CN102465098A - Culture medium composition for culturing chlorella - Google Patents
Culture medium composition for culturing chlorella Download PDFInfo
- Publication number
- CN102465098A CN102465098A CN2010105391855A CN201010539185A CN102465098A CN 102465098 A CN102465098 A CN 102465098A CN 2010105391855 A CN2010105391855 A CN 2010105391855A CN 201010539185 A CN201010539185 A CN 201010539185A CN 102465098 A CN102465098 A CN 102465098A
- Authority
- CN
- China
- Prior art keywords
- chlorella
- culture media
- media composition
- culture
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention relates to a culture medium composition suitable for culturing ordinary chlorella and a method for mixed culture of the chlorella by using the culture medium. The culture medium is obtained by adding 0.00126-0.063 g/L of Na2SO3 and an organic carbon source based on a common SE culture medium. The use of the culture medium in photoautotrophy-heterotrophy mixed culture of the ordinary chlorella can remarkably promote the growth of chlorella, and can allow the cell density of the ordinary chlorella to reach a level of high density culture in a short culture period, that is, the high density culture of the chlorella is realized. The use of the culture medium composition allows the ordinary chlorella to easily use the organic carbon source, especially a cheap carbon source for growing, can promote the later grease accumulation, and can greatly increase the quality of the ordinary chlorella, i.e. the grease content of the ordinary chlorella compared with heterotrophic culture to reach the level of photoautotrophic culture.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of high-density high-quality ordinary chlorella culture media composition of cultivating and method of cultivating chlorella of being suitable for specifically.
Background technology
It is longer that people must study history to chlorella, and wherein Chlorella vulgaris (Chlorella vulgaris) is a more algae kind of research, also is to commercially produce one of algae kind that generally adopts at present.
Nutritive ingredients such as chlorella rich in proteins, polysaccharide, unsaturated fatty acids can be used for food, medicine and energy aspect; Can accumulate lipid acid in a large number, some chlorella fatty acid content can account for 30%～60% of dry weight.Utilize the cultivation chlorella to accumulate oil resource, become the research field of utilizing the solar energy development renewable resources the most popular at present.Not only have powerful market potential, and have outstanding social value.
But most of wild chlorella growth speed is unhappy, and the stabilizing carbon dioxide ability is also limited, accomplish scale production, and just consumes lot of manpower and material resources, and gets little.
Chlorella is divided into two kinds of autotrophy and heterotrophism according to its nutritional mode.First kind of light autotrophy process is exactly that chlorella utilizes CO
2Gas carries out obtaining the energy when photosynthesis fixes carbon source, grows, and the accumulation grease.Because CO
2Solubleness is limited in nutrient solution, absorbs inefficiency, and the frustule growth velocity is limited, and mass-producing generally takes open pond to put in a suitable place to breed later on, but pollutes easily and has cell density and hang down and unfavorable factors such as floor space is big.Second kind is the heterotrophism training method, exactly the culturing process people for carbon source is provided, such as glucose etc.; Obviously, though, can increase substantially algae cell density through the heterotrophism carbon source is provided; But its frustule quality obviously descends, and albumen and fatty acid content are all on the low side.Glucose belongs to quick-acting carbon sources as the heterotrophism carbon source, can improve the speed of chlorella growth, but its oil and fat accumulation efficient do not improved accordingly, and such carbon source must increase the cultivation cost simultaneously, will receive the very big restriction of raw material.
Atev.A (Atev A.; Manova A.Heterotrophic cultivation of Chlorella vulgaris A2.Dok1.Bolg.Akad.Nauk, 687～690), magnify soldier etc. and (magnify the soldier (English) 1981,34 (5):; Wu Qingyu, the heterotrophism of chlorella cells transforms.Plant physiology circular, 1996,32 (2): 140～144) (Pan Xin, Li Jianhong wear the research that passes superfine chlorella heterotrophy cultivation with Pan Xin etc.Food science, 2002,23 (4): 28～33) all peculiar chlorella culturing process quality decline problem is set forth.Wherein research mainly is the variation of protein composition in the chlorella.
On the basis of chlorella culture condition and training method, people's sight turns to the training strategy that utilizes chlorella production biofuel.The single step training strategy: perfect medium is cultivated, though content of oil and grease can lower (increasing extraction cost), growth velocity is fast, can increase living weight apace; Incomplete culture medium culturing, though throughput rate is slow, high fat content can be offset the deficiency of living weight.Two step training strategies: utilize the perfect medium heterotrophism to cultivate earlier, its living weight is increased fast, utilize incomplete substratum autotrophy to cultivate as nitrogen restriction or change culture condition such as light intensity, temperature again, make frustule accumulate grease in a large number.
CN 200610025618.9 discloses a kind of method of culturing chlorella with high-density and high-quality, and this method comprises the dilution and the light autotrophy cultivation three phases of bio-reactor high Density Heterotrophic, high-density algae liquid.This method adds glucose as organic carbon source in the heterotrophism substratum, cost is higher.
CN200610024004.9 discloses a kind of method of cultivating Chlorella vulgaris, and this method adopts heterotrophism one smooth autotrophy series connection to cultivate Chlorella vulgaris.This method heterotrophism is cultivated the culture media composition of employing basically by KNO
3, glucose and small amounts of inorganic salt, trace element and water forms.Promptly added the more expensive glucose of price in the heterotrophism substratum in this method equally as organic carbon source.
