CN102517349A - Method for improving microalgae cultivation condition by using metabonomics so as to improve oil-producing capacity - Google Patents
Method for improving microalgae cultivation condition by using metabonomics so as to improve oil-producing capacity Download PDFInfo
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Abstract
The invention discloses a method for improving a microalgae cultivation condition by using metabonomics so as to improve the oil-producing capacity. The method comprises the following steps of: (1) detecting intracellular metabolites in microalgae; (2) analyzing the oil-producing capacity of the microalgae; (3) performing partial least squares (PLS) analysis; and (4) process analysis. The oil-producing process of the microalgae, an intracellular metabolism change rule and important metabolites in the oil-producing process can be researched wholly by the method, and the change rule of the metabolism levels lays a base for the internal mechanism of the oil-producing process, so a theoretical base is laid for further optimizing a microalgae cultivation process and improving the grease yield. A new concept and a new method are provided for the research on other oil-producing microorganism cultivation processes.
Description
Invention field
The invention belongs to the biofuel field, relate to a kind of metabolism group of utilizing and improve little algae culture condition to improve the method for oil-producing capacity.
Background technology
Along with the exhausted day by day and people of the fossil energy continuous attention to environmental protection problem, the green novel energy source of Sustainable Development is brought into schedule and has been attracted increasing concern.Bioenergy the earliest derives from food crop, but owing to strive ground with grain, has caused serious crisis in food, is replaced by s-generation bioenergy gradually at present.S-generation bioenergy mainly is through coming from the ethanol that depleted cellulose resource such as stalk is produced, Mierocrystalline cellulose wide material sources and enrich, but a large amount of toxicity inhibitory substance of pre-treatment process generation is a big limiting factor.And compare with gasoline or diesel oil, ethanol has higher corrodibility, renders a service also lower.At last, the alcoholic acid use need with diverse another set of dispensing nfrastructure of existing launch device and engine, suitability is relatively poor, its application has received bigger technical limitation.Thereby from the rich oil biomass, extract fatty acid methyl ester admixture, promptly biofuel is that the development of biofuel provides the prospect that more attracts.
And cetane value is the important evaluation index of the biofuel quality of generally acknowledging in the world, can characterising biological diesel oil pyrophoricity.Krisnangkura K proposed diesel-fuel cetane number and saponification value and iodine number in 1986 has linear dependence, and (Krisnangkura is simple method for estimation of cetane index of vegetable oil methylesters.Journal of the American Oil Chemists ' Society 63 (4) K.1986.A: 552-553.) to provide correlation formula.And big quantity research shows the cetane value of biofuel; The content of saponification value and iodine number and each different fatty acid methyl esters wherein and form closely related (AzamMM; Waris A; Nahar NM.2005.Prospects and potential of fatty acid methyl esters of somenon-traditional seed oils for use as biodiesel in India.Biomass & Bioenergy 29 (4): 293-302.), and set up the corresponding experience formula.The present invention has used for reference this formula the quality of biofuel has been analyzed.
And biofuel is mainly derived from bacterium, fungi and little algae, and especially little algae has and do not occupy cultivated land; Lower to the growth conditions requirement, growth cycle is short, and oleaginousness is higher relatively; Cultivate advantages such as relatively easy, thereby be called as one of the most potential biofuel biomass source.
At present; A large amount of laboratory culture and pilot plant test are used to little algae and produce the diesel oil field; Existing research shows, under situation such as non-autotrophy, nitrogen stress or high-density, helps oil and fat accumulation in little gonidium; But the metabolic mechanism to wherein relevant is but still fuzzy, and this also becomes industry and goes up an extensive critical limitation sexual factor that uses little algae production biofuel.
Summary of the invention
The purpose of this invention is to provide a kind of means and understand the metabolite Changing Pattern in little algae produce oil process through high-throughout metabolism group; Further disclose the metabotic change mechanism relevant, thereby improve little algae culture condition to improve the method for oil-producing capacity for existing technology provides the metabolism group of utilizing of optimizing direction with produce oil.
Technical scheme of the present invention is summarized as follows:
Utilize metabolism group to improve little algae culture condition, comprise the steps: to improve the method for little algae oil-producing capacity
(1) metabolite in little gonidium is measured:
1. cell harvesting and cancellation:
Little algae is cultivated, and at least one time point in index mid-term, later stage and three times of stationary phase of culturing process takes out algae liquid sample 100-150mL, and each time point takes out 3-5 part; 4 ℃ of following 3000-5000rpm; Centrifugal 3-4min collects the cell of lower floor, with 3-5mL metabolism stop buffer at-40 ℃ of following cancellation 5-10 minutes; Stop metabolic reaction, obtain FD at-80 ℃ of following lyophilize 4-6h;
Said metabolism stop buffer is for containing 1500mgL
-1NaNO
3, 36mgL
-1CaCl
22H
2O, 75mgL
-1MgSO
47H
2O and 40mgL
-1K
2HPO
43H
2The methanol aqueous solution of O, the volume ratio of first alcohol and water is 1: 2 in the said methanol aqueous solution;
2. extract the endocellular metabolism thing:
Get the FD 15-25mg that 1. step obtain and place centrifuge tube respectively, every pipe adds 1-2mL-40 ℃ metabolism extracting solution, mixing; Lid is tight and put into liquid nitrogen, and multigelation 3-5 time is at-20 ℃ of centrifugal 1-2min of following 4000-6000rpm; Collect supernatant, in residue, add 0.5-1mL metabolism extracting solution ,-20 ℃ of centrifugal 1-2min of following 4000-6000rpm in addition; After centrifugal, twice supernatant mixes, and adds the deuterium-labeled succsinic acid of 10-20 μ g; Get mixed solution, get 200 μ L mixed solutions, at-80 ℃ of following lyophilize 2-4 hours;
