, the algae is called direct carbon sink as long as the inorganic carbon of the source air is assimilated, and whether the algae adopts the carbon dioxide utilization path or the bicarbonate ion utilization path, the inorganic carbon inherent in the source water body is assimilated and called indirect carbon sink. The direct carbon sink directly removes carbon dioxide from the atmosphere, while the indirect carbon sink indirectly removes carbon dioxide from the atmosphere by removing inorganic carbon that is indigenous to the body of water to alter the equilibrium of the inorganic carbon in the body of water. Previously, microalgae carbon sink measured or estimated by people is direct carbon sink, while indirect carbon sink is ignored by people, and is not quantitative.
Carbon elements in nature have two stable isotopes:12c and13c, their natural average abundances are 98.89% and 1.11%, respectively. The composition of the stable carbon isotope is usually delta13C (‰) represents delta in nature13The change of C is-90 to +20 per mill. The strong fractionation characteristics of stable carbon isotopes are the basis for identifying the source of microalgal inorganic carbon. The mass balance principle, the isotope mixing model and the chemometric method are the basis for quantitatively identifying the source of the inorganic carbon in the microalgae.
The rock type of karst region is carbonateMainly comprises CaCO3And MgCO3And the like. The carbonate of the bedrock releases a large amount of HCO under the karst action3 -And Ca2+And the water enters a water body, so that the karst lake water body presents a high pH and high bicarbonate environment. The low concentration of bicarbonate can promote the growth of microalgae and the utilization of bicarbonate, and the high concentration of bicarbonate can inhibit the growth of microalgae and reduce the utilization portion of bicarbonate. The invention further couples out a relation equation of the bicarbonate utilization capacity and the bicarbonate concentration by constructing an equation of the microalgae protein increase multiple and the bicarbonate concentration and an equation of the fraction of the microalgae utilizing the bicarbonate and the bicarbonate concentration. The carbonate utilization capacity of the microalgae at any bicarbonate concentration can be predicted by substituting the bicarbonate concentration of the culture solution to be detected into the equation, and scientific support is provided for accurate estimation of the productivity and carbon sink of the microalgae in the karst lake.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for predicting the bicarbonate utilization capacity of microalgae, which not only can predict the bicarbonate utilization capacity of the microalgae under any bicarbonate concentration, but also can search the optimal bicarbonate concentration for the growth of the microalgae. The defect that the prior art cannot predict and evaluate the indirect carbon sequestration capacity of microalgae is overcome.
The invention adopts the following technical scheme: it comprises the following steps:
step one, selecting two kinds of delta13Sodium bicarbonate with the C value difference larger than 8 per mill is used as an isotope label 1 and an isotope label 2, different concentrations are set, and the sodium bicarbonate is respectively added into a culture solution to culture microalgae to be tested; delta of isotope-labeled 1 sodium bicarbonate13C value of deltaC1Delta. of isotopically labelled 2 sodium bicarbonate13C value of deltaC2；
Step two, measuring the biomass of the algae protein and the concentration c of bicarbonate of each culture solution when the culture time is 0 in the initial period of the culture;
step three, culturing the microalgae to be detected under the investigated culture condition for 4 days, harvesting the algae, and respectively determining and adding different concentrationsStable carbon isotope composition delta of the examined microalgae under corresponding culture conditions of culture solution culture of two isotope-labeled sodium bicarbonate13Value delta of CT1、δT2(ii) a Calculating the proliferation times of the processed microalgae protein biomass relative to the inoculated microalgae protein biomass, namely the multiplication times p of the algae protein;
step four, passing through an equation
Figure BDA0002774980880000031
Calculating the portion f of bicarbonate utilization under different bicarbonate concentrations of the microalgaeB；
Fitting an equation of the microalgae protein increase multiple p and the bicarbonate concentration c by a Gaussian equation to obtain various parameter values in the equation; fitting the equation of the microalgae protein increase multiple p and the bicarbonate concentration c by a Gaussian equation:
Figure BDA0002774980880000032
wherein the parameter m refers to the peak value of the Gaussian curve, c0N controls the width of the "clock" for its corresponding abscissa;
step six, fitting the fraction f of the microalgae using the bicarbonate by a Gaussian equationBObtaining the parameter values in the equation with the equation of the bicarbonate concentration c; fitting the fraction f of the microalgae using bicarbonate by a Gaussian equationBThe equation with bicarbonate concentration c is:
Figure BDA0002774980880000033
wherein the parameter q refers to the peak value of the Gaussian curve, c0R controls the width of the "clock" for its corresponding abscissa;
seventhly, further obtaining a relational equation between the bicarbonate utilization capacity BUC and the bicarbonate concentration c, wherein the relational equation between the bicarbonate utilization capacity BUC and the bicarbonate concentration c is as follows:
Figure BDA0002774980880000034
Figure BDA0002774980880000035
and step eight, substituting the concentration of the bicarbonate of the culture solution to be measured into the relational equation between the bicarbonate utilization capacity and the bicarbonate concentration, and calculating the carbonate utilization capacity of the microalgae under the bicarbonate concentration to be measured.