The document that above-mentioned difference is cultivated chlorella improves chlorella cultivation quality and all only is meant protein and chlorophyllous content in the frustule.Especially CN200610024004.9 though pass through to optimize chlorella culture group compound composition, does not mention the improve effect of this compsn cultivation chlorella to the chlorella fat content.
Therefore, in present research process,, can also obtain the lipid acid accumulative effect of high level, must start with from the minimum medium compsn for when reaching the cultivation chlorella and utilize the cheap carbon source high-density growth.Be necessary to solve chlorella heterotrophy high-density height and contain the culture group compound problem that oil quality is cultivated.
Summary of the invention
The technical problem that the present invention will solve provides a kind of culture media composition that is used to cultivate Chlorella vulgaris; This substratum can make chlorella reach under the prerequisite of high-density culture; The frustule oil and fat accumulation is cultivated to compare with existing two steps and is had a more substantial increase, and cultivates the level when reaching the cultivation of light autotrophy when finishing.
For realizing above-mentioned purpose, the invention provides a kind of culture media composition that is used to cultivate Chlorella vulgaris, said substratum consists essentially of Na
2SO
3, organic carbon source and small amounts of inorganic salt, trace element and water.
In one embodiment of the invention, the composition of every liter of said culture media composition comprises: Na
2SO
30.00126～0.063g, preferred 0.02～0.05g, organic carbon source 10～50g, NaNO
3Or KNO
30.1～0.5g, K
2HPO
43H
2O 0.05～0.1g, MgSO
47H
2O 0.05～0.1g, CaCl
22H
2O 0.01～0.05g, KH
2PO
40.05～0.2g, NaCl 0.05～0.1g, Soil extract (soil extract) 20～40mL, FeCl
36H
2O 0.001～0.01g, Fe-EDTA 0.5～1mL, trace element solution 0.5～1mL, the deionized water surplus.
Consisting of of 1 liter of trace element solution: H wherein
3BO
35.5～6.5g, ZnSO
47H
2O 4.0～5.0g, MnCl
2H
2O 0.5～1.0g, (NH
4)
6Mo
7O
244H
2O 0.20～0.40g, CuSO
45H
2O 0.50～1.00g, Co (NO
3)
26H
2O 0.10～0.20g and deionized water surplus.
According to culture media composition of the present invention, wherein said organic carbon source is selected from one or more in glucose, starch hydrolyzate, bacterium slag treatment solution and the algae-residue treatment solution.The organic carbon source consumption is to add this organic carbon source of 10～50g in every liter of culture medium solution, and the carbon source that wherein adds with hydrolyzed solution or treatment solution form also is to be added in the culture media composition by hydrolyzed solution or treatment solution weight that corresponding pretreatment process obtains.
Wherein said organic carbon source preferred bacterium slag treatment solution or algae-residue treatment solution.Described bacterium slag is selected from bacterium bacterium slag or yeast bacterium slag, the Cray Bai Shi pulmonitis strain of the preferred anaerobically fermenting of bacterium, the preferred candidiasis of yeast.Described bacterium slag or algae-residue treatment solution obtain through bacterium slag or algae-residue are carried out pre-treatment.The pretreatment process of said bacterium slag is: after bacterium or saccharomycetes to make fermentation finish; Behind 60 ℃～80 ℃ boiling 30min～60min; Filter at normal temperatures and obtain bacterium slag filter cake, the diluted alkaline that adds 0.01～0.1mol/L then is hydrolyzed, and used diluted alkaline is selected from NaOH and/or NaHCO
3, the solid-to-liquid ratio of hydrolytic process is 1: 10～1: 20 (mass ratio), and treatment temp is 15 ℃～30 ℃, and the treatment time is 5～10 days.The pretreatment process of said algae-residue is: obtain algal biscuit after the broken oil removing of frustule, the alkali lye that adds 0.01～0.1mol/L then is hydrolyzed, and the solid-to-liquid ratio of hydrolytic process is 1: 10～1: 20, and treatment temp is 15 ℃～30 ℃, and the treatment time is 5d～10d.
Glucose concn is 10～15g/L in the described substratum, and said starch or bacterium slag hydrolyzed solution concentration are 30～50g/L.
In another kind of performance of the present invention, every liter of culture media composition also comprises tomato extracting solution 5～15mL, preferred 6～10mL.Wherein the process for preparation of tomato extracting solution is: take by weighing 100% Normal juice tomato juice, be mixed with the solution of 100～200g/L concentration with dissolved in distilled water, with its centrifugal solid impurity that goes, obtain supernatant, be the tomato extracting solution.
The method that the present invention also provides a kind of acclimation shaking culture to raise together with the little algae of bead has simultaneously been used above-mentioned culture media composition in the domestication process.Described domestication and culture method comprises:
The algae kind of setting out is a kind of autotrophy chlorella, and the autotrophy chlorella is carried out heterotrophism condition acclimation shaking culture, and used substratum is above-mentioned substratum.Heterotrophism acclimation shaking culture condition is that following 25 ℃～30 ℃ of lucifuge situation, 100～150rmp, airbath shaking table are cultivated 7d～10d; After 5～10 batches of heterotrophism domestications; Heterotrophism domestication algae kind is carried out separation and purification: will cultivate in the exponential phase algae liquid dilution under the heterotrophism condition and coat on the solid plate that SE substratum (for minimum medium known in the art) done, low light condition cultivation 10d～20d obtains some single algaes to fall; This single algae is fallen behind the picking; Draw and in SE solid slant culture base, cultivate, after waiting to grow, can obtain the purebred algae kind of raising together with.