Said metabolism extracting solution is that volumn concentration is 50% methanol aqueous solution;
3. metabolite derivatize
In the sample that 2. step obtains, add 20mgmL
-1Methoxyl group ammonium salt hydrochlorate/pyridine solution 50 μ L; In 40 ℃ of water-baths, react 60-80min; Reaction finishes the back and adds the trimethyl silicon based trifluoroacetamide of 80 μ L N-methyl-N-, reacts 60-80min again in 40 ℃ of water-baths, the centrifugal 1min of 8000-12000rpm; Get supernatant 100 μ L and put into numbered GC sample introduction bottle, room temperature is placed 2h;
4. GC-MS detects
Adopt GC-MS that the sample that 3. step obtains is carried out qualitative and quantitative Treatment, condition is following:
Chromatographic column: DB-5 gas chromatographic column, its specification are 30m*0.25mm, 0.25 μ m;
Sample size: 1 μ L;
Splitting ratio: 10: 1;
Injector temperature: 280 ℃;
GC interface temperature: 270 ℃;
Helium flow velocity: constant voltage, 91KPa;
Heating schedule: 70 ℃ keep 2min, with 5 ℃ of min
-1Speed be raised to 290 ℃, and keep 6min at 290 ℃;
Ionization voltage: 70eV;
Source temperature: 250 ℃;
Sweep limit: 50-800m/z;
Sweep velocity: 2scans
-1
Thereby the qualitative and quantitative data of the metabolite that has obtained little algae;
(2) oil-producing capacity of little algae is analyzed:
1. cell harvesting and cancellation:
Little algae is cultivated, and at least one time point in index mid-term, later stage and three times of stationary phase of culturing process takes out algae liquid sample 100-150mL, and each time point takes out 3-5 part; 4 ℃ of following 3000-5000rpm; Centrifugal 3-4min collects the cell of lower floor, with 3-5mL metabolism stop buffer at-40 ℃ of following cancellation 5-10 minutes; Stop metabolic reaction, obtain FD at-80 ℃ of following lyophilize 4-6h;
Said metabolism stop buffer is for containing 1500mgL
-1NaNO
3, 36mgL
-1CaCl
22H
2O, 75mgL
-1MgSO
47H
2O and 40mgL
-1K
2HPO
43H
2The methanol aqueous solution of O, the volume ratio of first alcohol and water is 1: 2 in the said methanol aqueous solution;
2. extract lipid acid in the cell:
Get the FD 15-25mg that 1. step obtain and place centrifuge tube respectively, every pipe adds 0.75-1.5mL chloroform and 0.3-0.6mL ultrapure water, and 100rpm shakes 1h; Add 2-4mL fat extraction liquid again, with 100rpm concussion 0.5h, collect the chloroform phase under the room temperature; In residue, add 2-4mL fat extraction liquid, with 100rpm concussion 0.5h, collect the chloroform phase under the room temperature; Extract repeatedly 3 times, chloroform is merged mutually, add the 0.5-1mL 1M KCl aqueous solution, concussion, 3000-4000rpm; Centrifugal 3-5min abandons water, adds the 1-2mL ultrapure water again, concussion; 3000-4000rpm, centrifugal 3-5min abandons water, and 30-35 ℃ of vacuum-drying gets dry-matter;
Said fat extraction liquid is that volumn concentration is 66.7% chloroform methanol solution, and it is 0.1% butylated hydroxytoluene that said chloroform methanol solution contains mass percent;
3. methyl esterification of fatty acid:
It is in 14% the boron trifluoride-methanol solution that said dry-matter is dissolved in 600-1000 μ L mass percent; Add 10-20 μ g margaric acid as interior mark, place in the 15mL ST, at 100 ℃ of water-bath 20-30min; Temperature is reduced to room temperature; Add 500-1000 μ l normal hexane concussion extraction 30s, the centrifugal 2min of 4000-5000rpm obtains the normal hexane phase; Get 100-200 μ L normal hexane and put into numbered GC sample introduction bottle mutually;
4. GC-MS detects
Adopt GC-MS that the fatty acid methyl ester of normal hexane in mutually carried out qualitative and quantitative Treatment, condition is following:
Chromatographic column: DB-5 gas chromatographic column, its specification are 30m*0.25mm, 0.25 μ m;
Sample size: 1 μ L;
Splitting ratio: 10: 1;
Injector temperature: 280 ℃;
GC interface temperature: 270 ℃;
Helium flow velocity: constant voltage, 91KPa;
Heating schedule: 70 ℃ keep 2min, with 8 ℃ of min
-1Speed be raised to 290 ℃, and keep 6min at 290 ℃;
Ionization voltage: 70eV;
Source temperature: 250 ℃;
Sweep limit: 50-800m/z;
Sweep velocity: 2scans
-1
Thereby obtained the content of the single fatty acid methyl ester in the fatty acid methyl ester admixture;
5. day output
Calculate the day output of little algae fatty acid methyl ester admixture according to following formula:
P＝∑Wi/Day/CV
Wherein P is little algae fatty acid methyl ester day output, Wi be step 4. in the weight of each fatty acid methyl ester in the gained sample, to be that step is 1. said be inoculated into the time span of microalgae harvesting from little algae to Day, the volume of micro algae culturing liquid when CV is the 1. said results of step;
6. cetane value is confirmed
Calculate the cetane value of little algae fatty acid methyl ester admixture according to following formula:
SN＝∑(560×Pi)/MWi a
IN∑(254×D×Pi)/MWi b
CN＝46.3+5458/SN-0.225×IN c
Wherein SN is a saponification value; IN is an iodine number; CN is a cetane value; Pi is the dry weight percentage of each fatty acid methyl ester in the 4. middle gained sample of step, and MWi is the molecular weight of each fatty acid methyl ester in the 4. middle gained sample of step, and D is the double key number amount of each fatty acid methyl ester in the 4. middle gained sample of step.
(3) PLS analyzes
Little algae metabolite content that step (1) is obtained is an independent variable(s), and little algae fatty acid methyl ester day output and cetane value that step (2) is obtained are dependent variable, uses PLS to analyze, and obtains the metabolic markers relevant with produce oil;
(4) process analysis
The content of the metabolite mark that step (3) is obtained is processed chart according to culture condition difference; Observe and analyze the rule that these metabolites change; And then find metabolite and the associated metabolic network in little algae produce oil process, play a crucial role, thereby for being that little algae culture condition of purpose provides the optimization direction to improve little algae fatty acid methyl ester output and quality.
Described little algae relates to but is not limited to chlorella (Chlorella sorokiniana), grid algae (Scenedesmus obliquus), cytoalgae (Synechocystis sp.PCC6803) and anabena (Anabaena sp.PCC7120).
