CN112501237B - Method for predicting utilization capacity of microalgae bicarbonate - Google Patents

Method for predicting utilization capacity of microalgae bicarbonate Download PDF

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CN112501237B
CN112501237B CN202011265613.XA CN202011265613A CN112501237B CN 112501237 B CN112501237 B CN 112501237B CN 202011265613 A CN202011265613 A CN 202011265613A CN 112501237 B CN112501237 B CN 112501237B
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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 concentrations 13 Sodium bicarbonate with greatly different C values is used for simultaneously culturing the microalgae to be detected and measuring the algal body delta 13 C value and algae protein proliferation times, and acquiring the portions of the microalgae cultured by the bicarbonate with different concentrations 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. And substituting the bicarbonate concentration of the culture solution to be detected 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 detected.

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 2 2 Is a linear nonpolar molecule with neutral charge, can freely diffuse into cell bilayer lipid membrane, and enter CO in cell 2 Utilized for photosynthesis of microalgae cells; and (2) utilizing bicarbonate ions in the 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 anhydrase 2 Or directly in the form of bicarbonate ion, actively transported into chloroplast from chloroplast membrane protein, and converted into CO by carbonic anhydrase 2 For 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 CO 2 ,HCO 3 - ,H 2 CO 3 And CO 3 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 sequestration measured or estimated by people was direct carbon sequestration, while indirect carbon sequestration was ignored by people, not to mention quantification.
Carbon elements in nature have two stable isotopes: 12 c and 13 c, their natural average abundances were 98.89% and 1.11%, respectively. The composition of the stable carbon isotope is usually delta 13 C (‰) represents delta in nature 13 The 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 microalgae inorganic carbon.
The rock type of karst region is mainly carbonate, and the main component is CaCO 3 And MgCO 3 And so on. The carbonate of the bedrock releases a large amount of HCO under the karst action 3 - And Ca 2+ 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 bicarbonate concentration of the culture solution to be measured is substituted into the above equation, i.e.The carbonate utilization capacity of the microalgae under any bicarbonate concentration can be predicted, 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 delta 13 Sodium 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 bicarbonate 13 C value of delta C1 Isotopically labelled 2 sodium bicarbonate delta 13 C value of delta C2 ；
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 concentrations 13 Value delta of C T1 、δ T2 (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 microalgae B ；
Fifthly, fitting the microalgae by a Gaussian equationAn equation of protein increase times p and bicarbonate concentration c is obtained to obtain each parameter value in the equation; fitting the equation of the microalgae protein increase multiple p and the bicarbonate concentration c by using a Gaussian equation:
Figure BDA0002774980880000032
wherein the parameter m refers to the peak value of the Gaussian curve, c 0 N 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 equation B Obtaining 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 equation B The equation with bicarbonate concentration c is:
Figure BDA0002774980880000033
wherein the parameter q refers to the peak value of the Gaussian curve, c 0 R 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 the bicarbonate can promote the growth of the microalgae and the utilization of the bicarbonate, and the high concentration of the bicarbonate can inhibit the growth of the microalgae and reduce the utilization portion of the 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:
δ Ti ＝δ Ai -f Bi δ Ai +f Bi δ Bi (i＝1，2，3，------)(1)
where delta is Ti Is delta of microalgae 13 C value, δ Ai Delta of algal body assuming that microalgae completely utilizes inorganic carbon source of air 13 C value, δ Bi Delta of algal body assuming complete utilization of added inorganic carbon source by microalgae 13 C value, f Bi The proportion of the added inorganic carbon source utilized by the microalgae for the investigation.
It is clear that only delta is known Ti It is difficult to find f Bi Thus, the present invention employs δ with a large difference 13 And (3) simultaneously culturing the microalgae by using the sodium bicarbonate with the C value respectively, and identifying the share of the added inorganic carbon source utilized by the microalgae by using the stable carbon isotope double label.
For isotopic label 1 (i = 1), equation (1) is represented by the following formula:
δ T1 ＝δ A1 -f B1 δ A1 +f B1 δ B1 (2)
where delta is T1 To use the first known delta 13 Delta of microalgae cultured with sodium bicarbonate of C value 13 C value, δ A1 Delta of algal body assuming that microalgae completely utilizes inorganic carbon source of air 13 C value, δ B1 Delta of algal body assuming complete utilization of added inorganic carbon source by microalgae 13 C value, f B1 The proportion of the carbon source in the microalgae is determined by using the added inorganic carbon.