In the culture media composition that wherein domestication process is used, preferred organic carbon source comprises glucose, bacterium bacterium slag treatment solution or algae-residue treatment solution simultaneously, and glucose concn is 10～15g/L, and bacterium slag or algae-residue treatment solution concentration are 30～50g/L.
Under the situation of some embodiment, the present invention also provides a kind of method of cultivating chlorella, and this method adopts light autotrophy-little algae of heterotrophism mixed culture bead, it is characterized in that said method has adopted culture media composition of the present invention.
The method of described cultivation chlorella comprises: adding pH value is 6.0～7.0 above-mentioned substratum in the bio-reactor; Insert Chlorella vulgaris algae kind by 5%～15% of working volume and cultivate, culture condition is: stirring velocity is 80～100rpm, the mixed gas of bubbling air and carbonic acid gas; Air flow is 0.1～1.0vvm; Intensity of illumination is 2000～3000lx, and culture temperature is 25 ℃～32 ℃, and control pH value is less than 10 in the culturing process.
After being mixed with culture media composition, regulating pH value to 6.0～7.0 of said culture media composition as required, and sterilized 30～60 minutes down at 121 ℃～130 ℃.
Compare with existing chlorella substratum, culture media composition of the present invention has following characteristics:
1, owing to added Na
2SO
3Culture media composition of the present invention is used for light autotrophy-heterotrophism mixed culture Chlorella vulgaris, can promote the growth of chlorella, can make Chlorella vulgaris cell density in short culture cycle reach the level of high-density culture; Promptly can realize the high-density culture of chlorella, than no Na
2SO
3Living weight can improve 80%～140% during interpolation.
2, Na
2SO
3With being used of tomato extracting solution, micro-mother liquor; Make Chlorella vulgaris utilize organic carbon source to grow more easily; And the oil and fat accumulation to self also has bigger promoter action in later stage; To be fat content with heterotrophism cultivate to compare the quality that can make Chlorella vulgaris has a more substantial increase, and reached the level when the light autotrophy is cultivated.
3, in culture media composition of the present invention, can add cheap bacterium slag or algae-residue treatment solution as organic carbon source, such organic carbon source and Na
2SO
3Match with tomato extracting solution, micro-mother liquor, can either realize the high-density culture of chlorella, the higher fat content that obtains has also significantly reduced the cultivation cost of chlorella simultaneously.
Embodiment
(this substratum is known in the art at first to adopt the SE substratum of reporting in the document; Can be used for chlorella grows under light autotrophy condition; Realize the heterotrophism cultivation through adding carbon source), it is to use in the foregoing invention content to remove Na in the substratum that the SE substratum is revised slightly
2SO
3With the organic carbon source composition.Cultivate the chlorella that sets out with this amended SE substratum, though this chlorella cells growth of setting out can realize cultivating in the SE substratum, frustule concentration is not high when cultivating end, and the fat content in the frustule is also lower.Explain that this substratum needs further to optimize, could realize the purpose that high-density and high-quality is cultivated.
The present invention is the basis with this SE substratum, through adding Na therein
2SO
3And organic carbon source, change wherein organic carbon source action effect, at carbon source nitrogenous source and Na
2SO
3Effect down; And other constituent contents in the adjustment substratum; Comprise and add the tomato extracting solution, obtained to be applicable to that the chlorella that sets out optimizes the substratum of the mutagenesis chlorella mixed culture that obtains through heterotrophism, make this substratum can realize the high-density and high-quality cultivation of Chlorella vulgaris or mutagenesis chlorella.
Frustule quality parameter described in the present invention is a unit frond fat content, and the living weight index of said frond is a frond dry weight in the unit nutrient solution.Those skilled in the art can adopt routine techniques that said frond fat content and frond dry weight are measured, and concrete grammar is known in the art.
Below will be further described technology contents of the present invention through embodiment.Only if having saidly in addition, the concentration of the substance described in the inventive method and the embodiment all is to be benchmark with the substratum TV, and promptly the content of substance accounts for per-cent or the mole number or the quality etc. of nutrient solution preparation total nutrient solution volume after accomplishing.
Embodiment 1
Shake adding 100mL substratum as follows in the bottle at 250mL; Inoculative proportion is 10% (be seed liquor volume account for volume of culture 10%), and wherein seed is the chlorella through domestication, and heterotrophism is cultivated (liquid amount 100mL; Temperature 30 degree; Shaking speed 100rpm) 48 hours, the result recorded cell density and is 14.24g/L to the maximum, and fat content is 30.8% (total oil quantity accounts for the percentage ratio of total dried cell weight in the cell).