The basic principle of the invention is as follows:
the low concentration of bicarbonate can promote the growth of microalgae and the utilization of bicarbonate, and the high concentration of bicarbonate can inhibit the growth of microalgae and reduce the utilization portion of bicarbonate. The inorganic carbon source used by the microalgae is inorganic carbon in air and inorganic carbon added in the culture solution. Therefore, the proportion of microalgae using inorganic carbon sources from air and using added inorganic carbon sources can be obtained by using an isotope mixing model of two end members.
The isotope mixture model of the two end-members can be expressed as:
δTi＝δAi-fBiδAi+fBiδBi(i＝1，2，3，------)(1)
where delta isTiIs delta of microalgae13C value, δAiDelta of algal body assuming that microalgae completely utilizes inorganic carbon source of air13C value, δBiDelta of algal body assuming complete utilization of added inorganic carbon source by microalgae13C value, fBiThe proportion of the added inorganic carbon source utilized by the microalgae for the investigation.
It is clear that only delta is knownTiIt is difficult to find fBiThus, the present invention employs δ with a large difference13And (3) simultaneously culturing the microalgae by using the sodium bicarbonate with the C value, and identifying the share of the added inorganic carbon source utilized by the microalgae by using the stable carbon isotope double marker.
For isotopic label 1(i ═ 1), equation (1) is expressed as follows:
δT1＝δA1-fB1δA1+fB1δB1 (2)
where delta isT1To use the first known delta13Carbon of C valueDelta of microalgae cultured with sodium hydrogen acid13C value, δA1Delta of algal body assuming that microalgae completely utilizes inorganic carbon source of air13C value, δB1Delta of algal body assuming complete utilization of added inorganic carbon source by microalgae13C value, fB1The proportion of the carbon source in the microalgae is determined by using the added inorganic carbon.
For isotopically labeled 2(i ═ 2), equation (1) is expressed as follows:
δT2＝δA2-fB2δA2+fB2δB2 (3)
where delta isT2To use the first known delta13Delta of microalgae cultured with sodium bicarbonate of C value13C value, δA2Delta of algal body assuming that microalgae completely utilizes inorganic carbon source of air13C value, δB2Delta of algal body assuming complete utilization of added inorganic carbon source by microalgae13C value, fB2The proportion of the added inorganic carbon is utilized for the investigation of the microalgae.
(2) And (3) δ in the two equationsA1＝δA2，fB＝fBi＝fB1＝fB2Simultaneous solution
Figure BDA0002774980880000041
(4) In the formula ofB1-δB2Then the delta can be converted to the isotopically labelled 1 sodium bicarbonate13C value deltaC1Delta. with isotopically labelled 2 sodium bicarbonate13C value deltaC2The difference of (a) is then:
Figure BDA0002774980880000042
thus, the delta of isotopically labelled 1 sodium bicarbonate can be determined13C value deltaC1Delta. with isotopically labelled 2 sodium bicarbonate13C value deltaC2Simultaneous determination using corresponding markersSodium bicarbonate-cultured microalgae delta of13C value, i.e. determining deltaT1And deltaT2And (5) calculating the share of the added inorganic carbon used by the microalgae to be examined.
The net photosynthetic rate of the microalgal protein fold increase p versus bicarbonate concentration c can be expressed in terms of a gaussian equation. Fitting the equation of the microalgae protein increase multiple p and the bicarbonate concentration c by a Gaussian equation:
Figure BDA0002774980880000043
wherein the parameter m refers to the peak value of the Gaussian curve, c0N controls the width of the "clock" for its corresponding abscissa.