The invention provides a kind of means of analyzing little algae produce oil mechanism; This means relate to metabolism group method associating multivariate statistics; Through oil-producing capacity and the metaboilic level of little algae under different growing environments analyzed; Find the metabolite relevant with produce oil, thus be culture process the optimization provider to.
Utilize method of the present invention can study on the whole in little algae produce oil process; The endocellular metabolism Changing Pattern; Find the important metabolite in the produce oil process; The Changing Pattern of these metaboilic levels provides foundation for the inherent mechanism of understanding the produce oil process, thereby is further to optimize little algae culture process, improves grease yield and provides fundamental basis.Also new thinking and method is provided simultaneously for the research of other oleaginous microorganism culture process processes.
Description of drawings
Fig. 1 is the oil-contg variation of following stationary phase of chlorella different vaccination density;
Fig. 2 is the chlorella different vaccination density fatty acid methyl ester quantitative changeization of following stationary phase;
Fig. 3 is the cetane value changing conditions of the gained fatty acid methyl ester of chlorella different vaccination density following stationary phase;
Fig. 4 is the chlorella different vaccination density metabolism collection of illustrative plates of following stationary phase;
Fig. 5 is that the oil-producing capacity of chlorella different vaccination density following stationary phase and PLS that corresponding metaboilic level changes analyze shot chart;
Fig. 6 is that the oil-producing capacity of chlorella different vaccination density following stationary phase and PLS that corresponding metaboilic level changes analyze VIP figure;
Fig. 7 is that crucial metabolite relevant with produce oil in the chlorella is in the different vaccination density variation tendency of following stationary phase.
Embodiment
Following embodiment can make those skilled in the art more comprehensively understand the present invention, but does not limit the present invention in any way.Below in conjunction with specific embodiment the present invention is described further:
A kind of metabolism group of utilizing is improved little algae culture condition to improve the method for little algae oil-producing capacity, comprises the steps:
(1) metabolite in chlorella (Chlorella sorokiniana) born of the same parents is measured:
1. chlorella (Chlorella sorokiniana) cell harvesting and cancellation:
Said little algae is carried out the cultivation of different vaccination density, and initial inoculation density is set to 1 * 10 respectively
4, 1 * 10
5, 1 * 10
6And 1 * 10
7CellsmL
-1, to stationary phase, each inoculum density takes out 5 parts of algae liquid samples, every part of 100mL respectively in cell cultures; 4 ℃ of following 3000rpm, centrifugal 3min collects the cell of lower floor;-40 ℃ of following cancellation 5 minutes, stop metabolic reaction with 4mL metabolism stop buffer, obtain FD at-80 ℃ of following lyophilize 4h;
Said metabolism stop buffer is for containing 1500mgL
-1NaNO
3, 36mgL
-1CaCl
22H
2O, 75mgL
-1MgSO
47H
2O and 40mgL
-1K
2HPO
43H
2The methanol aqueous solution of O, the volume ratio of first alcohol and water is 1: 2 in the said methanol aqueous solution;
2. extract the endocellular metabolism thing:
Get each 5 parts of the FDs of each inoculum density that 1. step obtain, every part of 15mg places centrifuge tube respectively, and every pipe adds 1mL-40 ℃ metabolism extracting solution, mixing; Lid is tight and put into liquid nitrogen, and multigelation 3 times is at-20 ℃ of centrifugal 1min of following 4000rpm; Collect supernatant, in residue, add 0.5mL metabolism extracting solution ,-20 ℃ of centrifugal 1min of following 4000rpm in addition; After centrifugal, twice supernatant mixes, and adds the deuterium-labeled succsinic acid of 10 μ g; Get mixed solution, get 200 μ L mixed solutions ,-80 ℃ of following lyophilizes 2 hours;
Said metabolism extracting solution is that volumn concentration is 50% methanol aqueous solution;
3. metabolite derivatize
In the sample that 2. step obtains, add 20mgmL
-1Methoxyl group ammonium salt hydrochlorate/pyridine solution 50 μ L; In 40 ℃ of water-baths, react 60min; Reaction finishes the back and adds 80 μ L N-methyl N-trimethyl silicon based trifluoroacetamides, reacts 60min again in 40 ℃ of water-baths, the centrifugal 1min of 8000rpm; Get supernatant 100 μ L and put into numbered GC sample introduction bottle, room temperature is placed 2h;
4. GC-MS detects
Adopt GC-MS that the sample that 3. step obtains is carried out qualitative and quantitative Treatment, condition is following:
Chromatographic column: DB-5 gas chromatographic column, its specification are 30m*0.25mm, 0.25 μ m;
Sample size: 1 μ L;
Splitting ratio: 10: 1;
Injector temperature: 280 ℃;
GC interface temperature: 270 ℃;
Helium flow velocity: constant voltage, 91KPa;
Heating schedule: 70 ℃ keep 2min, with 5 ℃ of min
-1Speed be raised to 290 ℃, and keep 6min at 290 ℃;
Ionization voltage: 70eV;
Source temperature: 250 ℃;
Sweep limit: 50-800m/z;
Sweep velocity: 2scans
-1
Thereby the qualitative and quantitative data of the metabolite that has obtained little algae;
(2) oil-producing capacity of little algae is analyzed:
1. cell harvesting and cancellation:
1. cell harvesting and the cancellation metabolite in chlorella (Chlorella sorokiniana) born of the same parents measured with the step (1) of present embodiment;
2. extract lipid acid in the cell:
Get each 5 parts of the FDs of the different vaccination density that 1. step obtain, every part of 15mg places centrifuge tube respectively, and every pipe adds 0.75mL chloroform and 0.3mL ultrapure water, and 100rpm shakes 1h; Add 2mL fat extraction liquid again, with 100rpm concussion 0.5h, collect the chloroform phase under the room temperature; In residue, add 2mL fat extraction liquid, with 100rpm concussion 0.5h, collect the chloroform phase under the room temperature; Extract repeatedly 3 times, chloroform is merged mutually, add the 0.5mL 1M KCl aqueous solution, concussion, 3000rpm, centrifugal 3min abandons water, adds the 1mL ultrapure water again, concussion, 3000rpm, centrifugal 3min abandons water, and 30 ℃ of vacuum-dryings get dry-matter;
Said fat extraction liquid is that volumn concentration is 66.7% chloroform methanol solution, and it is 0.1% butylated hydroxytoluene that said chloroform methanol solution contains mass percent;
