CN104593314A - Method for improving micro algae growth rate and cell fat content employing high-salinity domestication - Google Patents

Method for improving micro algae growth rate and cell fat content employing high-salinity domestication Download PDF

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CN104593314A
CN104593314A CN201410854241.2A CN201410854241A CN104593314A CN 104593314 A CN104593314 A CN 104593314A CN 201410854241 A CN201410854241 A CN 201410854241A CN 104593314 A CN104593314 A CN 104593314A
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程军
岑可法
刘建忠
周俊虎
张彦威
周志军
王智化
黄镇宇
杨卫娟
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Zhejiang University ZJU
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Abstract

The invention relates to a biomass energy utilization technology, and aims at providing a method for improving micro-algae growth rate and cell fat content employing high-salinity domestication. The method comprises the following steps: inoculating a micro algae liquid into a culture medium of a triangular flask and carrying out enlarge cultivation; and carrying out gradient increase according to 0.3% salinity until inoculating algae species into the culture medium of which the initial salinity is 2.9%-3.1%, and continuously cultivating for 3-5 generations, so as to obtain the high-fat content algae species capable of rapidly growing. By virtue of a high-salinity gradient domestication method, a carbon-nitrogen metabolism pathway of micro-algae cells and the synthesis pathway are reconstructed; the metabolic pathway change of proline, ABC transportprotein, proteasome and glutathione is relatively large; and the key biological enzyme expression quantities of acetyl-coa carboxylase (ACCase), beta-ketoacyl-ACP synthase I and II and the like are regulated upwards, so that the micro algae growth rate and the cell oil-enrichment capacity are effectively improved.

Description

High salinity domestication improves the method for micro algae growth speed and cell grease content
Technical field
The invention relates to biomass energy utilization technologies, particularly high salinity domestication improves the method for micro algae growth speed and cell grease content.
Background technology
Utilize marine microalgae to absorb the clean reproducible liquid fuels such as sun power preparing biological diesel oil, for solving, the fossil energy worsening shortages such as oil and the serious crisis of Air–pollution From Combustion and contradiction are significant.Micro algae growth is very fast, and physiological metabolism is powerful, and photosynthetic efficiency is very high, utilizes seawater resources and absorbs a large amount of CO 2carry out photosynthesis, by Biochemical processes synthesis enrichment grease (Sheehan et al., 1998 that green is clean; Huntley et al., 2007), can transform and produce the higher-grade biofuel of calorific value up to 41MJ/kg, therefore, it is possible to substitute petrifaction diesel to be applied to the vehicles, and not discharge SO when burning 2deng dusty gas.According to American Studies mechanism prediction (Benemann et al., 2008), the potentiality of the annual production biofuel of energy microalgae of every square kilometre are 36000-150000 bucket/years, its oil production potential is 144-600 times of soybean oil (250 barrels/year), it is 30-125 times of jatropha curcas oil (1200 barrels/year), be 15-41 times of plam oil (3600 barrels/year), therefore micro-algae is a kind of very promising biodiesel raw material.
But exist very large conflicting between micro algae growth speed and grease enrichment, when the micro-algae of the overwhelming majority reaches optimum growh speed, in its cell, fat content is lower; And manual control growth conditions make micro-algae per dry wt fat content increase time, the growth velocity of micro-algae and solar conversion efficiency is then made obviously to reduce (Dempster et al., 1998), so fast and that grease yield the is high new algae kind of exploitation growth velocity is key point.Advantage Strain selection method mainly contains three aspects: (1) natural Strain selection: USDOE has definitely confirmed that the algae fat content of diatom is relative to other algae kind fat content higher (Sheehan et al., 1998; ), algae fat content 10-30% under natural condition; Photosynthetic productivity through the micro-algae of domestication some of them reaches 50gm -2d -1, oil length reaches 80%.(2) cell engineering: the people such as Zaslavskaia (Zaslavskaia et al., 2001) by genetic engineering technique, the gene of encodes glucose transporter being introduced a kind of cannot carrying out, in the diatom Phaeodactylum tricomutum cell of heterotrophic growth, can utilize Exogenous Glucose carry out heterotrophic growth and reach the cell density of 100g/L in the dark; (3) macroscopic growth factor regulation and control: the main research institution such as renewable energy source National Laboratory of the U.S., University of California Berkeley (Sheehan et al., 1998; Hu et al., 2008) research and develop the grease-contained diatom of multiple richness and green alga in laboratory conditions, under the condition of substratum nitrogen stress, scarce silicon, the highest fat content reaches 40-66%, is 3-12 times of microalgae grease content under natural condition.