Every liter of substratum is formed:
Glucose 15g Na
2SO
30.040g
KNO
30.35g micro-mother liquor 0.5mL
K
2HPO
4·3H
2O?0.075g MgSO
4·7H
2O?0.075g
CaCl
2·2H
2O 0.025g KH
2PO
4?0.175g
NaCl 0.025 Soil extract (soil extract) 30mL
FeCl
3·6H
2O?0.005g Fe-EDTA 1mL
Distilled water surplus
1 liter of micro-mother liquor prescription: H wherein
3BO
36.0g, ZnSO
47H
2O 4.5g, MnCl
2H
2O0.75g, (NH
4)
6Mo
7O
244H
2O 0.35g, CuSO
45H
2O 0.75g, Co (NO
3)
26H
2O 0.15g, distilled water surplus.
Comparative example 1
The substratum essentially consist does not add Na with embodiment 1
2SO
3This component, culture condition and process and embodiment 1 are consistent.The cell density that the cultivation results analysis records has only 6.11g/L, and the content that grease accounts for dried cell weight also has only 27.6%.
Can find out according to above-mentioned experimental result, under same basic medium condition, use Na
2SO
3Culture effect obvious effect to chlorella is having Na
2SO
3Exist down, chlorella utilizes organic carbon source to grow more easily, and the oil and fat accumulation in self is also had bigger promoter action in later stage.
Embodiment 2
Shake adding 100mL substratum as follows in the bottle at 250mL; Inoculative proportion is 10% (be seed liquor volume account for volume of culture 10%), and wherein seed is the chlorella through the heterotrophism domestication, and heterotrophism is cultivated (liquid amount 100mL; Temperature 30 degree; Shaking speed 100rpm) 48 hours, the result recorded cell density and is 19.41g/L to the maximum, and fat content is 35.2% (total oil quantity accounts for the percentage ratio of total dried cell weight in the cell).
Wherein bacterium slag treatment solution is to obtain through bacterium or yeast bacterium slag being hydrolyzed handle.Wherein preferred Cray Bai Shi pneumobacillus, it is unique substrate with glycerine, under anaerobic fermentative prodn 1, ammediol.After the fermentation ends, behind 80 ℃ of boiling 40min, filter at normal temperatures and obtain bacterium slag filter cake, bacterium slag filter cake through after drying, is hydrolyzed under the NaOH of 0.1mol/L solution, the solid-to-liquid ratio of hydrolytic process is 1: 20, and the treatment time is 7d, and treatment temp is 25 ℃.Bacterium slag treatment solution after treatment can be used as organic carbon source and is used for the preparing culture medium compsn.
Every liter of substratum is formed as follows:
Bacterium slag treatment solution 30g Na
2SO
30.040g
NaNO
30.35g micro-mother liquor 0.7mL
K
2HPO
4·3H
2O?0.075g MgSO
4·7H
2O?0.075g
CaCl
2·2H
2O?0.025g KH
2PO
4?0.175g
NaCl 0.025g Soil extract (soil extract) 30mL
FeCl
3·6H
2O?0.005g Fe-EDTA 1mL
Distilled water surplus
1 liter of micro-mother liquor prescription: H wherein
3BO
36.0g, ZnSO
47H
2O 4.5g, MnCl
2H
2O0.75g, (NH
4)
6Mo
7O
244H
2O 0.35g, CuSO
45H
2O 0.75g, Co (NO
3)
26H
2O 0.15g, distilled water surplus.
Comparative example 2
The substratum essentially consist is with embodiment 2, and different is not add Na in the substratum
2SO
3This component, culture condition and process and embodiment 2 are consistent.The cell density that the cultivation results analysis records has only 8.11g/L, and the content that grease accounts for dried cell weight also has only 28.2%.
Can find out according to above-mentioned experimental result, under same basic medium condition, use Na
2SO
3Culture effect obvious effect to chlorella is having Na
2SO
3Exist down, especially under the situation of using bacterium slag pretreatment fluid, chlorella utilizes organic carbon source to grow more easily, and the oil and fat accumulation in self is also had bigger promoter action in later stage.
The change of nitrogenous source and trace element simultaneously also can be played certain promoter action to the cultivation of chlorella, and their acting in conjunction improves culture effect.
Embodiment 3
Shake in the bottle at 250mL and to add 100mL substratum as follows, inoculative proportion is 10% (be seed liquor volume account for volume of culture 10%), and used seed is from Chlorella vulgaris; This chlorella does not pass through heterotrophism acclimation shaking culture process; Directly its heterotrophism was cultivated (liquid amount 100mL, temperature 30 degree, shaking speed 100rpm) 48 hours; The result records cell density and is 11.30g/L to the maximum, and fat content is 28.6% (total oil quantity accounts for the percentage ratio of total dried cell weight in the cell).
Every liter of substratum is formed as follows:
Glucose 30g Na
2SO
30.020g
KNO
30.25g micro-mother liquor 0.1mL
K
2HPO
4·3H
2O?0.075g MgSO
4·7H
2O?0.075g
CaCl
2·2H
2O?0.025g KH
2PO
4?0.175g
NaCl 0.025 Soil extract (soil extract) 30mL
FeCl
3·6H
2O?0.005g Fe-EDTA 1mL
Distilled water surplus
1 liter of micro-mother liquor prescription: H wherein
3BO
36.0g, ZnSO
47H
2O 4.5g, MnCl
2H
2O0.75g, (NH
4)
6Mo
7O
244H
2O 0.35g, CuSO
45H
2O 0.75g, Co (NO
3)
26H
2O 0.15g, distilled water surplus.