3. methyl esterification of fatty acid:
It is in 14% the boron trifluoride-methanol solution that said dry-matter is dissolved in 600 μ L mass percents; Add 10 μ g margaric acids as interior mark, place in the 15mL ST, at 100 ℃ of water-bath 20min; Temperature is reduced to room temperature; Add 600 μ l normal hexanes concussion extraction 30s, the centrifugal 2min of 4000rpm obtains the normal hexane phase; Get 100 μ L normal hexanes and put into numbered GC sample introduction bottle mutually;
4. GC-MS detects
Adopt GC-MS that the fatty acid methyl ester of normal hexane in mutually carried out qualitative and quantitative Treatment, condition is following:
Chromatographic column: DB-5 gas chromatographic column, its specification are 30m*0.25mm, 0.25 μ m;
Sample size: 1 μ L;
Splitting ratio: 10: 1;
Injector temperature: 280 ℃;
GC interface temperature: 270 ℃;
Helium flow velocity: constant voltage, 91KPa;
Heating schedule: 70 ℃ keep 2min, with 8 ℃ of min
-1Speed be raised to 290 ℃, and keep 6min at 290 ℃;
Ionization voltage: 70eV;
Source temperature: 250 ℃;
Sweep limit: 50-800m/z;
Sweep velocity: 2scans
-1
Thereby obtained the content of the single fatty acid methyl ester in the fatty acid methyl ester admixture;
5. day output
Calculate the day output of little algae fatty acid methyl ester admixture according to following formula:
P＝∑Wi/Day/CV
Wherein P is little algae fatty acid methyl ester day output, Wi be step 4. in the weight of each fatty acid methyl ester in the gained sample, to be that step is 1. said be inoculated into the time span of microalgae harvesting from little algae to Day, the volume of micro algae culturing liquid when CV is the 1. said results of step;
6. cetane value is confirmed
Calculate the cetane value of little algae fatty acid methyl ester admixture according to following formula:
SN＝∑(560×Pi)/MWi a
IN∑(254×D×Pi)/MWi b
CN＝46.3+5458/SN-0.225×IN c
Wherein SN is a saponification value; IN is an iodine number; CN is a cetane value; Pi is the dry weight percentage of each fatty acid methyl ester in the 4. middle gained sample of step, and MWi is the molecular weight of each fatty acid methyl ester in the 4. middle gained sample of step, and D is the double key number amount of each fatty acid methyl ester in the 4. middle gained sample of step.
(3) PLS analyzes
The chlorella metabolite content that step (1) is obtained is an independent variable(s), and chlorella fatty acid methyl ester day output and cetane value that step (2) is obtained are dependent variable, uses PLS to analyze, and obtains the metabolic markers relevant with produce oil;
(4) process analysis
The content of the metabolite mark that step (3) is obtained is processed chart according to culture condition difference; Observe and analyze the rule that these metabolites change; And then find metabolite and the associated metabolic network in chlorella produce oil process, play a crucial role, thereby for being that little algae culture condition of purpose provides the optimization direction to improve chlorella fatty acid methyl ester output and quality.
As shown in Figure 1, different vaccination density is little to the oil-contg influence of chlorella stationary phase, but Fig. 2 shows, along with the increase of inoculum density, the oil yield of chlorella stationary phase constantly increases, and especially inoculum density is 10
7CellsmL
-1The time, the fatty acid methyl ester day output in the chlorella is than 10
4CellsmL
-1Shi Tigao 64.5%.Can find out that by Fig. 3 working as inoculum density reaches 10
5CellsmL
-1After, the cetane value of chlorella gained biofuel is along with the increase of inoculum density improves constantly, when inoculum density reaches 10
7CellsmL
-1After, its cetane value is than having improved 8.6% at least under other inoculum density culture condition.This shows that inoculum density has remarkable influence to the chlorella oil-producing capacity, therefore according to according to the invention, the associating metaboilic level is analyzed oil-producing capacity.Fig. 4 has provided the metabolism collection of illustrative plates of associated sample, detects 88 kinds of metabolites altogether, and the result is shown in table 1.1.
Table 1.1 chlorella intracellular metabolite thing
Metaboilic level with chlorella under the different vaccination density is the independent variable(s) matrix, is the Y matrix with the chlorella oil-producing capacity (fatty acid methyl ester day output and cetane value) of same sample, data is carried out PLS analyze; The R2X (cum) that obtains model is 0.765; R2Y (cum) is 0.786, and Q2 (cum) is 0.692, explains that model is credible; Decryption well, and higher predictive ability is arranged.Can find out that by Fig. 5 inoculum density is 10
7CellsmL
-1Chlorella and other samples obviously separate, and and t1, u1 positive correlation respectively.And Fig. 6 has provided the VIP figure of this PLS analytical model, can find the potential metabolite relevant with oil-producing capacity through VIP figure.Can find out that by figure one to have 15 metabolites are possible biomarkers (VIP＞1), these metabolites are analyzed.
As shown in Figure 7, these can be divided into three major types with the closely-related metabolite of oil-producing capacity in chlorella: relevant sucrose and the trisaccharide maltose with starch metabolism; The palmitinic acid relevant, hexadecadienoic acid, hiragonic acid, linolenic acid, Triple Pressed Stearic Acid, octadecadienoic acid, 1-glyceryl linoleate, 1-palmitin with metabolism of fat; Glycerine in other metabolism, phosphoric acid and lactic acid.Because the biological meaning of unknown material 14 and hexadecadienoic acid methyl esters is indeterminate, will not discuss at this place.Can find out that by Fig. 7 inoculum density is 10
7CellsmL
-1The metabolic activity of chlorella be starkly lower than other inoculum density systems.All to follow the nuclear carbon metabolism closely related with Kelvin for these pathways metabolisms, is 10 although can infer inoculum density thus
7CellsmL
-1The oil-producing capacity of chlorella be higher than other culture systems, but its environment of living in is not an optimum, therefore also has room for promotion.The then explanation that descends of these metabolic pathway flux relevant with the carbon metabolism improves light intensity and has that to help improve inoculum density be 10
7CellsmL
-1The oil-producing capacity of chlorella.