At present morely in the world trend towards Natural Selection and transgenic approach (Jarvis et al., 2008), also have great interest to mutagenesis approach but rarely seen research report.The people such as Ben-Amotz (Ben-Amotz et al., 1985) confirm that some algae kind finds that there is very high fat content under high salinity, the people such as Cohen (Cohen et al., 1988) also find that the Porphyridium Cruentum frustule of growth under high salinity contains more a high proportion of linolic acid and arachidonic acid, both are the main fatty acids forming store triglycerides three ester together with Palmiticacid, but can high salinity domestication improve micro algae growth speed still little bibliographical information at present.Therefore how to adopt high salinity gradient acclimation method to select the algae kind that growth velocity is fast and fat content is high be a technical barrier, significant for exploitation micro-algae new forms of energy reduction production cost.
Summary of the invention
Main purpose of the present invention is to overcome deficiency of the prior art, provides a kind of high salinity to tame the method improving micro algae growth speed and cell grease content.For solving the problems of the technologies described above, solution of the present invention is:
There is provided high salinity to tame the method improving micro algae growth speed and cell grease content, specifically comprise the steps:
(1) be 5% ~ 10% by the triangular flask of micro-algae liquid inoculation to 1L by inoculum size, in triangular flask, fill the substratum of 800mL in advance; Under the condition of constant temperature 20 ~ 25 DEG C, every day successively illumination 12h and dark 12h, pass into air amplification culture 10 ~ 15 days, and intensity of illumination during illumination is 3500 ~ 4000lux;
(2) get the algae kind after enlarged culturing and be inoculated into capacity in 400mL cylindrical bottle with 5 ~ 10% inoculum sizes, substratum in cylindrical bottle is 250 ~ 350mL, initial salinity is 0%, pass into the air of 70 ~ 90mL/min, cultivate after 8 ~ 12 days with the inoculum size of 5 ~ 10% turn be linked into salinity be in the substratum of 0.2 ~ 0.4% cultivate 8 ~ 12 days;
(3) get the algae liquid after cultivating in step (2) with 5 ~ 10% inoculum size turn that to be linked into salinity be cultivate 8 ~ 12 days in the substratum of 0.5 ~ 0.7%, be that 0.3% repetition above-mentioned steps is until micro-algae being seeded to salinity is in the substratum of 2.9 ~ 3.1% by salinity gradient;
(4) by above-mentioned algae kind in salinity be 2.9 ~ 3.1% substratum in cultured continuously 3 ~ 5 generation, namely obtain the high fat content algae kind that can grow fast.
In the present invention, the component ratio of described substratum is: 0.15g NaHCO 3, 0.02g KH 2pO 4, 0.027gVB 1, 1.5 × 10-6g VB 12, 0.2g Na 2siO 39H 2o, 1.0g NaNO 3, 0.0005g Biotin, 1mL trace element and 980mL artificial seawater.
In the present invention, the component ratio of described trace element is: containing 4.35g Na in 1000mL distilled water 2eDTA, 7.3mg Na 2moO 42H 2o, 12mgCoCl 26H 2o, 3.9g FeC 6h 5o 75H 2o, 10mgCuSO 45H 2o, 23mg ZnSO 4, 178mg MnCl 24H 2o and 600mg H 3bO 3.
In the present invention, the component ratio (salinity=3.0%) of described artificial seawater is: containing 21.2157g NaCl, 3.407g Na in 1000mL distilled water 2sO 4, 0.3577g KCl, 9.3042g MgCl 26H 2o, 1.3044g CaCl 2, 0.0862g KBr, 0.0226g H 3bO 3, 0.2760g NaF and 0.0219g SrCl 26H 2o.