Fraction f of bicarbonate utilized by microalgaeBThe equation with bicarbonate concentration c can be expressed as a gaussian equation. Fitting the fraction f of the microalgae using bicarbonate by a Gaussian equationBThe equation with bicarbonate concentration c is:
Figure BDA0002774980880000044
Figure BDA0002774980880000051
wherein the parameter q refers to the peak value of the Gaussian curve, c0R controls the width of the "clock" for its corresponding abscissa.
The utilization capacity of the microalgae bicarbonate is the fraction f of the microalgae using the bicarbonateBThe product of the increase factor p of microalgae protein. The equation for the bicarbonate utilization capacity BUC versus the bicarbonate concentration c is therefore:
Figure BDA0002774980880000052
and substituting the bicarbonate concentration of the culture solution to be measured into the relational equation between the bicarbonate utilization capacity and the bicarbonate concentration, so as to calculate the carbonate utilization capacity of the microalgae under the bicarbonate concentration to be measured.
The invention has the following advantages:
1) the method can quantitatively predict the bicarbonate utilization capacity of the microalgae under any bicarbonate concentration.
2) The method can conveniently and accurately quantify the optimal bicarbonate concentration for the growth of the microalgae.
3) The method can quantitatively research the influence of bicarbonate on the growth and development and carbon sink action of microalgae.
4) The method overcomes the defect that the prior art cannot predict and evaluate the indirect carbon sink capacity of the microalgae, and provides scientific and technological support for accurately estimating the productivity and the carbon sink of the microalgae in the karst lake.
Detailed Description
The embodiment of the invention comprises the following steps: which comprises the following steps of,
step one, selecting two kinds of delta13Sodium bicarbonate with the C value difference larger than 8 per mill is used as an isotope label 1 and an isotope label 2, different concentrations are set, and the sodium bicarbonate is respectively added into a culture solution to culture microalgae to be tested; delta of isotope-labeled 1 sodium bicarbonate13C value of deltaC1Delta. of isotopically labelled 2 sodium bicarbonate13C value of deltaC2；
Step two, measuring the biomass of the algae protein and the concentration c of bicarbonate of each culture solution when the culture time is 0 in the initial period of the culture;
step three, culturing the microalgae to be tested under the investigated culture conditions, harvesting the microalgae after 4 days of culture, and respectively determining the stable carbon isotope composition delta of the microalgae to be tested under each corresponding culture condition of culture solution culture by adding two kinds of isotope-labeled sodium bicarbonate with different concentrations13Value delta of CT1、δT2(ii) a Calculating the proliferation times of the processed microalgae protein biomass relative to the inoculated microalgae protein biomass, namely the multiplication times p of the algae protein;
step four, passing through an equation
Figure BDA0002774980880000053
Calculating the portion f of bicarbonate utilization under different bicarbonate concentrations of the microalgaeB；
Step five, fitting the microalgae protein increase times p and weight by a Gaussian equationThe equation of the carbonate concentration c is used for obtaining various parameter values in the equation; fitting the equation of the microalgae protein increase multiple p and the bicarbonate concentration c by a Gaussian equation:
Figure BDA0002774980880000061
wherein the parameter m refers to the peak value of the Gaussian curve, c0N controls the width of the "clock" for its corresponding abscissa;
step six, fitting the fraction f of the microalgae using the bicarbonate by a Gaussian equationBObtaining the parameter values in the equation with the equation of the bicarbonate concentration c; fitting the fraction f of the microalgae using bicarbonate by a Gaussian equationBThe equation with bicarbonate concentration c is:
Figure BDA0002774980880000062
wherein the parameter q refers to the peak value of the Gaussian curve, c0R controls the width of the "clock" for its corresponding abscissa;
seventhly, further obtaining a relational equation between the bicarbonate utilization capacity BUC and the bicarbonate concentration c, wherein the relational equation between the bicarbonate utilization capacity BUC and the bicarbonate concentration c is as follows:
Figure BDA0002774980880000063
Figure BDA0002774980880000064
and step eight, substituting the concentration of the bicarbonate of the culture solution to be measured into the relational equation between the bicarbonate utilization capacity and the bicarbonate concentration, and calculating the carbonate utilization capacity of the microalgae under the bicarbonate concentration to be measured.
