CN102465098B - Culture medium composition for culturing chlorella - Google Patents
Culture medium composition for culturing chlorella Download PDFInfo
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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 specifically a kind of the high-density high-quality ordinary chlorella culture media composition of cultivating and method of cultivating chlorella of being suitable for.
Background technology
It is longer that people get research history to chlorella, and wherein Chlorella vulgaris (Chlorella vulgaris) is a more algae kind of research, is also to commercially produce at present one of algae kind that generally adopts.
The nutritive ingredients such as chlorella rich in proteins, polysaccharide, unsaturated fatty acids can be used for food, medicine and energy aspect; Can accumulate in a large number lipid acid, some chlorella fatty acid content can account for 30%～60% of dry weight.Utilize the cultivation chlorella to accumulate oil resource, become and utilized at present the most popular research field of solar energy development renewable resources.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 a large amount of manpower and materials, and gets little.
Chlorella is divided into two kinds of autotrophy and heterotrophism according to its nutritional mode.The first 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.Due to CO
2Solubleness is limited in nutrient solution, absorbs inefficiency, and the frustule growth velocity is limited, and mass-producing generally takes later on open pond to put in a suitable place to breed, but easily pollutes and has cell density and hang down and the unfavorable factors such as floor space is large.The second is the heterotrophism training method, exactly the culturing process people for carbon source is provided, such as glucose etc., obviously, although by the heterotrophism carbon source is provided, can increase substantially algae cell density, 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 be subject to the very big restriction of raw material.
Atev.A (Atev A., Manova A.Heterotrophic cultivation of Chlorella vulgaris A2.Dok1.Bolg.Akad.Nauk, (English) 1981,687～690), magnify soldier etc. and (magnify the soldier 34 (5):, Wu Qingyu, the heterotrophism of chlorella cells transforms.Plant physiology circular, 1996,32 (2): 140～144) and Pan Xin etc. (Pan Xin, Li Jianhong wear the research that passes superfine chlorella heterotrophy cultivation.Food science, 2002,23 (4): 28～33) all peculiar chlorella culturing process quality decline problem is set forth.Wherein research is mainly the variation of protein composition in 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, although the content of grease can lower (increasing extraction cost), growth velocity is fast, can increase rapidly biomass; Incomplete culture medium culturing, although throughput rate is slow, high fat content can be offset the deficiency of biomass.Two step training strategies: first utilize the perfect medium heterotrophism to cultivate, its biomass is increased fast, recycle that incomplete substratum autotrophy is cultivated as nitrogen limits or change culture condition such as light intensity, temperature, make frustule accumulate in a large number grease.
CN 200610025618.9 discloses a kind of method of culturing chlorella with high-density and high-quality, and the method comprises dilution and the light autotrophy cultivation three phases of bio-reactor high Density Heterotrophic, high-density algae liquid.The method adds glucose as organic carbon source in the heterotrophism substratum, and cost is higher.
CN200610024004.9 discloses a kind of method of cultivating Chlorella vulgaris, and the method adopts the smooth autotrophy series connection of heterotrophism one to cultivate Chlorella vulgaris.The method heterotrophism is cultivated the culture media composition of employing basically by KNO
3, glucose and a small amount of inorganic salt, trace element and water forms.Be to have added equally the more expensive glucose of price as organic carbon source in the heterotrophism substratum in the method.
The document that above-mentioned difference is cultivated chlorella improves chlorella cultivation quality and all only refers to protein and chlorophyllous content in frustule.Especially CN200610024004.9, although cultivate the composition composition by optimizing chlorella, do not mention that this composition cultivation chlorella is to the effect of improving of chlorella fat content.
Therefore, for when reaching the cultivation chlorella and utilize the cheap carbon source high-density growth, can also obtain the lipid acid accumulative effect of high level in present research process, must start with from the minimum medium composition.Be necessary to solve the cultivation composition problem of the high oil-containing quality training of chlorella heterotrophy high-density.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of culture media composition for cultivating 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 achieving the above object, the invention provides a kind of culture media composition for cultivating Chlorella vulgaris, described substratum consists essentially of Na
2SO
3, organic carbon source and a small amount of inorganic salt, trace element and water.