In the present invention, the culture condition of described step (2) ~ (4) is: artificial greenhouse 20 ~ 30 DEG C, and initial pH value is 8 ~ 10, and every day is illumination 12h and dark 12h successively, and intensity of illumination during illumination is 6000 ~ 10000lux.
In the present invention, described micro-algae liquid refers to: the transgenic alga kind of screening the natural algae kind obtained, algae kind mutant that physics and chemistry behavior obtains or obtaining through improvement of genes from physical environment.
Compared with prior art, the invention has the beneficial effects as follows:
Adopt high salinity gradient acclimation method, C N metabolism approach and the oil synthesis approach of microalgae cell are rebuild, the metabolic pathway of proline(Pro), abc transport albumen, proteasome, gsh changes greatly, the key organism expression of enzymes amounts such as acetyl-CoA carboxylase (ACCase), β-one acyl-ACP synthase I and II raise, thus effectively improve the ability of micro algae growth speed and cell enrichment grease.After the domestication of high salinity gradient, micro algae growth speed adds 53.8%, and in cell, fat content increases by 130.5%.
Accompanying drawing explanation
Fig. 1 is process flow sheet of the present invention.
Embodiment
Below in conjunction with accompanying drawing and embodiment, the present invention is described in further detail:
A kind of high salinity domestication as shown in Figure 1 improves the method for micro algae growth speed and cell grease content, specifically comprises the steps:
(1) be 5% ~ 10% by the triangular flask (substratum of 800mL) of micro-algae liquid inoculation to 1L by inoculum size, described original algae liquid comprises from physical environment, to screen the natural algae kind obtained, algae kind mutant that physics and chemistry behavior obtains, the transgenic alga kind that obtains through improvement of genes.Air enlarged culturing is passed into 10 ~ 15 days under the condition of constant temperature 20 ~ 25 DEG C, intensity of illumination 3500 ~ 4000lux, every day successively illumination 12h dark 12h.
(2) get the algae kind after enlarged culturing and be inoculated into capacity in 400mL cylindrical bottle with 5 ~ 10% inoculum sizes, substratum in cylindrical bottle is 250 ~ 350mL, initial salinity is 0%, pass into the air of 70 ~ 90mL/min, it is 20 ~ 30 DEG C in temperature, intensity of illumination 6000 ~ 10000lux, Light To Dark Ratio 12h:12h, initial pH value is cultivate after 8 ~ 12 days under the condition of 8 ~ 10, inoculum size with 5 ~ 10% turns that to access to salinity be in the substratum of 0.2 ~ 0.4%, continues with same CMC model 8 ~ 12 days.
Described substratum consists of: 0.15g NaHCO 3, 0.02g KH 2pO 4, 0.027g VB 1, 1.5 × 10 -6g VB 12, 0.2g Na 2siO 39H 2o, 1.0g NaNO 3, 0.0005g Biotin, 1mL trace element and 980mL salinity be 0.2 ~ 3.0% artificial seawater or deionized water.Wherein, described trace element consists of: containing 4.35gNa in 1000mL distilled water 2eDTA, 7.3mg Na 2moO 42H 2o, 12mgCoCl 26H 2o, 3.9g FeC 6h 5o 75H 2o, 10mgCuSO 45H 2o, 23mg ZnSO 4, 178mg MnCl 24H 2o and 600mg H 3bO 3.Described artificial seawater formula (salinity=3.0%) is: containing 21.2157g NaCl, 3.407g Na in 1000mL distilled water 2sO 4, 0.3577g KCl, 9.3042g MgCl 26H 2o, 1.3044g CaCl 2, 0.0862g KBr, 0.0226g H 3bO 3, 0.2760g NaF and 0.0219g SrCl 26H 2o.
(3) get the algae liquid after cultivating in step (2) with 5 ~ 10% inoculum size turn that to access to salinity be in the substratum of 0.5 ~ 0.7%, it is 20 ~ 30 DEG C in temperature, intensity of illumination 6000 ~ 10000lux, Light To Dark Ratio 12h:12h, initial pH value is cultivate 8 ~ 12 days under the condition of 8 ~ 10.Above-mentioned steps is repeated until micro-algae being seeded to salinity is in the substratum of 2.9 ~ 3.1% by salinity increment 0.3%.