For isotopically labeled 2 (i = 2), equation (1) represents the following formula:
δ T2 ＝δ A2 -f B2 δ A2 +f B2 δ B2 (3)
where delta is T2 To use the first known delta 13 Cultured with sodium bicarbonate of C valueDelta of microalgae 13 C value, δ A2 Delta of algal body assuming that microalgae completely utilizes inorganic carbon source of air 13 C value, δ B2 Delta of algal body assuming complete utilization of added inorganic carbon source by microalgae 13 C value, f B2 The proportion of the added inorganic carbon is utilized for the investigation of the microalgae.
(2) And (3) delta in both equations A1 ＝δ A2 ，f B ＝f Bi ＝f B1 ＝f B2 Simultaneous solution
Figure BDA0002774980880000041
(4) In the formula of B1 -δ B2 Then the delta can be converted to the isotopically labelled 1 sodium bicarbonate 13 C value delta C1 Delta. With isotopically labelled 2 sodium bicarbonate 13 C value delta C2 The difference of (a) is then:
Figure BDA0002774980880000042
thus, the delta of isotopically labelled 1 sodium bicarbonate can be determined 13 C value delta C1 Delta. With isotopically labelled 2 sodium bicarbonate 13 C value delta C2 Simultaneous determination of microalgal delta cultured with correspondingly labelled sodium bicarbonate 13 C value, i.e. determining delta T1 And delta T2 And (5) calculating the share of the added inorganic carbon used by the microalgae to be examined.
The net photosynthetic rate of microalgal protein increase by factor 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, c 0 N controls the width of the "clock" for its corresponding abscissa.
Fraction f of bicarbonate utilized by microalgae B The equation with bicarbonate concentration c can be expressed as a gaussian equation. Fitting the proportion f of the bicarbonate utilized by the microalgae by using a Gaussian equation B The equation with bicarbonate concentration c is:
Figure BDA0002774980880000044
Figure BDA0002774980880000051
wherein the parameter q refers to the peak value of the Gaussian curve, c 0 R controls the width of the "bell" for its corresponding abscissa.
The utilization capacity of the bicarbonate of the microalgae is the proportion f of the bicarbonate utilized by the microalgae B And the product of the microalgae protein increase by the factor p. The equation for the bicarbonate utilization capacity BUC as a function of the bicarbonate concentration c is therefore:
Figure BDA0002774980880000052
and substituting the bicarbonate concentration of the culture solution to be detected 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 detected.
The invention has the advantages that:
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 of microalgae and carbon sink effect.
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 the accurate estimation of 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 delta 13 Sodium 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 isotopically labelled 1 sodium bicarbonate 13 C value of delta C1 Isotopically labelled 2 sodium bicarbonate delta 13 C value of delta C2 ；
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 concentrations 13 Value delta of C T1 、δ T2 (ii) a Calculating the proliferation multiple of the processed microalgae protein biomass relative to the inoculated microalgae protein biomass, namely the multiplication multiple 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 microalgae B ；
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 BDA0002774980880000061
wherein the parameter m refers to the peak value of the Gaussian curve, c 0 N controls the width of the "clock" for its corresponding abscissa;
step six, fitting the proportion f of the bicarbonate utilized by the microalgae by a Gaussian equation B Obtaining each parameter value in the equation with the equation of the concentration c of the bicarbonate; fitting the fraction f of the microalgae using bicarbonate by a Gaussian equation B The equation with bicarbonate concentration c is:
Figure BDA0002774980880000062
wherein the parameter q refers to the peak value of the Gaussian curve, c 0 R 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/12h; the temperature is 25 ℃; the illumination intensity is 100 mu mol.m -2 ·s -1 pH 8.0 (adjusted with hydrochloric acid and sodium hydroxide). Sodium bicarbonate was added to the culture solution at a concentration of 0, 0.5, 2.0, 4.0, 8.0 and 16.0mmol/L, and delta of sodium bicarbonate was added 13 C is-28.4 ‰ (PDB) (delta) respectively C1 ) 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 algae 13 C value (Table 2). The method of the invention is used for obtaining the share f of the inorganic carbon source added by the microalgae B (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 constructed B The equation of-c), the parameters of the obtained equation (see table 4), and the relation between the bicarbonate utilization capacity BUC and the bicarbonate concentration cEquation, the bicarbonate concentration of 2.75, 3, 4.45, 5, 8, 8.30, 10, 15.70mmol/L was substituted into the equation of the relationship between the bicarbonate utilization capacity BUC and the bicarbonate concentration c, and the results of predicting 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, 15.70mmol/L were obtained as shown in Table 5.