In sum; Inoculum density has material impact to the chlorella oil-producing capacity, changes and the oil-producing capacity analysis in conjunction with associated metabolic, though explain that high inoculum density helps oil-producing capacity and improves; But the cellular metabolism vigor descends; And relevant with photosynthesis through analyze finding, the change light intensity may be improved the metabolic capacity of the chlorella under the high inoculum density, thereby regulatable site is provided for the raising of its oil-producing capacity.
A kind of metabolism group of utilizing is improved little algae culture condition to improve the method for little algae oil-producing capacity, comprises the steps:
(1) metabolite in chlorella (Chlorella sorokiniana) born of the same parents is measured:
1. chlorella (Chlorella sorokiniana) cell harvesting and cancellation:
Said little algae is carried out the cultivation of different vaccination density, and initial inoculation density is set to 1 * 10 respectively
4, 1 * 10
5, 1 * 10
6And 1 * 10
7CellsmL
-1, in cell cultures to index later stage and stationary phase, each time point takes out 4 parts of algae liquid samples respectively in each inoculum density; Every part of 120mL, 4 ℃ of following 5000rpm, centrifugal 3min; Collect the cell of lower floor;-40 ℃ of following cancellation 7 minutes, stop metabolic reaction with 3mL metabolism stop buffer, obtain FD at-80 ℃ of following lyophilize 6h;
The metabolism stop buffer is with embodiment 1;
2. extract the endocellular metabolism thing:
Get each 4 parts of the FDs that different vaccination density that 1. step obtain cultivates different sample times, every part of 25mg places centrifuge tube respectively, and every pipe adds 2mL-40 ℃ metabolism extracting solution, mixing; Lid is tight and put into liquid nitrogen, and multigelation 5 times is at-20 ℃ of centrifugal 1min of following 6000rpm; Collect supernatant, in residue, add 1mL metabolism extracting solution ,-20 ℃ of centrifugal 1min of following 6000rpm in addition; After centrifugal, twice supernatant mixes, and adds the deuterium-labeled succsinic acid of 20 μ g; Get mixed solution, get 200 μ L mixed solutions ,-80 ℃ of following lyophilizes 4 hours;
The metabolism extracting solution is with embodiment 1;
3. metabolite derivatize
In the sample that 2. step obtains, add 20mgmL
-1Methoxyl group ammonium salt hydrochlorate/pyridine solution 50 μ L; In 40 ℃ of water-baths, react 80min; Reaction finishes the back and adds the trimethyl silicon based trifluoroacetamide of 80 μ L N-methyl-N-, reacts 80min again in 40 ℃ of water-baths, the centrifugal 1min of 12000rpm; Get supernatant 100 μ L and put into numbered GC sample introduction bottle, room temperature is placed 2h;
4. GC-MS detects
Adopt GC-MS that sample is carried out qualitative and quantitative Treatment, condition is with embodiment 1;
(2) oil-producing capacity of little algae is analyzed:
1. cell harvesting and cancellation:
1. cell harvesting and the cancellation metabolite in chlorella (Chlorella sorokiniana) born of the same parents measured with the step (1) of present embodiment;
2. extract lipid acid in the cell:
Get each 4 parts of the FDs that different vaccination density that 1. step obtain cultivates different sample times, every part of 25mg places centrifuge tube respectively, and every pipe adds 1.5mL chloroform and 0.6mL ultrapure water, and 100rpm shakes 1h; Add 4mL fat extraction liquid again, with 100rpm concussion 0.5h, collect the chloroform phase under the room temperature; In residue, add 4mL fat extraction liquid, with 100rpm concussion 0.5h, collect the chloroform phase under the room temperature; Extract repeatedly 3 times, chloroform is merged mutually, add the 1mL 1M KCl aqueous solution, concussion, 4000rpm, centrifugal 5min abandons water, adds the 2mL ultrapure water again, concussion, 4000rpm, centrifugal 5min abandons water, and 35 ℃ of vacuum-dryings get dry-matter;
Fat extraction liquid is with embodiment 1;
3. methyl esterification of fatty acid:
It is in 14% the boron trifluoride-methanol solution that said dry-matter is dissolved in 1000 μ L mass percents; Add 20 μ g margaric acids as interior mark, place in the 15mL ST, at 100 ℃ of water-bath 30min; Temperature is reduced to room temperature; Add 1000 μ l normal hexanes concussion extraction 30s, the centrifugal 2min of 5000rpm obtains the normal hexane phase; Get 150 μ L normal hexanes and put into numbered GC sample introduction bottle mutually;
4. GC-MS detects
Adopt GC-MS that the fatty acid methyl ester of normal hexane in mutually carried out qualitative and quantitative Treatment, condition is with embodiment 1, thereby obtained the content of single fatty acid methyl ester in little algae fatty acid methyl ester admixture;
5. day output
Calculate the day output of little algae fatty acid methyl ester admixture according to following formula:
P＝∑Wi/Day/CV
Wherein P is little algae fatty acid methyl ester day output, Wi be step 4. in the weight of each fatty acid methyl ester in the gained sample, to be that step is 1. said be inoculated into the time span of microalgae harvesting from little algae to Day, the volume of micro algae culturing liquid when CV is the 1. said results of step;
6. cetane value is confirmed
Calculate the cetane value of little algae fatty acid methyl ester admixture according to following formula:
SN＝∑(560×Pi)/MWi a
IN∑(254×D×Pi)/MWi b
CN＝46.3+5458/SN-0.225×IN c
Wherein SN is a saponification value; IN is an iodine number; CN is a cetane value; Pi is the dry weight percentage of each fatty acid methyl ester in the 4. middle gained sample of step, and MWi is the molecular weight of each fatty acid methyl ester in the 4. middle gained sample of step, and D is the double key number amount of each fatty acid methyl ester in the 4. middle gained sample of step.
(3) PLS analyzes
Little algae metabolite content that step (1) is obtained is an independent variable(s), and little algae fatty acid methyl ester day output and cetane value that step (2) is obtained are dependent variable, uses PLS to analyze, and obtains the metabolic markers relevant with produce oil;
(4) process analysis
The content of the metabolite mark that step (3) is obtained is processed chart according to culture condition difference; Observe and analyze the rule that these metabolites change; And then find metabolite and the associated metabolic network in little algae produce oil process, play a crucial role, thereby for being that little algae culture condition of purpose provides the optimization direction to improve little algae fatty acid methyl ester output and quality.