In one embodiment of the invention, the composition of every liter of described 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 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 is also to be added in 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 Bacteria 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 by bacterium slag or algae-residue are carried out pre-treatment.The pretreatment process of described bacterium slag is: after bacterium or saccharomycetes to make fermentation finish, after 60 ℃～80 ℃ boiling 30min～60min, filter at normal temperatures and obtain bacterium slag filter cake, then add the diluted alkaline of 0.01～0.1mol/L to be hydrolyzed, diluted alkaline used 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 described algae-residue is: obtain algal biscuit after the broken oil removing of frustule, then add the alkali lye of 0.01～0.1mol/L to be 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.
In described substratum, glucose concn is 10～15g/L, and described 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 100% Normal juice tomato juice, be mixed with the solution of 100～200g/L concentration with the distilled water dissolving, with its centrifugal solid impurity that goes, obtain supernatant liquor, be the tomato extracting solution.
The present invention also provides a kind of the domestication to cultivate the method for raising together with the little algae of bead simultaneously, has 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, the autotrophy chlorella is carried out the domestication of heterotrophism condition cultivate, and used medium is above-mentioned substratum.Heterotrophism domestication culture condition is that lower 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 under the heterotrophism condition exponential phase algae liquid dilution spread on the solid plate that SE substratum (being minimum medium known in the art) is done, low light condition is cultivated 10d～20d, obtaining some single algaes falls, this single algae is fallen after picking, draw and cultivate in SE solid slant culture base, after growing, 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, Bacteria 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.
In the situation that some embodiment, the present invention also provides a kind of method of cultivating chlorella, and the method adopts light autotrophy-little algae of heterotrophism mixed culture bead, it is characterized in that, described method has adopted culture media composition of the present invention.
The method of described cultivation chlorella comprises: adding the pH value in the bio-reactor is 6.0～7.0 above-mentioned substratum, 5%～15% access Chlorella vulgaris algae kind by working volume is cultivated, culture condition is: stirring velocity is 80～100rpm, pass into the mixed gas of air and carbonic acid gas, air flow is 0.1～1.0vvm, intensity of illumination is 2000～3000lx, and culture temperature is 25 ℃～32 ℃, controls the pH value in culturing process less than 10.
After being mixed with culture media composition, regulating as required the pH value to 6.0 of described culture media composition～7.0, and sterilized 30～60 minutes under 121 ℃～130 ℃.
Compare with existing chlorella substratum, culture media composition of the present invention has following features:
1, owing to having 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 shorter culture cycle reach the level of high-density culture, namely can realize the high-density culture of chlorella, than nothing Na
2SO
3During interpolation, biomass can improve 80%～140%.
2, Na
2SO
3With being used in conjunction with of tomato extracting solution, micro-mother liquor, make Chlorella vulgaris more easily utilize organic carbon source to grow, and the oil and fat accumulation to self also has larger promoter action in later stage, to be fat content cultivate to compare with heterotrophism the quality that can make Chlorella vulgaris has a more substantial increase, and reached the level when the light autotrophy is cultivated.
3, can add cheap bacterium slag or algae-residue treatment solution as organic carbon source, such organic carbon source and Na in culture media composition of the present invention
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
At first (this substratum is known in the art to adopt the SE substratum of reporting in document, can be used for chlorella grows under the light autotrophic condition, realize the heterotrophism cultivation by adding carbon source), it is to use in the foregoing invention content to remove Na in substratum that the SE substratum is revised slightly
2SO
3With the organic carbon source composition.Cultivate with this amended SE substratum the chlorella that sets out, although this chlorella cells growth of setting out can realize cultivating in the SE substratum, when cultivating end, frustule concentration is not high, and the fat content in frustule is also lower.Illustrate that this substratum needs to be optimized, could realize the purpose that high-density and high-quality is cultivated.