TABLE 1 fold increase (p) of microalgal protein by bicarbonate treatment at different concentrations
[HCO 3 - ] a [HCO 3 - ] 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
a Sodium bicarbonate concentration to the culture broth
b Bicarbonate ion (bicarbonate) concentration actually measured in the initial stage of the culture solution
p is the multiplication times of the treated microalgae biomass relative to the inoculated microalgae biomass, namely the multiplication times of the algae protein
TABLE 2 carbon isotope composition of microalgae treated with bicarbonate at different concentrations
Figure BDA0002774980880000071
b Measured initial bicarbonate ion (bicarbonate) concentration in the culture
δ T1 Adding delta 13 NaHCO with C of-17.4 ‰ 3 The culture solution of (2) to obtain microalgae delta 13 C value
δ T2 Adding delta 13 NaHCO with C of-28.4% 3 The microalgae delta cultured by the culture solution 13 C value
TABLE 3 proportion of bicarbonate used by microalgae treated with different concentrations of bicarbonate (f) B )
Figure BDA0002774980880000072
Figure BDA0002774980880000081
b Bicarbonate ion (bicarbonate) concentration actually measured in the initial stage of the culture solution
f B Use of bicarbonate fraction for microalgae
TABLE 4 fold increase of protein and bicarbonate concentration (p-c) in Chlamydomonas reinhardtii and Chlorella pyrenoidosa, proportion of bicarbonate and bicarbonate concentration (f) B Equation parameters of-c)
Microalgae species Type of equation Parameter m (q) Parameter n (r) Parameter c 0
Chlamydomonas reinhardtii p-c 4.9884 12.3151 10.5390
Chlamydomonas reinhardtii f B -c 0.3725 4.8032 11.1324
Chlorella pyrenoidosa p-c 4.9919 15.6987 9.2193
Chlorella pyrenoidosa f B -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 optimal 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 share are 11.1324mmol/L and 11.0310mmol/L respectively, which are very close to each other, thereby showing 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 (3)

1. A method for predicting the utilization capacity of bicarbonate in microalgae, comprising:
step one, selecting two kinds of delta 13 Sodium 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 bicarbonate 13 C value of delta C1 Delta. Of isotopically labelled 2 sodium bicarbonate 13 C value of delta C2 ；
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 tested under the investigated culture conditions for 4 days, harvesting the microalgae, and respectively determining the stable carbon isotope composition delta of the microalgae to be tested under the corresponding culture conditions of the culture solution culture by adding two kinds of isotope labeled sodium bicarbonate with different concentrations 13 Value delta of C T1 、δ T2 (ii) a And the fold increase p of algal proteins;
step four, passing through an equation
Figure FDA0003791796940000011
Calculating the portion f of bicarbonate utilization under different concentrations of the bicarbonate in the microalgae B ；
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; wherein the content of the first and second substances,
fitting the equation of the microalgae protein increase multiple p and the bicarbonate concentration c by a Gaussian equation:
Figure FDA0003791796940000012
Figure FDA0003791796940000013
wherein the parameter m refers to the peak value of the Gaussian curve, c 0 N 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 equation B Obtaining the parameter values in the equation with the equation of the bicarbonate concentration c; wherein, the first and the second end of the pipe are connected with each other,
fitting the fraction f of the microalgae using bicarbonate by a Gaussian equation B The equation with bicarbonate concentration c is:
Figure FDA0003791796940000014
wherein the parameter q refers to the peak value of the Gaussian curve, c 0 R controls the width of the "bell" for its corresponding abscissa;
seventhly, further obtaining a relational equation between the bicarbonate utilization capacity BUC and the bicarbonate concentration c:
Figure FDA0003791796940000015
and step eight, substituting the concentration of the bicarbonate of the culture solution to be measured into the relational equation between the utilization capacity of the heptad carbonate and the concentration of the bicarbonate in the step eight, and calculating the utilization capacity of the carbonate of the microalgae under the concentration of the bicarbonate to be measured.
2. The method of claim 1, wherein the method comprises the following steps: 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 time of 0.
3. The method of claim 1, wherein the method comprises the steps of: in step three, the fold increase p of the algal protein is the multiplication fold of the treated microalgae protein biomass relative to the inoculation time.
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