Similar through experiment showed, the result with embodiment 1.
A kind of metabolism group of utilizing is improved little algae culture condition to improve the method for little algae oil-producing capacity, comprises the steps:
(1) metabolite in chlorella (Chlorella sorokiniana) born of the same parents is measured:
1. chlorella (Chlorella sorokiniana) cell harvesting and cancellation:
Said little algae is carried out the cultivation of different vaccination density, and initial inoculation density is set to 1 * 10 respectively
4, 1 * 10
5, 1 * 10
6And 1 * 10
7CellsmL
-1, in cell cultures to index mid-term, index latter stage and stationary phase; Each time point under each inoculum density is cultivated respectively takes out 3 parts of algae liquid samples, every part of 150mL, 4 ℃ of following 3000rpm; Centrifugal 4min collects the cell of lower floor, with 5mL metabolism stop buffer-40 ℃ of following cancellation 10 minutes; Stop metabolic reaction, obtain FD at-80 ℃ of following lyophilize 6h;
The metabolism stop buffer is with embodiment 1;
2. extract the endocellular metabolism thing:
Get each 3 parts of the FDs of the different vaccination density different time points that 1. step obtain, every part of 20mg places centrifuge tube respectively, and every pipe adds 2mL-40 ℃ metabolism extracting solution, mixing; Lid is tight and put into liquid nitrogen, and multigelation 5 times is at-20 ℃ of centrifugal 2min of following 6000rpm; Collect supernatant, in residue, add 1mL metabolism extracting solution ,-20 ℃ of centrifugal 2min of following 6000rpm in addition; After centrifugal, twice supernatant mixes, and adds the deuterium-labeled succsinic acid of 20 μ g; Get mixed solution, get 200 μ L mixed solutions ,-80 ℃ of following lyophilizes 4 hours;
The metabolism extracting solution is with embodiment 1;
3. metabolite derivatize
In the sample that 2. step obtains, add 20mgmL
-1Methoxyl group ammonium salt hydrochlorate/pyridine solution 50 μ L; In 40 ℃ of water-baths, react 70min; Reaction finishes the back and adds the trimethyl silicon based trifluoroacetamide of 80 μ L N-methyl-N-, reacts 60min again in 40 ℃ of water-baths, the centrifugal 1min of 12000rpm; Get supernatant 100 μ L and put into numbered GC sample introduction bottle, room temperature is placed 2h;
4. GC-MS detects
Adopt GC-MS that sample is carried out the qualitative and quantitative Treatment of metabolite, condition is with embodiment 1:
(2) oil-producing capacity of little algae is analyzed:
1. cell harvesting and the cancellation metabolite in chlorella (Chlorella sorokiniana) born of the same parents measured with the step (1) of present embodiment;
2. extract lipid acid in the cell:
Get each 3 parts of the FDs of the different vaccination density different time points that 1. step obtain, every part of 20mg places centrifuge tube respectively, and every pipe adds 1mL chloroform and 0.4mL ultrapure water, and 100rpm shakes 1h; Add 3mL fat extraction liquid again, with 100rpm concussion 0.5h, collect the chloroform phase under the room temperature; In residue, add 3mL fat extraction liquid, with 100rpm concussion 0.5h, collect the chloroform phase under the room temperature; Extract repeatedly 3 times, chloroform is merged mutually, add the 0.75mL 1M KCl aqueous solution, concussion, 3000rpm, centrifugal 3min abandons water, adds the 1.5mL ultrapure water again, concussion, 3000rpm, centrifugal 3min abandons water, and 35 ℃ of vacuum-dryings get dry-matter;
Fat extraction liquid is with embodiment 1;
3. methyl esterification of fatty acid:
It is in 14% the boron trifluoride-methanol solution that said dry-matter is dissolved in 600 μ L mass percents; Add 20 μ g margaric acids as interior mark, place in the 15mL ST, at 100 ℃ of water-bath 20min; Temperature is reduced to room temperature; Add 600 μ l normal hexanes concussion extraction 30s, the centrifugal 2min of 4000rpm obtains the normal hexane phase; Get 200 μ L normal hexanes and put into numbered GC sample introduction bottle mutually;
4. GC-MS detects
Adopt GC-MS that the fatty acid methyl ester of normal hexane in mutually carried out qualitative and quantitative Treatment, condition is with embodiment 1, thereby obtained the content of the single fatty acid methyl ester in the fatty acid methyl ester admixture;
5. day output
Calculate the day output of little algae fatty acid methyl ester admixture according to following formula:
P＝∑Wi/Day/CV
Wherein P is little algae fatty acid methyl ester day output, and Wi is the weight of each fatty acid methyl ester in the sample, and Day is the time span that is inoculated into results from little algae, the volume number of micro algae culturing liquid when CV is results.
6. cetane value is confirmed
Calculate the cetane value of little algae fatty acid methyl ester admixture according to following formula:
SN＝∑(560×Pi)/MWi a
IN∑(254×D×Pi)/MWi b
CN＝46.3+5458/SN-0.225×IN c
Wherein SN is a saponification value; IN is an iodine number; CN is a cetane value; Pi is the dry weight percentage of each fatty acid methyl ester in the 4. middle gained sample of step, and MWi is the molecular weight of each fatty acid methyl ester in the 4. middle gained sample of step, and D is the double key number amount of each fatty acid methyl ester in the 4. middle gained sample of step.
(3) PLS analyzes
Little algae metabolite content that step (1) is obtained is an independent variable(s), and little algae fatty acid methyl ester day output and cetane value that step (2) is obtained are dependent variable, uses PLS to analyze, and obtains the metabolic markers relevant with produce oil;
(4) process analysis
The content of the metabolite mark that step (3) is obtained is processed chart according to culture condition difference; Observe and analyze the rule that these metabolites change; And then find metabolite and the associated metabolic network in little algae produce oil process, play a crucial role, thereby for being that little algae culture condition of purpose provides the optimization direction to improve little algae fatty acid methyl ester output and quality.
Similar through experiment showed, the result with embodiment 1.