Comparative example 3
The substratum essentially consist is not wherein added Na with embodiment 3
2SO
3With micro-mother liquor, used algae kind is from Chlorella vulgaris, and this chlorella does not pass through heterotrophism acclimation shaking culture process, directly its heterotrophism is cultivated, and culture condition and process and embodiment 3 are consistent.The cell density that the cultivation results analysis records has only 4.26g/L, and the content that grease accounts for dried cell weight also has only 25.4%.
Can find out according to above-mentioned experimental result, under same basic medium condition, use Na
2SO
3Be used culture effect obvious effect with micro-mother liquor, Na is being arranged Chlorella vulgaris
2SO
3Exist down, chlorella utilizes organic carbon source to grow more easily, and the oil and fat accumulation in self is also had bigger promoter action in later stage.
Compare with 2 with embodiment 1, chlorella more can be effectively utilized organic carbon source under the situation that heterotrophism and autotrophy condition all possess through after the heterotrophism domestication, tames chlorella and can more effectively realize the mixed culture process.
Embodiment 4
Shake adding 100mL substratum as follows in the bottle at 250mL; Inoculative proportion is 10% (be seed liquor volume account for volume of culture 10%), and wherein seed is the chlorella through the heterotrophism domestication, and heterotrophism is cultivated (liquid amount 100mL; Temperature 30 degree; Shaking speed 100rpm) 48 hours, the result recorded cell density and is 20.30g/L to the maximum, and fat content is 32.8% (total oil quantity accounts for the percentage ratio of total dried cell weight in the cell).
Wherein the treatment process of bacterium slag treatment solution is with embodiment 2.
Every liter of substratum is formed as follows:
Bacterium slag treatment solution 35g Na
2SO
30.050g
NaNO
30.35g micro-mother liquor 0.7mL
K
2HPO
4·3H
2O?0.075g MgSO
4·7H
2O?0.075g
CaCl
2·2H
2O?0.025g KH
2PO
40.175g
NaCl 0.025g Soil extract (soil extract) 30mL
FeCl
3·6H
2O?0.005g Fe-EDTA?1mL
Tomato extracting solution 10mL distilled water surplus
1 liter of micro-mother liquor prescription: H wherein
3BO
36.0g, ZnSO
47H
2O 4.5g, MnCl
2H
2O0.75g, (NH
4)
6Mo
7O
244H
2O 0.35g, CuSO
45H
2O 0.75g, Co (NO
3)
26H
2O 0.15g, distilled water surplus.
Tomato extracting solution preparation: take by weighing 100% Normal juice tomato juice, be mixed with the solution of 150g/L concentration,, obtain supernatant, be the tomato extracting solution with its centrifugal solid impurity that goes with dissolved in distilled water.
Embodiment 5
Shake in the bottle at 250mL and to add 100mL substratum as follows, inoculative proportion is 10% (be seed liquor volume account for volume of culture 10%), and wherein seed is the chlorella through the heterotrophism domestication; Carry out mixed culture (liquid amount 100mL, temperature 30 degree, shaking speed 100rpm; The mixed gas of bubbling air and carbonic acid gas; Air flow is 0.5vvm, and intensity of illumination is 3000lx, and control pH value is less than 10 in the culturing process) 48 hours; The result records cell density and is 22.15g/L to the maximum, and fat content is 38.0% (total oil quantity accounts for the percentage ratio of total dried cell weight in the cell).
Wherein the treatment process of bacterium slag treatment solution is with embodiment 2.
Every liter of substratum is formed as follows:
Bacterium slag treatment solution 50g Na
2SO
30.020g
NaNO
30.35g micro-mother liquor 0.7mL
K
2HPO
4·3H
2O?0.075g MgSO
4·7H
2O?0.075g
CaCl
2·2H
2O?0.025g KH
2PO
4?0.175g
NaCl 0.025g Soil extract (soil extract) 30mL
FeCl
3·6H
2O?0.005g Fe-EDTA 1mL
Tomato extracting solution 10mL distilled water surplus
1 liter of micro-mother liquor prescription: H wherein
3BO
36.0g, ZnSO
47H
2O 4.5g, MnCl
2H
2O0.75g, (NH
4)
6Mo
7O
244H
2O 0.35g, CuSO
45H
2O 0.75g, Co (NO
3)
26H
2O 0.15g, distilled water surplus.The preparation of tomato extracting solution is with embodiment 4.
From embodiment 1,2,4 and 5, and comparative example 1 and 2, can find out, at preferred Na
2SO
3In the amount ranges, Na is being arranged
2SO
3Exist down, especially under the situation of using bacterium slag pretreatment fluid, chlorella utilizes organic carbon source to grow more easily, and also has bigger promotion to create a bad precedent to the oil and fat accumulation in self in later stage.And can be known that by embodiment 5 this domestication chlorella can be implemented heterotrophism and autotrophy mixed culture process under the compsn culture medium condition, the cultivation quality of chlorella has all obtained corresponding raising.