(4) by above-mentioned algae kind in salinity be 2.9 ~ 3.1% substratum in, temperature is 20 ~ 30 DEG C, intensity of illumination 6000 ~ 10000lux, Light To Dark Ratio 12h:12h, initial pH value be under the condition of 8 ~ 10 cultivate 3 ~ 5 generations.Namely the high fat content algae kind that can grow fast is obtained.
The following examples can make professional and technical personnel's comprehend the present invention of this specialty, but do not limit the present invention in any way.
Embodiment 1
(1) be 5% by the triangular flask (substratum of 800mL) of rhombus algae liquid inoculation to 1L by inoculum size, at constant temperature 20 DEG C, intensity of illumination 3500lux, under the condition of dark 12h every day successively illumination 12h, pass into air amplification culture 10 days.
(2) get the algae kind after enlarged culturing and be inoculated into capacity in 400mL cylindrical bottle with 5% inoculum size, substratum in cylindrical bottle is 250mL, initial salinity is 0%, pass into the air of 70mL/min, be 25 DEG C in temperature, intensity of illumination 8000lux, Light To Dark Ratio 12h:12h, initial pH value is cultivate after 8 days under the condition of 9, and the inoculum size with 5 ~ 10% turns that to access to salinity be cultivate 8 days in the substratum of 0.2%.
Described substratum consists of: 0.15g NaHCO 3, 0.02g KH 2pO 4, 0.027g VB 1, 1.5 × 10 -6g VB 12, 0.2g Na 2siO 39H 2o, 1.0g NaNO 3, 0.0005g Biotin, 1mL trace element and 980mL salinity be the artificial seawater of 0.2%.Described trace element consists of: containing 4.35g Na in 1000mL distilled water 2eDTA, 7.3mgNa 2moO 42H 2o, 12mgCoCl 26H 2o, 3.9g FeC 6h 5o 75H 2o, 10mg CuSO 45H 2o, 23mgZnSO 4, 178mg MnCl 24H 2o and 600mg H 3bO 3.Described artificial seawater formula is: containing 21.2157g NaCl, 3.407g Na in 1000mL distilled water 2sO 4, 0.3577g KCl, 9.3042g MgCl 26H 2o, 1.3044g CaCl 2, 0.0862g KBr, 0.0226g H 3bO 3, 0.2760g NaF and 0.0219g SrCl 26H 2o.
(3) get the algae liquid in step (2) with 5% inoculum size turn that to access to salinity be in the substratum of 0.5%, be 20 DEG C in temperature, intensity of illumination 6000lux, Light To Dark Ratio 12h:12h, initial pH value is cultivate 8 days under the condition of 9.Be that 0.3% repetition above-mentioned steps is until micro-algae being seeded to salinity is in the substratum of 2.9% by salinity increment.
(4) by above-mentioned algae kind in salinity be 2.9% substratum in, temperature is 20 DEG C, intensity of illumination 6000lux, Light To Dark Ratio 12h:12h, and initial pH value is cultivated for 3 generations under the condition of 9.Namely obtain micro algae growth speed and reach the algae kind that 1.8g/L and cell grease content reach 50%.
Embodiment 2
(1) be 8% by the triangular flask (substratum of 800mL) of chlorella liquid inoculation to 1L by inoculum size, at constant temperature 23 DEG C, intensity of illumination 3750lux, under the condition of dark 12h every day successively illumination 12h, pass into air amplification culture 12 days.
(2) get the algae kind after enlarged culturing and be inoculated into capacity in 400mL cylindrical bottle with 8% inoculum size, substratum in cylindrical bottle is 300mL, initial salinity is 0%, pass into the air of 80mL/min, be 25 DEG C in temperature, intensity of illumination 8000lux, Light To Dark Ratio 12h:12h, initial pH value is cultivate after 10 days under the condition of 8, and the inoculum size with 8% turns that to access to salinity be cultivate 10 days in the substratum of 0.3%.
Described substratum consists of: 0.15g NaHCO 3, 0.02g KH 2pO 4, 0.027g VB 1, 1.5 × 10 -6g VB 12, 0.2g Na 2siO 39H 2o, 1.0g NaNO 3, 0.0005g Biotin, 1mL trace element and 980mL salinity be the artificial seawater of 0.3%.Described trace element consists of: containing 4.35g Na in 1000mL distilled water 2eDTA, 7.3mgNa 2moO 42H 2o, 12mgCoCl 26H 2o, 3.9g FeC 6h 5o 75H 2o, 10mg CuSO 45H 2o, 23mgZnSO 4, 178mg MnCl 24H 2o and 600mg H 3bO 3.Described artificial seawater formula is: containing 21.2157g NaCl, 3.407g Na in 1000mL distilled water 2sO 4, 0.3577g KCl, 9.3042g MgCl 26H 2o, 1.3044g CaCl 2, 0.0862g KBr, 0.0226g H 3bO 3, 0.2760g NaF and 0.0219g SrCl 26H 2o.
(3) get the algae liquid in step (2) with 8% inoculum size turn that to access to salinity be in the substratum of 0.6% be 25 DEG C in temperature, intensity of illumination 8000lux, Light To Dark Ratio 12h:12h, initial pH value is cultivate 10 days under the condition of 8.Be that 0.3% repetition above-mentioned steps is until micro-algae being seeded to salinity is in the substratum of 3.0% by salinity increment.
(4) by above-mentioned algae kind in salinity be 3.0% substratum in, temperature is 25 DEG C, intensity of illumination 8000lux, Light To Dark Ratio 12h:12h, and initial pH value is cultivated for 4 generations under the condition of 8.Namely obtain micro algae growth speed and reach the algae kind that 3.2g/L and cell grease content reach 55%.
Embodiment 3
(1) be 10% by the triangular flask (substratum of 800mL) of micro-plan ball algae liquid inoculation to 1L by inoculum size, under the condition of constant temperature 25 DEG C, intensity of illumination 4000lux, every day successively illumination 12h dark 12h, pass into air enlarged culturing 15 days.
(2) get the algae kind after enlarged culturing and be inoculated into capacity in 400mL cylindrical bottle with 10% inoculum size, substratum in cylindrical bottle is 350mL, initial salinity is 0%, pass into the air of 90mL/min, be 29 DEG C in temperature, intensity of illumination 9000lux, Light To Dark Ratio 12h:12h, initial pH value is cultivate after 12 days under the condition of 10, and the inoculum size with 10% turns that to access to salinity be cultivate 12 days in the substratum of 0.4%.
Described substratum consists of: 0.15g NaHCO 3, 0.02g KH 2pO 4, 0.027g VB 1, 1.5 × 10 -6g VB 12, 0.2g Na 2siO 39H 2o, 1.0g NaNO 3, 0.0005g Biotin, 1mL trace element and 980mL salinity be the artificial seawater of 0.4%.Described trace element consists of: containing 4.35g Na in 1000mL distilled water 2eDTA, 7.3mgNa 2moO 42H 2o, 12mgCoCl 26H 2o, 3.9g FeC 6h 5o 75H 2o, 10mg CuSO 45H 2o, 23mgZnSO 4, 178mg MnCl 24H 2o and 600mg H 3bO 3.Described artificial seawater formula is: containing 21.2157g NaCl, 3.407g Na in 1000mL distilled water 2sO 4, 0.3577g KCl, 9.3042g MgCl 26H 2o, 1.3044g CaCl 2, 0.0862g KBr, 0.0226g H 3bO 3, 0.2760g NaF and 0.0219g SrCl 26H 2o.
(3) get the algae liquid in step (2) with 10% inoculum size turn that to access to salinity be in the substratum of 0.7%, be 30 DEG C in temperature, intensity of illumination 10000lux, Light To Dark Ratio 12h:12h, initial pH value is cultivate 12 days under the condition of 10.Be that 0.3% repetition above-mentioned steps is until micro-algae being seeded to salinity is in the substratum of 3.1% by salinity increment.
(4) by above-mentioned algae kind in salinity be 3.1% substratum in, temperature is 30 DEG C, intensity of illumination 10000lux, Light To Dark Ratio 12h:12h, and initial pH value is cultivated for 5 generations under the condition of 10.Namely obtain micro algae growth speed and reach the algae kind that 2.5g/L and cell grease content reach 48%.
Finally, it should be noted that above what enumerate is only specific embodiments of the invention.Obviously, the invention is not restricted to above embodiment, a lot of distortion can also be had.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.

Claims (6)

1. high salinity domestication improves the method for micro algae growth speed and cell grease content, it is characterized in that, specifically comprises the steps:
(1) be 5% ~ 10% by the triangular flask of micro-algae liquid inoculation to 1L by inoculum size, in triangular flask, fill the substratum of 800mL in advance; Under the condition of constant temperature 20 ~ 25 DEG C, every day successively illumination 12h and dark 12h, pass into air amplification culture 10 ~ 15 days, and intensity of illumination during illumination is 3500 ~ 4000lux;
(2) get the algae kind after enlarged culturing and be inoculated into capacity in 400mL cylindrical bottle with 5 ~ 10% inoculum sizes, substratum in cylindrical bottle is 250 ~ 350mL, initial salinity is 0%, pass into the air of 70 ~ 90mL/min, cultivate after 8 ~ 12 days with the inoculum size of 5 ~ 10% turn be linked into salinity be in the substratum of 0.2 ~ 0.4% cultivate 8 ~ 12 days;
(3) get the algae liquid after cultivating in step (2) with 5 ~ 10% inoculum size turn that to be linked into salinity be cultivate 8 ~ 12 days in the substratum of 0.5 ~ 0.7%, be that 0.3% repetition above-mentioned steps is until micro-algae being seeded to salinity is in the substratum of 2.9 ~ 3.1% by salinity gradient;
(4) by above-mentioned algae kind in salinity be 2.9 ~ 3.1% substratum in cultured continuously 3 ~ 5 generation, namely obtain the high fat content algae kind that can grow fast.
2. method according to claim 1, is characterized in that, the component ratio of described substratum is: 0.15gNaHCO 3, 0.02g KH 2pO 4, 0.027g VB 1, 1.5 × 10-6g VB 12, 0.2g Na 2siO 39H 2o, 1.0gNaNO 3, 0.0005g Biotin, 1mL trace element and 980mL artificial seawater.
3. method according to claim 2, is characterized in that, the component ratio of described trace element is: containing 4.35g Na in 1000mL distilled water 2eDTA, 7.3mg Na 2moO 42H 2o, 12mgCoCl 26H 2o, 3.9g FeC 6h 5o 75H 2o, 10mg CuSO 45H 2o, 23mg ZnSO 4, 178mg MnCl 24H 2o and 600mgH 3bO 3.
4. method according to claim 2, is characterized in that, the component ratio of described artificial seawater is: containing 21.2157g NaCl, 3.407g Na in 1000mL distilled water 2sO 4, 0.3577g KCl, 9.3042gMgCl 26H 2o, 1.3044g CaCl 2, 0.0862g KBr, 0.0226g H 3bO 3, 0.2760g NaF and 0.0219gSrCl 26H 2o.
5. method according to claim 1, it is characterized in that, the culture condition of described step (2) ~ (4) is: artificial greenhouse 20 ~ 30 DEG C, and initial pH value is 8 ~ 10, every day is illumination 12h and dark 12h successively, and intensity of illumination during illumination is 6000 ~ 10000lux.
6. method according to claim 1, is characterized in that, described micro-algae liquid refers to: the transgenic alga kind of screening the natural algae kind obtained, algae kind mutant that physics and chemistry behavior obtains or obtaining through improvement of genes from physical environment.
CN201410854241.2A 2014-12-31 2014-12-31 Method for improving micro algae growth rate and cell fat content employing high-salinity domestication Pending CN104593314A (en)

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CN106754388A (en) * 2016-12-27 2017-05-31 新奥科技发展有限公司 One kind intends Nannochloropsis oculata and its acclimation method and application
CN113005041A (en) * 2021-03-23 2021-06-22 山东大学 Diamond algae, culture method thereof and application thereof in super-salt oil production

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