The chlorella that the present invention adopted (Chlorella sorokiniana) only is used to explain the present invention; But and be not used in qualification the present invention; Experiment showed, that grid algae (Scenedesmus obliquus), cytoalgae (Synechocystis sp.PCC6803) and anabena (Anabaena sp.PCC7120) also can be used for the present invention.
Claims (2)
1. utilize metabolism group to improve little algae culture condition, it is characterized in that comprising the steps: to improve the method for little algae oil-producing capacity
(1) metabolite in little gonidium is measured:
1. cell harvesting and cancellation:
Little algae is cultivated, and at least one time point in index mid-term, later stage and three times of stationary phase of culturing process takes out algae liquid sample 100-150mL, and each time point takes out 3-5 part; 4 ℃ of following 3000-5000rpm; Centrifugal 3-4min collects the cell of lower floor, with 3-5mL metabolism stop buffer at-40 ℃ of following cancellation 5-10 minutes; Stop metabolic reaction, obtain FD at-80 ℃ of following lyophilize 4-6h;
Said metabolism stop buffer is for containing 1500mgL
-1NaNO
3, 36mgL
-1CaCl
22H
2O, 75mgL
-1MgSO
47H
2O and 40mgL
-1K
2HPO
43H
2The methanol aqueous solution of O, the volume ratio of first alcohol and water is 1: 2 in the said methanol aqueous solution;
2. extract the endocellular metabolism thing:
Get the FD 15-25mg that 1. step obtain and place centrifuge tube respectively, every pipe adds 1-2mL-40 ℃ metabolism extracting solution, mixing; Lid is tight and put into liquid nitrogen, and multigelation 3-5 time is at-20 ℃ of centrifugal 1-2min of following 4000-6000rpm; Collect supernatant, in residue, add 0.5-1mL metabolism extracting solution ,-20 ℃ of centrifugal 1-2min of following 4000-6000rpm in addition; After centrifugal, twice supernatant mixes, and adds the deuterium-labeled succsinic acid of 10-20 μ g; Get mixed solution, get 200 μ L mixed solutions, at-80 ℃ of following lyophilize 2-4 hours;
Said metabolism extracting solution is that volumn concentration is 50% methanol aqueous solution;
3. metabolite derivatize
In the sample that 2. step obtains, add 20mgmL
-1Methoxyl group ammonium salt hydrochlorate/pyridine solution 50 μ L; In 40 ℃ of water-baths, react 60-80min; Reaction finishes the back and adds the trimethyl silicon based trifluoroacetamide of 80 μ L N-methyl-N-, reacts 60-80min again in 40 ℃ of water-baths, the centrifugal 1min of 8000-12000rpm; Get supernatant 100 μ L and put into numbered GC sample introduction bottle, room temperature is placed 2h;
4. GC-MS detects
Adopt GC-MS to step 3. the gained sample carry out qualitative and quantitative Treatment, condition is following:
Chromatographic column: DB-5 gas chromatographic column, its specification are 30m*0.25mm, 0.25 μ m;
Sample size: 1 μ L;
Splitting ratio: 10: 1;
Injector temperature: 280 ℃;
GC interface temperature: 270 ℃;
Helium flow velocity: constant voltage, 91KPa;
Heating schedule: 70 ℃ keep 2min, with 5 ℃ of min
-1Speed be raised to 290 ℃, and keep 6min at 290 ℃;
Ionization voltage: 70eV;
Source temperature: 250 ℃;
Sweep limit: 50-800m/z;
Sweep velocity: 2scans
-1
Thereby the qualitative and quantitative data of the metabolite that has obtained little algae;
(2) oil-producing capacity of little algae is analyzed:
1. cell harvesting and cancellation:
Little algae is cultivated, and at least one time point in index mid-term, later stage and three times of stationary phase of culturing process takes out algae liquid sample 100-150mL, and each time point takes out 3-5 part; 4 ℃ of following 3000-5000rpm; Centrifugal 3-4min collects the cell of lower floor, with 3-5mL metabolism stop buffer at-40 ℃ of following cancellation 5-10 minutes; Stop metabolic reaction, obtain FD at-80 ℃ of following lyophilize 4-6h;
Said metabolism stop buffer is for containing 1500mgL
-1NaNO
3, 36mgL
-1CaCl
22H
2O, 75mgL
-1MgSO
47H
2O and 40mgL
-1K
2HPO
43H
2The methanol aqueous solution of O, the volume ratio of first alcohol and water is 1: 2 in the said methanol aqueous solution;
2. extract lipid acid in the cell:
Get the FD 15-25mg that 1. step obtain and place centrifuge tube respectively, every pipe adds 0.75-1.5mL chloroform and 0.3-0.6mL ultrapure water, and 100rpm shakes 1h; Add 2-4mL fat extraction liquid again, with 100rpm concussion 0.5h, collect the chloroform phase under the room temperature; In residue, add 2-4mL fat extraction liquid, with 100rpm concussion 0.5h, collect the chloroform phase under the room temperature; Extract repeatedly 3 times, chloroform is merged mutually, add the 0.5-1mL 1M KCl aqueous solution, concussion, 3000-4000rpm; Centrifugal 3-5min abandons water, adds the 1-2mL ultrapure water again, concussion; 3000-4000rpm, centrifugal 3-5min abandons water, and 30-35 ℃ of vacuum-drying gets dry-matter;
Said fat extraction liquid is that volumn concentration is 66.7% chloroform methanol solution, and it is 0.1% butylated hydroxytoluene that said chloroform methanol solution contains mass percent;
3. methyl esterification of fatty acid:
It is in 14% the boron trifluoride-methanol solution that said dry-matter is dissolved in 600-1000 μ L mass percent; Add 10-20 μ g margaric acid as interior mark, place in the 15mL ST, at 100 ℃ of water-bath 20-30min; Temperature is reduced to room temperature; Add 500-1000 μ l normal hexane concussion extraction 30s, the centrifugal 2min of 4000-5000rpm obtains the normal hexane phase; Get 100-200 μ L normal hexane and put into numbered GC sample introduction bottle mutually;
4. GC-MS detects
Adopt GC-MS that the fatty acid methyl ester of normal hexane in mutually carried out qualitative and quantitative Treatment, condition is following:
Chromatographic column: DB-5 gas chromatographic column, its specification are 30m*0.25mm, 0.25 μ m;
Sample size: 1 μ L;
Splitting ratio: 10: 1;
Injector temperature: 280 ℃;
GC interface temperature: 270 ℃;
Helium flow velocity: constant voltage, 91KPa;
Heating schedule: 70 ℃ keep 2min, with 8 ℃ of min
-1Speed be raised to 290 ℃, and keep 6min at 290 ℃;
Ionization voltage: 70eV;
Source temperature: 250 ℃;
Sweep limit: 50-800m/z;
Sweep velocity: 2scans
-1
Thereby obtained the content of the single fatty acid methyl ester in the fatty acid methyl ester admixture;
5. day output
Calculate the day output of little algae fatty acid methyl ester admixture according to following formula:
P＝∑Wi/Day/CV
Wherein P is little algae fatty acid methyl ester day output, Wi be step 4. in the weight of each fatty acid methyl ester in the gained sample, to be that step is 1. said be inoculated into the time span of microalgae harvesting from little algae to Day, the volume of micro algae culturing liquid when CV is the 1. said results of step;
6. cetane value is confirmed
Calculate the cetane value of little algae fatty acid methyl ester admixture according to following formula:
SN＝∑(560×Pi)/MWi a
IN∑(254×D×Pi)/MWi b
CN＝46.3+5458/SN-0.225×IN c
Wherein SN is a saponification value; IN is an iodine number; CN is a cetane value; Pi is the dry weight percentage of each fatty acid methyl ester in the 4. middle gained sample of step, and MWi is the molecular weight of each fatty acid methyl ester in the 4. middle gained sample of step, and D is the double key number amount of each fatty acid methyl ester in the 4. middle gained sample of step.