Embodiment 6
Among this embodiment Chlorella vulgaris is carried out the heterotrophism domestication.The algae kind of setting out is a kind of autotrophy chlorella, and the autotrophy chlorella is carried out heterotrophism condition acclimation shaking culture:
Every liter of substratum is formed as follows:
Bacterium slag treatment solution 20g Na
2SO
30.020g
NaNO
30.30g micro-mother liquor 0.5mL
K
2HPO
4·3H
2O?0.070g MgSO
4·7H
2O?0.075g
CaCl
2·2H
2O?0.025g KH
2PO
4?0.2g
NaCl 0.025g Soil extract (soil extract) 40mL
FeCl
3·6H
2O?0.005g Fe-EDTA?1mL
Glucose 10g distilled water surplus
1 liter of micro-mother liquor prescription: H wherein
3BO
36.0g, ZnSO
47H
2O 4.0g, MnCl
2H
2O0.70g, (NH
4)
6Mo
7O
244H
2O 0.40g, CuSO
45H
2O 0.75g, Co (NO
3)
26H
2O 0.15g, distilled water surplus.
Heterotrophism acclimation shaking culture condition is that following 30 ℃ of lucifuge situation, 120rmp, airbath shaking table are cultivated 10d; After the domestication of 8 batches of heterotrophism, heterotrophism domestication algae kind is carried out separation and purification: will cultivate exponential phase algae liquid dilution under the heterotrophism condition and coat on the solid plate that SE substratum (for the minimum medium of being known in this field) done low light condition cultivation 15d; Obtaining some single algaes falls; This single algae is fallen behind the picking, draw and in SE solid slant culture base, cultivate, after waiting to grow; Can obtain the purebred algae kind of raising together with, promptly tame chlorella.
Can confirm that by above-mentioned a plurality of embodiment and comparative example this chlorella substratum is using bacterium slag treatment solution and Na
2SO
3, the chlorella quality of cultivating out when tomato extracting solution and micro-mother liquor is the most outstanding; Especially use under the situation of chlorella as the chlorella algae kind of setting out after this heterotrophism substratum is tamed, the growth that the domestication chlorella is cultivated the back acquisition at this optimization culture group compound is all improved a lot with the cultivation quality of throughput than original Chlorella vulgaris.Can find out through embodiment 5 that simultaneously the said composition domestication obtains chlorella after passing through heterotrophism autotrophy mixed culture, the cultivation quality of chlorella is the most outstanding.
Claims (14)
1. a culture media composition that is used to cultivate chlorella is characterized in that, every liter of said culture media composition comprises:
Na
2SO
30.00126～0.063g, organic carbon source 10～50g, NaNO
3Or KNO
30.1～0.5g, K
2HPO
43H
2O 0.05～0.1g, MgSO
47H
2O 0.05～0.1g, CaCl
22H
2O 0.01～0.05g, KH
2PO
40.05～0.2g, NaCl 0.05～0.1g, Soil extract 20～40mL, FeCl
36H
2O0.001～0.01g, Fe-EDTA 0.5～1mL, trace element solution 0.5～1mL and deionized water surplus.
2. according to the described culture media composition of claim 1, it is characterized in that every liter of said culture media composition comprises 0.02～0.05g Na
2SO
3
3. according to the described culture media composition of claim 1, it is characterized in that the composition of 1 liter of said trace element solution comprises: H
3BO
35.5～6.5g, ZnSO
47H
2O 4.0～5.0g, MnCl
2H
2O 0.5～1.0g, (NH
4)
6Mo
7O
244H
2O 0.20～0.40g, CuSO
45H
2O 0.50～1.00g, Co (NO
3)
26H
2O0.10～0.20g and deionized water surplus.
4. according to the described culture media composition of claim 1, it is characterized in that described organic carbon source is one or more in glucose, starch hydrolyzate, bacterium slag treatment solution and the algae-residue treatment solution.
5. according to the described culture media composition of claim 1, it is characterized in that described organic carbon source is bacterium slag treatment solution or algae-residue treatment solution, its content in culture media composition is 30～50g/L
6. according to claim 4 or 5 described culture media compositions, it is characterized in that described bacterium slag is selected from bacterium bacterium slag or yeast bacterium slag.
7. according to the described culture media composition of claim 6, it is characterized in that described bacterium is selected from the Cray Bai Shi pulmonitis strain of anaerobically fermenting, described yeast is selected from candidiasis.
8. according to any described culture media composition of claim 1～7, it is characterized in that described bacterium slag treatment solution makes through following method:
After bacterium or saccharomycetes to make fermentation finish; Behind 60 ℃～80 ℃ boiling 30min～60min; Filter at normal temperatures and obtain bacterium slag filter cake, the alkaline solution that adds 0.01～0.1mol/L then is hydrolyzed, and the solid-to-liquid ratio of hydrolytic process is 1: 10～1: 20; Treatment temp is 15 ℃～30 ℃, and the treatment time is 5d～10d;
Described algae-residue treatment solution prepares through following method: obtain algal biscuit after the broken oil removing of frustule; The alkali lye that adds 0.01～0.1mol/L then is hydrolyzed; The solid-to-liquid ratio of hydrolytic process is 1: 10～1: 20, and treatment temp is 15 ℃～30 ℃, and the treatment time is 5d～10d.