The present invention is take this SE substratum as the basis, by adding therein Na
2SO
3And organic carbon source, change wherein organic carbon source action effect, at carbon source nitrogenous source and Na
2SO
3Under effect, and other constituent contents in the adjustment substratum, comprise and add the tomato extracting solution, the chlorella that obtained to be applicable to set out is optimized the substratum of the mutagenesis chlorella mixed culture that obtains by 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 unit frond fat content, and the biomass indexes of described frond is frond dry weight in the unit nutrient solution.Those skilled in the art can adopt routine techniques that described 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 by embodiment.Unless separately have describedly, the concentration of the substance described in the inventive method and embodiment is all take the substratum cumulative volume as benchmark, and namely the content of substance accounts for per-cent or mole number or the quality etc. of nutrient solution preparation total nutrient solution volume after completing.
Embodiment 1
Add 100mL substratum as follows in the 250mL shaking flask, inoculative proportion is 10% (be seed liquor volume account for volume of culture 10%), wherein seed is the chlorella through domestication, heterotrophism is cultivated (liquid amount 100mL, temperature 30 degree, shaking speed 100rpm) 48 hours, result recorded cell density and is 14.24g/L to the maximum, and fat content is 30.8% (in cell, total oil quantity accounts for the percentage ratio of total dry cell weight).
Every liter of substratum forms:
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 formula: 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 are consistent with embodiment 1.The cell density that the cultivation results analysis records only has 6.11g/L, and the content that grease accounts for dry cell weight also only has 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
3Under existence, chlorella more easily utilizes organic carbon source to grow, and the oil and fat accumulation in self is also had larger promoter action in later stage.
Embodiment 2
Add 100mL substratum as follows in the 250mL shaking flask, inoculative proportion is 10% (be seed liquor volume account for volume of culture 10%), wherein seed is the chlorella through the heterotrophism domestication, heterotrophism is cultivated (liquid amount 100mL, temperature 30 degree, shaking speed 100rpm) 48 hours, result recorded cell density and is 19.41g/L to the maximum, and fat content is 35.2% (in cell, total oil quantity accounts for the percentage ratio of total dry cell weight).
Wherein bacterium slag treatment solution is to obtain by bacterium or yeast bacterium slag being hydrolyzed process.Preferred Cray Bai Shi pneumobacillus wherein, it is take glycerine as unique substrate, under anaerobic producing 1,3-propanediol through fermentation.After fermentation ends, after 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 preparation culture media composition.
Every liter of substratum is composed 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 formula: 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 substratum
2SO
3This component, culture condition and process are consistent with embodiment 2.The cell density that the cultivation results analysis records only has 8.11g/L, and the content that grease accounts for dry cell weight also only has 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
3Under existence, especially in the situation that use bacterium slag pretreatment fluid, chlorella more easily utilizes organic carbon source to grow, and the oil and fat accumulation in self is also had larger promoter action in later stage.
The simultaneously change of nitrogenous source and trace element also can be played certain promoter action to the cultivation of chlorella, and their acting in conjunction improves culture effect.
Embodiment 3
Add 100mL substratum as follows in the 250mL shaking flask, inoculative proportion is 10% (be seed liquor volume account for volume of culture 10%), seed used is from Chlorella vulgaris, this chlorella is not through heterotrophism domestication culturing process, directly its heterotrophism was cultivated (liquid amount 100mL, temperature 30 degree, shaking speed 100rpm) 48 hours, result records cell density and is 11.30g/L to the maximum, and fat content is 28.6% (in cell, total oil quantity accounts for the percentage ratio of total dry cell weight).
Every liter of substratum is composed 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 formula: 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, algae kind used is from Chlorella vulgaris, and this chlorella through heterotrophism domestication culturing process, is not directly cultivated its heterotrophism, and culture condition and process are consistent with embodiment 3.The cell density that the cultivation results analysis records only has 4.26g/L, and the content that grease accounts for dry cell weight also only has 25.4%.