Example (b):
the culture material is as follows: chlamydomonas reinhardtii and Chlorella pyrenoidosa. The basic culture solution adopts an SE culture medium, and the basic culture conditions are as follows: light period L/D: 12h/12 h; the temperature is 25 ℃; the illumination intensity is 100 mu mol.m-2·s-1pH 8.0 (adjusted with hydrochloric acid and sodium hydroxide). Adding 0, 0.5, 2.0, 4.0, 8.0, 16.0mmol/L sodium bicarbonate into the culture solution, adding carbonDelta of sodium hydrogen carbonate13C is-28.4 ‰ (PDB) (delta) respectivelyC1) And-17.4% o (PDB) (. delta.) (delta.)C2). The algal protein biomass and the bicarbonate concentration c of each culture solution were measured at 0 cultivation time in the initial stage of cultivation (Table 1); harvesting the algae after culturing for 4 days, and respectively determining the protein biomass and the delta of the algae13C value (Table 2). The method of the invention is used for obtaining the share f of the inorganic carbon source added by the microalgaeB(Table 3), the protein fold increase and bicarbonate concentration (p-c) of Chlamydomonas reinhardtii and Chlorella pyrenoidosa, and the proportion of bicarbonate and bicarbonate concentration (f) were constructedB-c), obtaining parameters of the equation (as shown in table 4), further obtaining a relation equation of the bicarbonate utilization capacity BUC and the bicarbonate concentration c, and substituting the bicarbonate concentrations of 2.75, 3, 4.45, 5, 8, 8.30, 10 and 15.70mmol/L into the relation equation of the bicarbonate utilization capacity BUC and the bicarbonate concentration c to obtain the prediction results of the Bicarbonate Utilization Capacity (BUC) of Chlamydomonas reinhardtii and Chlorella pyrenoidosa at the bicarbonate concentrations of 2.75, 3, 4.45, 5, 8, 8.30, 10 and 15.70mmol/L as shown in table 5.
TABLE 1 fold increase (p) of microalgal protein by bicarbonate treatment at different concentrations
[HCO3 -]a [HCO3 -]b Chlamydomonas reinhardtii Chlorella pyrenoidosa
mmol/L mmol/L p p
0.00 0.65 3.51±0.27 4.24±0.22
0.50 1.55 4.00±0.30 4.31±0.22
2.00 2.75 4.11±0.18 4.51±0.23
4.00 4.45 4.26±0.20 5.28±0.17
8.00 8.30 4.97±0.23 4.67±0.21
16.00 15.70 4.56±0.29 4.64±0.23
aSodium bicarbonate concentration to the culture broth
bBicarbonate ion (bicarbonate) observed during the initial phase of the cultureSalt) concentration
p is the multiplication factor of the treated microalgae biomass relative to the inoculated microalgae biomass, namely the multiplication factor of the algae protein
TABLE 2 carbon isotope composition of microalgae treated with bicarbonate at different concentrations
Figure BDA0002774980880000071
bThe measured initial bicarbonate ion (bicarbonate) concentration in the culture solution
δT1Adding delta13NaHCO with C of-17.4 ‰3The culture solution of (2) to obtain microalgae delta13C value
δT2Adding delta13NaHCO with C of-28.4 ‰3The culture solution of (2) to obtain microalgae delta13C value
TABLE 3 proportion of bicarbonate utilization by microalgae treated with different concentrations of bicarbonate (f)B)
Figure BDA0002774980880000072
Figure BDA0002774980880000081
bBicarbonate ion (bicarbonate) concentration actually measured in the initial stage of the culture solution
fBUtilization of bicarbonate fraction for microalgae
TABLE 4 Chlamydomonas reinhardtii and Chlorella pyrenoidosa protein fold increase and bicarbonate concentration (p-c), proportion of bicarbonate used and bicarbonate concentration (f)BThe equation parameters of-c)
Microalgae species Type of equation Parameter m (q) Parameter n (r) Parameter c0
Chlamydomonas reinhardtii p-c 4.9884 12.3151 10.5390
Chlamydomonas reinhardtii fB-c 0.3725 4.8032 11.1324
Chlorella pyrenoidosa p-c 4.9919 15.6987 9.2193
Chlorella pyrenoidosa fB-c 0.5095 4.2519 11.0310
TABLE 5 prediction and measurement of Bicarbonate Utilization Capacity (BUC) of Chlamydomonas reinhardtii and Chlorella pyrenoidosa at bicarbonate concentrations of 2.75, 3, 4.45, 5, 8, 8.30, 10, 15.70mmol/L
Figure BDA0002774980880000082
Figure BDA0002774980880000091
The implementation effect of the invention is as follows: as can be seen from Table 4, the optimum bicarbonate concentrations for the growth of Chlamydomonas reinhardtii and Chlorella pyrenoidosa are 10.5390mmol/L and 9.2193mmol/L respectively, and the bicarbonate concentrations with the largest bicarbonate fraction are 11.1324mmol/L and 11.0310mmol/L respectively, which are very close to each other, indicating that the invention has better credibility. As can be seen from Table 5, the ranges of 2.75mmol/L to 15.7mmol/L of bicarbonate have good prediction capability, and the prediction errors are 0.0051 to 0.0823BUC, which shows that the method can well predict the bicarbonate utilization capability of the microalgae in the karst lake and provide scientific support for accurate estimation of the productivity and carbon sink of the microalgae in the karst lake.

Claims (6)

1. A method for predicting microalgae bicarbonate utilization capacity, comprising:
step one, selecting two kinds of delta13Sodium bicarbonate with the C value difference larger than 8 per mill is used as an isotope label 1 and an isotope label 2, different concentrations are set, and the sodium bicarbonate is respectively added into a culture solution to culture microalgae to be tested; delta of isotope-labeled 1 sodium bicarbonate13C value of deltaC1Delta. of isotopically labelled 2 sodium bicarbonate13C value of deltaC2；
Step two, measuring the biomass of the algae protein and the concentration c of bicarbonate of each culture solution in the initial period of culture;
step three, culturing the microalgae to be detected under the investigated culture condition for 4 days, harvesting the algae, respectively measuring and adding two kinds of same substances with different concentrationsStable carbon isotope composition delta of the examined microalgae under corresponding culture conditions of the culture solution culture of the sodium bicarbonate marked by the site element13Value delta of CT1、δT2(ii) a And the fold increase p of algal proteins;
step four, passing through an equation
Figure FDA0002774980870000011
Calculating the portion f of bicarbonate utilization under different bicarbonate concentrations of the microalgaeB；
Fitting an equation of the microalgae protein increase multiple p and the bicarbonate concentration c by a Gaussian equation to obtain various parameter values in the equation;
step six, fitting the fraction f of the microalgae using the bicarbonate by a Gaussian equationBObtaining the parameter values in the equation with the equation of the bicarbonate concentration c;
and seventhly, further obtaining a relational equation between the bicarbonate utilization capacity BUC and the bicarbonate concentration c.
And step eight, substituting the concentration of the bicarbonate of the culture solution to be measured into the relational equation between the bicarbonate utilization capacity and the bicarbonate concentration, and calculating the carbonate utilization capacity of the microalgae under the bicarbonate concentration to be measured.
2. The method of claim 1, wherein the method comprises the steps of: in the second step, the bicarbonate concentration c of each culture solution measured at the initial stage of the culture is the concentration of bicarbonate in the culture solution at the time of the culture of 0.
3. The method of claim 1, wherein the method comprises the steps of: in the third step, the so-called fold increase p of algal protein is the multiplication of the treated microalgae protein biomass relative to the time of inoculation.
4. The method of claim 1, wherein the method is used for predicting the utilization capacity of the microalgae bicarbonateThe method is characterized in that: and step five, fitting an equation of the microalgae protein increase multiple p and the bicarbonate concentration c by using a Gaussian equation, wherein the equation comprises the following steps:
Figure FDA0002774980870000012
wherein the parameter m refers to the peak value of the Gaussian curve, c0N controls the width of the "clock" for its corresponding abscissa.
5. The method of claim 1, wherein the method comprises the steps of: the sixth step of fitting the fraction f of the microalgae using the bicarbonate by using a Gaussian equationBThe equation with bicarbonate concentration c is:
Figure FDA0002774980870000021
wherein the parameter q refers to the peak value of the Gaussian curve, c0R controls the width of the "clock" for its corresponding abscissa.
6. The method of claim 1, wherein the method comprises the steps of: the relation equation of the bicarbonate utilization capacity BUC and the bicarbonate concentration c in the step seven is as follows:
Figure FDA0002774980870000022
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