9. according to the described culture media composition of claim 8, it is characterized in that described alkali is selected from NaOH and/or NaHCO
3
10. according to claim 1 or 2 described culture media compositions, it is characterized in that, also comprise tomato extracting solution 5～15mL in every liter of said culture media composition.
11. according to the described culture media composition of claim 10; It is characterized in that; The process for preparation of said tomato extracting solution is: take by weighing 100% Normal juice tomato juice, be mixed with the solution of 100～200g/L concentration with dissolved in distilled water, with its centrifugal solid impurity that goes; Obtain supernatant, be the tomato extracting solution.
12. the method for the little algae of acclimation shaking culture bead is characterized in that, the domestication process has been used any described culture media composition of claim 1～11.
13. a method of cultivating chlorella, this method adopts light autotrophy-heterotrophism mixed culture Chlorella vulgaris, it is characterized in that said method has been used any described culture media composition of claim 1～11.
14. method according to the described cultivation of claim 13 chlorella; It is characterized in that said method comprises: add pH value in the bio-reactor and be 6.0～7.0 any described culture media composition of claim 1～11, cultivate by 5%～15% access Chlorella vulgaris algae kind of working volume; Culture condition is: stirring velocity is 80～100rpm; The mixed gas of bubbling air and carbonic acid gas, air flow are 0.1～1.0vvm, and intensity of illumination is 2000～3000lx; Culture temperature is 25 ℃～32 ℃, and control pH value is less than 10 in the culturing process.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010539185 CN102465098B (en) | 2010-11-05 | 2010-11-05 | Culture medium composition for culturing chlorella |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010539185 CN102465098B (en) | 2010-11-05 | 2010-11-05 | Culture medium composition for culturing chlorella |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102465098A true CN102465098A (en) | 2012-05-23 |
| CN102465098B CN102465098B (en) | 2013-06-19 |
Family
ID=46069224
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010539185 Active CN102465098B (en) | 2010-11-05 | 2010-11-05 | Culture medium composition for culturing chlorella |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102465098B (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103114041A (en) * | 2012-12-19 | 2013-05-22 | 盐城工学院 | Method for rapidly cultivating chlorella |
| CN103396953A (en) * | 2013-08-20 | 2013-11-20 | 甘肃富民生态农业科技有限公司 | Batch feeding method for cultivating chlorella |
| WO2014199220A1 (en) | 2013-06-12 | 2014-12-18 | Solarvest BioEnergy Inc. | Methods of producing algal cell cultures and biomass, lipid compounds and compositions, and related products |
| CN104498362A (en) * | 2014-12-26 | 2015-04-08 | 甘肃德福生物科技有限公司 | Biomass cultivation method for chlorella |
| CN107198038A (en) * | 2017-05-24 | 2017-09-26 | 甘肃凯源生物技术开发中心 | The feed of cultivation health pig prepared by chlorella powder and spirulina powder composition and said composition |
| CN107746809A (en) * | 2017-12-13 | 2018-03-02 | 何红娣 | The method for improving algae bio amount |
| CN107841464A (en) * | 2017-12-15 | 2018-03-27 | 何红娣 | A kind of cultural method of algae |
| CN108004190A (en) * | 2018-01-19 | 2018-05-08 | 何红娣 | Bacillus is used for the method for increasing bead algae biomass |
| CN108085283A (en) * | 2018-01-19 | 2018-05-29 | 何红娣 | A kind of helotism high density Algaculture method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20040101854A (en) * | 2003-05-27 | 2004-12-03 | 학교법인 한양학원 | Development of high density mass culture of freshwater green alga Chlorella without silicate and iron |
| CN101173215A (en) * | 2007-11-01 | 2008-05-07 | 上海交通大学 | Low-phosphor culture medium for chlorella heterotrophy culture |
-
2010
- 2010-11-05 CN CN 201010539185 patent/CN102465098B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20040101854A (en) * | 2003-05-27 | 2004-12-03 | 학교법인 한양학원 | Development of high density mass culture of freshwater green alga Chlorella without silicate and iron |
| CN101173215A (en) * | 2007-11-01 | 2008-05-07 | 上海交通大学 | Low-phosphor culture medium for chlorella heterotrophy culture |
Non-Patent Citations (2)
| Title |
|---|
| WEI DONG 等: "Optimization of Culture Medium for Heterotrophic Chlorella Protothecoids Producing Total Fatty Acids", 《CHEMISTRY & BIOENGINEERING》, vol. 25, no. 3, 31 December 2008 (2008-12-31), pages 35 - 40 * |
| 李兴武 等: "普通小球藻异养-光自养串联培养的培养基", 《过程工程学报》, vol. 6, no. 2, 31 December 2006 (2006-12-31), pages 277 - 280 * |
Cited By (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103114041A (en) * | 2012-12-19 | 2013-05-22 | 盐城工学院 | Method for rapidly cultivating chlorella |
| EP3030647A4 (en) * | 2013-06-12 | 2017-05-03 | Solarvest Bioenergy Inc. | Methods of producing algal cell cultures and biomass, lipid compounds and compositions, and related products |