Can find out according to above-mentioned experimental result, under same basic medium condition, use Na
2SO
3Be used in conjunction with culture effect obvious effect to Chlorella vulgaris with micro-mother liquor, Na is being arranged
2SO
3Under existence, chlorella more easily utilizes organic carbon source to grow, and the oil and fat accumulation in self is also had larger promoter action in later stage.
Compare with 2 with embodiment 1, after chlorella is tamed by heterotrophism, more can effectively utilize organic carbon source in the situation that heterotrophism and autotrophic condition all possess, the domestication chlorella can more effectively be realized mixed cultivation process.
Embodiment 4
Add 100mL substratum as follows in the 250mL shaking flask, inoculative proportion is 10% (be seed liquor volume account for volume of culture 10%), wherein seed is the chlorella through the heterotrophism domestication, heterotrophism is cultivated (liquid amount 100mL, temperature 30 degree, shaking speed 100rpm) 48 hours, result recorded cell density and is 20.30g/L to the maximum, and fat content is 32.8% (in cell, total oil quantity accounts for the percentage ratio of total dry cell weight).
Wherein the treatment process of bacterium slag treatment solution is with embodiment 2.
Every liter of substratum is composed 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 formula: 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 100% Normal juice tomato juice, be mixed with the solution of 150g/L concentration with the distilled water dissolving, with its centrifugal solid impurity that goes, obtain supernatant liquor, be the tomato extracting solution.
Embodiment 5
add 100mL substratum as follows in the 250mL shaking flask, inoculative proportion is 10% (be seed liquor volume account for volume of culture 10%), wherein seed is the chlorella through the heterotrophism domestication, carry out mixed culture (liquid amount 100mL, temperature 30 degree, shaking speed 100rpm, pass into the mixed gas of air and carbonic acid gas, air flow is 0.5vvm, intensity of illumination is 3000lx, control the pH value in culturing process less than 10) 48 hours, result records cell density and is 22.15g/L to the maximum, fat content is 38.0% (in cell, total oil quantity accounts for the percentage ratio of total dry cell weight).
Wherein the treatment process of bacterium slag treatment solution is with embodiment 2.
Every liter of substratum is composed 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 formula: 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 amount ranges, Na is being arranged
2SO
3Under existence, especially in the situation that use bacterium slag pretreatment fluid, chlorella more easily utilizes organic carbon source to grow, and also has larger promotion to create a bad precedent to the oil and fat accumulation in self in later stage.And by embodiment 5 as can be known, this domestication chlorella can be implemented heterotrophism and autotrophy mixed cultivation process under the composition culture medium condition, and the cultivation quality of chlorella has all obtained corresponding raising.
Embodiment 6
In this embodiment, Chlorella vulgaris is carried out the heterotrophism domestication.The algae kind of setting out is a kind of autotrophy chlorella, the autotrophy chlorella is carried out the domestication of heterotrophism condition cultivate:
Every liter of substratum is composed 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 formula: 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 domestication culture condition is that lower 30 ℃ of lucifuge situation, 120rmp, airbath shaking table are cultivated 10d; After 8 batches of heterotrophism domestications, heterotrophism domestication algae kind is carried out separation and purification: will cultivate under the heterotrophism condition exponential phase algae liquid dilution spread on the solid plate that SE substratum (being known minimum medium in this field) is done, low light condition is cultivated 15d, obtaining some single algaes falls, this single algae is fallen after picking, draw and cultivate in SE solid slant culture base, after growing, can obtain the purebred algae kind of raising together with, namely tame chlorella.
Can be determined 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 in the situation of chlorella as the chlorella algae kind of setting out after this heterotrophism substratum is tamed, the domestication chlorella is cultivated the rear growth that obtains of composition cultivation in this optimization and all improves a lot with the cultivation quality of throughput than original Chlorella vulgaris.Can find out by embodiment 5 simultaneously, the said composition domestication obtains chlorella after passing through heterotrophism autotrophy mixed culture, and the cultivation quality of chlorella is the most outstanding.
Claims (11)
1. a culture media composition that is used for cultivating chlorella, is characterized in that, every liter of described culture media composition comprises:
Na
2SO
30.02～0.05g, 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 20～40mL, FeCl
36H
2O 0.001～0.01g, Fe-EDTA 0.5～1mL, trace element solution 0.5～1mL and deionized water surplus;
Wherein the composition of 1 liter of described trace element solution comprises: H
3BO
35.5～6.5g, ZnSO
4.7H
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.
2. according to culture media composition claimed in claim 1, it is characterized in that, described organic carbon source is one or more in glucose, starch hydrolyzate, bacterium slag treatment solution and algae-residue treatment solution.
3. according to culture media composition claimed in claim 1, it is characterized in that, described organic carbon source is bacterium slag treatment solution or algae-residue treatment solution, and its content in culture media composition is 30～50g/L.
4. according to the described culture media composition of claim 2 or 3, it is characterized in that, described bacterium slag is selected from Bacteria slag or yeast bacterium slag.
5. according to culture media composition claimed in claim 4, it is characterized in that, described bacterium is selected from the Cray Bai Shi pulmonitis strain of anaerobically fermenting, and described yeast is selected from candidiasis.
6. according to culture media composition claimed in claim 3, it is characterized in that, described bacterium slag treatment solution makes by the following method:
After bacterium or saccharomycetes to make fermentation finish, after 60 ℃～80 ℃ boiling 30min～60min, filter at normal temperatures and obtain bacterium slag filter cake, then add the alkaline solution of 0.01～0.1mol/L to be hydrolyzed, the solid-liquid mass 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 by the following method: obtain algal biscuit after the broken oil removing of frustule, then add the alkali lye of 0.01～0.1mol/L to be hydrolyzed, the solid-liquid mass ratio of hydrolytic process is 1: 10～1: 20, and treatment temp is 15 ℃～30 ℃, and the treatment time is 5d～10d.
7. according to culture media composition claimed in claim 6, it is characterized in that, described alkali is selected from NaOH and/or NaHCO
3
8. according to culture media composition claimed in claim 1, it is characterized in that, also comprise tomato extracting solution 5～15mL in every liter of described culture media composition; The process for preparation of described tomato extracting solution is: take 100% Normal juice tomato juice, be mixed with the solution of 100～200g/L concentration with the distilled water dissolving, with its centrifugal solid impurity that goes, obtain supernatant liquor, be the tomato extracting solution.
9. tame the method for cultivating the little algae of bead for one kind, it is characterized in that, the domestication process has been used the described culture media composition of claim 1～8 any one.
10. method of cultivating chlorella, the method adopts light autotrophy-heterotrophism mixed culture Chlorella vulgaris, it is characterized in that, and described method has been used the described culture media composition of claim 1～8 any one.
11. the method according to cultivation chlorella claimed in claim 10, it is characterized in that, described method comprises: adding the pH value in the bio-reactor is 6.0～7.0 the described culture media composition of claim 1～8 any one, 5%～15% access Chlorella vulgaris algae kind by working volume is cultivated, culture condition is: stirring velocity is 80～100rpm, pass into the mixed gas of air and carbonic acid gas, air flow is 0.1～1.0vvm, intensity of illumination is 2000～30001x, culture temperature is 25 ℃～32 ℃, controls the pH value in culturing process less than 10.
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| BR112015031263B1 (en) * | 2013-06-12 | 2022-10-11 | Solarvest Bioenergy Inc | METHODS TO PRODUCE AN ALGAE BIOMASS OR ALGAE CELL CULTURE AND AT LEAST ONE LIPID COMPOUND |
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| 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 |
| 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 |
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| 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 |
| 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 |
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