(3) PLS analyzes
Little algae metabolite content that step (1) is obtained is an independent variable(s), and little algae fatty acid methyl ester day output and cetane value that step (2) is obtained are dependent variable, uses PLS to analyze, and obtains the metabolic markers relevant with produce oil;
(4) process analysis
The content of the metabolite mark that step (3) is obtained is processed chart according to culture condition difference; Observe and analyze the rule that these metabolites change; And then find metabolite and the associated metabolic network in little algae produce oil process, play a crucial role, thereby for being that little algae culture condition of purpose provides the optimization direction to improve little algae fatty acid methyl ester output and quality.
2. the metabolism group of utilizing according to claim 1 is improved little algae culture condition to improve the method for little algae oil-producing capacity, it is characterized in that said little algae is chlorella (Chlorella sorokiniana), grid algae (Scenedesmus obliquus), cytoalgae (Synechocystis sp.PCC6803) and anabena (Anabaena sp.PCC7120).
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103675181A (en) * | 2013-12-05 | 2014-03-26 | 陈东 | Detection method for organic acid, amino acid and sugar in intracellular and extracellular fluids by using gas chromatography-mass spectrometry |
| CN104561146A (en) * | 2013-10-18 | 2015-04-29 | 天津大学 | PH two-stage adjustment culture method for increasing micro-algal grease yield |
| CN106488985A (en) * | 2014-01-27 | 2017-03-08 | 原子能和替代能源委员会 | The inhibitor of steroid metabolism makes the application of triglycerides accumulation and its method in microalgae |
| CN107860857A (en) * | 2017-10-27 | 2018-03-30 | 中国科学院青岛生物能源与过程研究所 | A kind of extraction and analytical method of yellow silk frustule intracellular metabolite thing |
| CN112540133A (en) * | 2020-11-02 | 2021-03-23 | 暨南大学 | Detection and analysis method for marine microalgae intracellular metabolome |
| CN112540134A (en) * | 2020-11-02 | 2021-03-23 | 暨南大学 | Extraction and detection method of marine microalgae fatty acid |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1699516A (en) * | 2005-06-01 | 2005-11-23 | 清华大学 | Process for preparing bio-diesel-oil by using microalgae fat |
| CN101532991A (en) * | 2009-03-21 | 2009-09-16 | 国家海洋局第一海洋研究所 | Method for quickly detecting content of fatty acid in marine microalgae |
-
2011
- 2011-11-28 CN CN201110384759.0A patent/CN102517349B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1699516A (en) * | 2005-06-01 | 2005-11-23 | 清华大学 | Process for preparing bio-diesel-oil by using microalgae fat |
| CN101532991A (en) * | 2009-03-21 | 2009-09-16 | 国家海洋局第一海洋研究所 | Method for quickly detecting content of fatty acid in marine microalgae |
Non-Patent Citations (2)
| Title |
|---|
| 廖启斌等: "海洋微藻脂肪酸的气相色谱分析", 《海洋通报》, vol. 19, no. 6, 31 December 2000 (2000-12-31), pages 66 - 71 * |
| 李文权等: "三种海洋微藻生长期脂肪酸组成研究", 《海洋环境科学》, vol. 21, no. 2, 31 May 2002 (2002-05-31), pages 10 - 13 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561146A (en) * | 2013-10-18 | 2015-04-29 | 天津大学 | PH two-stage adjustment culture method for increasing micro-algal grease yield |
| CN103675181A (en) * | 2013-12-05 | 2014-03-26 | 陈东 | Detection method for organic acid, amino acid and sugar in intracellular and extracellular fluids by using gas chromatography-mass spectrometry |
| CN106488985A (en) * | 2014-01-27 | 2017-03-08 | 原子能和替代能源委员会 | The inhibitor of steroid metabolism makes the application of triglycerides accumulation and its method in microalgae |
| CN106488985B (en) * | 2014-01-27 | 2020-03-20 | 原子能和替代能源委员会 | Use of inhibitors of sterol metabolism to accumulate triglycerides in microalgae and methods thereof |
| CN107860857A (en) * | 2017-10-27 | 2018-03-30 | 中国科学院青岛生物能源与过程研究所 | A kind of extraction and analytical method of yellow silk frustule intracellular metabolite thing |
| CN112540133A (en) * | 2020-11-02 | 2021-03-23 | 暨南大学 | Detection and analysis method for marine microalgae intracellular metabolome |
| CN112540134A (en) * | 2020-11-02 | 2021-03-23 | 暨南大学 | Extraction and detection method of marine microalgae fatty acid |
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