| JP2020185012A (en) * | 2013-06-12 | 2020-11-19 | ソーラーベスト バイオエナジー インク | Methods of producing algal cell cultures and biomass, lipid compounds and compositions, and related products |
| WO2014199220A1 (en) | 2013-06-12 | 2014-12-18 | Solarvest BioEnergy Inc. | Methods of producing algal cell cultures and biomass, lipid compounds and compositions, and related products |
| AU2014279815B2 (en) * | 2013-06-12 | 2020-10-22 | Solarvest BioEnergy Inc. | Methods of producing algal cell cultures and biomass, lipid compounds and compositions, and related products |
| CN105452442A (en) * | 2013-06-12 | 2016-03-30 | 索拉维斯特生物能源公司 | Methods of producing algal cell cultures and biomass, lipid compounds and compositions, and related products |
| EP3030647A1 (en) * | 2013-06-12 | 2016-06-15 | Solarvest Bioenergy Inc. | Methods of producing algal cell cultures and biomass, lipid compounds and compositions, and related products |
| JP2016523529A (en) * | 2013-06-12 | 2016-08-12 | ソーラーベスト バイオエナジー インク | Algal cell culture medium and biomass, lipid compound and composition, and related product manufacturing method |
| CN103396953B (en) * | 2013-08-20 | 2015-02-25 | 甘肃富民生态农业科技有限公司 | Batch feeding method for cultivating chlorella |
| CN103396953A (en) * | 2013-08-20 | 2013-11-20 | 甘肃富民生态农业科技有限公司 | Batch feeding method for cultivating chlorella |
| CN104498362A (en) * | 2014-12-26 | 2015-04-08 | 甘肃德福生物科技有限公司 | Biomass cultivation method for chlorella |
| CN107198038A (en) * | 2017-05-24 | 2017-09-26 | 甘肃凯源生物技术开发中心 | The feed of cultivation health pig prepared by chlorella powder and spirulina powder composition and said composition |
| CN107198038B (en) * | 2017-05-24 | 2021-01-15 | 河西学院 | Chlorella powder and spirulina powder composition and feed prepared from composition and used for breeding healthy pigs |
| CN107746809A (en) * | 2017-12-13 | 2018-03-02 | 何红娣 | The method for improving algae bio amount |
| CN107746809B (en) * | 2017-12-13 | 2020-04-24 | 杭州渔森农业技术开发有限公司 | Method for increasing algae biomass |
| CN107841464B (en) * | 2017-12-15 | 2020-04-24 | 杭州渔森农业技术开发有限公司 | Algae culture method |
| CN107841464A (en) * | 2017-12-15 | 2018-03-27 | 何红娣 | A kind of cultural method of algae |
| CN108004190B (en) * | 2018-01-19 | 2019-12-06 | 杭州渔森农业技术开发有限公司 | Method for increasing chlorella biomass by using bacillus |
| CN108085283B (en) * | 2018-01-19 | 2019-12-06 | 杭州渔森农业技术开发有限公司 | method for culturing high-density algae through symbiosis of bacteria and algae |
| CN108085283A (en) * | 2018-01-19 | 2018-05-29 | 何红娣 | A kind of helotism high density Algaculture method |
| CN108004190A (en) * | 2018-01-19 | 2018-05-08 | 何红娣 | Bacillus is used for the method for increasing bead algae biomass |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102465098B (en) | 2013-06-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102465098B (en) | Culture medium composition for culturing chlorella | |
| CN103459585B (en) | Process for production of microalgae, cyanobacteria and metabolites thereof | |
| CN103249831A (en) | Method for culturing mixotrophic single-ell algae in the presence of a discontinuous provision of light in the form of flashes | |
| CN102311920B (en) | Culture method for chlorella | |
| CN105441524A (en) | Method for increasing yield of microalgae grease with saccharose as carbon source through co-culture | |
| CN102311921B (en) | Method for culturing chlorella | |
| CN105648023B (en) | A kind of method that microalgae mixed culture prepares grease | |
| Agwa et al. | Heterotrophic cultivation of Chlorella sp. using different waste extracts | |
| Bhateria et al. | Algae as biofuel | |
| Magdaong et al. | Effect of aeration rate and light cycle on the growth characteristics of Chlorella sorokiniana in a photobioreactor | |
| Min et al. | Pilot-scale cultivation of water-net in secondary effluent using an open pond raceway for nutrient removal and bioethanol production | |
| WO2015085631A1 (en) | Method for culturing botryococcus spp. with high yield | |
| CN106554976B (en) | Method for producing microalgae grease by using monoraphidium | |
| CN107937276B (en) | Method for promoting carbon sequestration growth of chlorella by mixing and regulating carbon dioxide and acetic acid | |
| Dwivedi et al. | Valuable product from water hyacinth–review paper | |
| CN102443562B (en) | A kind of substratum and cultural method promoting proliferation of microalgae papidly | |
| CN107841464A (en) | A kind of cultural method of algae | |
| CN103103126A (en) | Production method for lipid through coupled culture of microbes | |
| CN103086582A (en) | Methane preparation method | |
| CN105713934B (en) | A method of producing microalgae grease | |
| CN107746809A (en) | The method for improving algae bio amount | |
| CN108004190A (en) | Bacillus is used for the method for increasing bead algae biomass | |
| CN105713935B (en) | A kind of method of microalgae mixed culture production grease | |
| CN104232559A (en) | Microalgae culturing method and grease producing method | |
| CN109321510B (en) | Application of strigolactone in promoting accumulation of grease of monocladium algae |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |