CN109355322A - The method for improving heterotrophic microalgae lipid-producing using epiphysin joint salt stress - Google Patents
The method for improving heterotrophic microalgae lipid-producing using epiphysin joint salt stress Download PDFInfo
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- 239000000654 additive Substances 0.000 claims description 2
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- 239000012467 final product Substances 0.000 description 1
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- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 1
- 239000003448 gibberellin Substances 0.000 description 1
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- DRLFMBDRBRZALE-UHFFFAOYSA-N melatonin Chemical compound COC1=CC=C2NC=C(CCNC(C)=O)C2=C1 DRLFMBDRBRZALE-UHFFFAOYSA-N 0.000 description 1
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- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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Abstract
The present invention relates to a kind of methods for improving heterotrophic microalgae lipid-producing using epiphysin joint salt stress, belong to technical field of microalga biology.The present invention is under the conditions of temperature is 24 ~ 26 DEG C, using glucose as the BG-11 basal medium Heterotrophic culture single needle algae of carbon source, logarithmic growth phase later period collection frustule is grown to single needle algae, the BG-11 culture medium containing NaCl is used to dilute resuspension frustule as induction algae solution；Epiphysin mother liquor is prepared with dehydrated alcohol, it is 1-100 μm of ol/L that epiphysin mother liquor, which is added dilution epiphysin concentration in induction algae solution, is placed in 24 ~ 26 DEG C, Fiber differentiation under illumination condition；The grease in frustule is extracted using organic solvent；The method of the present invention is easy to operate, can shorten the period of frustule induction, while improving microalgae grease content and lipid-producing, and can solve microalgae existing biomass during salt stress reduces the problems such as low with lipid-producing.
Description
Technical field
The present invention relates to a kind of methods for improving heterotrophic microalgae lipid-producing using epiphysin joint salt stress, belong to microalgae
Field of biotechnology.
Background technique
Microalgae can utilize CO under different training modes2Or other carbon sources produce sustainability biological products and life
Object fuel.Exploitation microalgae production biodiesel has great importance, and restricts the main factor of microalgae biodiesel industrialization
First is that the fat content and lipid-producing of microalgae are too low.The low method of Biomass yield is solved mainly using addition external source carbon source
Heterotrophic culture microalgae, the in a short time biomass of Rapid Accumulation microalgae；The method of traditional promotion microalgae accumulation grease relies on mostly
In Abiotic stress conditions, such as bloom photograph, nutritive salt defect, extreme temperature stress, salt stress and heavy metal stress etc..Wherein
Salt stress can effectively facilitate the accumulation of grease in microalgae as a kind of common abiotic stress, but under salt stress microalgae life
Length will receive certain inhibition.How to improve the resistance under microalgae salt stress with and meanwhile improve microalgae fat content and grease produce
Rate has become a hot topic of research.
The enrichment of grease relies primarily on the application of Abiotic stress conditions in microalgae at present and the external source of plant hormone adds
Add.The application of the Abiotic stress conditions such as bloom photograph, nitrogen defect can improve the content of grease in microalgae to a certain extent, but
Stress conditions can inhibit the growth of frustule, so that the lipid-producing in microalgae be made to reduce.Plant hormone is to adjust growth and development
The chemical messenger of various aspects has very high activity at very low concentrations；Some researches show that different plant hormones pair
Single needle algae production of biodiesel has apparent influence, and indole-3-acetic acid, gibberellin, kallikrein, tri- rhizome of Chinese monkshood of 1- are pure and mild de-
It falls the plant hormones such as acid and single needle algae fat content can be improved.Under abiotic stress action condition, external source adds plant hormone
It is expected to alleviate the bring negative interaction that abiotic stress grows frustule to a certain extent, enhances frustule to the abiotic side of body
Urgent resistance effectively improves the content of grease in microalgae.
Summary of the invention
The present invention provides a kind of method for improving heterotrophic microalgae lipid-producing using epiphysin joint salt stress, side of the present invention
Method using can heterotrophism microalgae, carry out algae heterotrophism culture make frustule fast-growth in a short time, be then diluted to suitable
The concentration of amount carries out the salt stress culture of light autotrophy, largely accumulates grease in combination with epiphysin induction frustule, and utilize organic
Grease in solvent extraction frustule；Operation of the present invention is simple, can improve fat content in a short time, guarantee microalgae growth and
Improve microalgae grease yield.
The present invention is as follows using the method that epiphysin joint salt stress improves heterotrophic microalgae lipid-producing:
(1) at 24 ~ 26 DEG C, using 8 ~ 12g/L glucose as the BG-11 basal medium Heterotrophic culture single needle algae of carbon source, to list
Needle algae grows to the logarithmic growth phase later period, collects frustule, is resuspended with the BG-11 culture medium dilution of the NaCl Han 15 ~ 25g/L single
Needle frustule is to 0.9 ~ 1.2g/L as induction algae solution；
(2) prepare epiphysin mother liquor with dehydrated alcohol, epiphysin mother liquor is added in the induction algae solution of step (1) dilution take off it is black
Plain concentration is 1 μm of ol/L ~ 100 μm ol/L, is placed in 24 ~ 26 DEG C, Fiber differentiation under illumination condition；
(3) grease in frustule is extracted using organic solvent after cultivating.
The single needle algae is the strain of single needle phycomyceteMonoraphidium spQLY-1(NCBI:KM199735)；
Intensity of illumination is 29 ~ 31 μm of ol m in the step (2)−2 s−1；
Organic solvent is chloroform-methanol in step by step rapid (3), the volume ratio of chloroform and methanol in chloroform-methanol
For 1:2；The method that organic solvent extracts the grease in frustule is that medium centrifugal is enriched with to and is collected frond, is washed with distillation
Dry algae powder is made in freeze-drying after washing 3 times；Quartz sand is added in dry algae powder and is ground, adds chloroform-methanol repetition
Extracting is whitened to frond, and organic phase is collected by centrifugation, and is dried in vacuo up to grease, wherein quartz sand additive amount is dry algae powder quality
2 times.
Epiphysin (Melatonin, MT) is as a kind of amine bormones being widely present in plant, to plant non-
There is important regulating and controlling effect in terms of growth and development under biotic.It can be used as natural quenching plant
The active oxygen that internal cause abiotic stress bursts out removes interior free yl, so that body is from oxidative damage.Research shows that epiphysin
The functional state of cytoskeleton can also be adjusted in conjunction with calmodulin, to execute specific function.External source addition epiphysin can mention
The activity of high anti-oxidation enzyme system and the content of antioxidant reduce membrane lipid peroxidation level, protect the integrality of adipose membrane, reduce
The extravasation of electrolyte mitigates injury of the stress from outside to plant, improves body resistance.In addition, can be mentioned under Abiotic stress conditions
The level of endogenous epiphysin in high plant, being overexpressed the relevant gene of epiphysin synthesis also can be improved plants against abiotic stress
Resistance.
The invention has the benefit that
(1) process of the present invention is simple and convenient, at low cost, and only need to add micro epiphysin can ensure microalgae under salt stress
Growth, and can be further improved the accumulation of grease in microalgae, to significantly improve the lipid-producing of microalgae；
(2) lipid-producing invention significantly improves microalgae under salt stress is added 1,10 and 100 μm of ol/L when external source and is taken off
Lipid-producing improves 14.55%, 21.28% and 14.82% than control group respectively under melanocyte joint salt stress；
(3) epiphysin molecular weight of the invention is small, is easily absorbed by plants, it can also be ensured that growth and hair of the plant in adverse circumstance
It educates；In the culture of oil-producing microalgae, epiphysin can be used as exogenous plant hormones and promote microalgae largely to accumulate grease and improve microalgae
Resistance.
Specific embodiment
The present invention is described in further details combined with specific embodiments below, the present invention is not limited only to these embodiments.
Comparative example 1:
(1) at 25 DEG C, using 10g/L glucose as the BG-11 basal medium Heterotrophic culture single needle algae of carbon sourceMonoraphidiumSp. QLY-1, to single needle algaeMonoraphidiumSp. QLY-1 grows to the logarithmic growth phase later period
(biomass reaches 5g/L) collects frustule, and fresh BG-11 culture medium is used to dilute resuspension frustule to 0.9g/L as inducing
Algae solution；
(2) the induction algae solution of step (1) is placed in temperature is 25 DEG C, intensity of illumination is 30 μm of ol m−2 s−1Under the conditions of, cold light
Lamp continuous light Fiber differentiation, is collected by centrifugation frustule daily；
(3) grease in organic solvent extraction step (2) frustule is utilized；Wherein organic solvent is chloroform-methanol, chlorine
The imitative volume ratio with methanol of imitation-carbinol Chlorine in Solution is 1:2；The method that organic solvent extracts the grease in frustule is that will cultivate
Liquid is through 5000 r/min centrifugal enrichment, 5 min, and dry algae powder is made in freeze-drying after washing 3 times repeatedly with distilled water, weighs；Dry algae is added
2 times of quartz sand of silty amount is simultaneously ground, and is added chloroform-methanol and is repeated to extract and whiten to frond, is collected by centrifugation
Organic phase merges organic dry grease to obtain the final product that is concerned with；Single needle algae biomass, fat content, lipid-producing be respectively 0.97 g/L,
36.43% and 88.34mg/L.1) d(is shown in Table.
Comparative example 2:
(1) raw to single needle algae using 10g/L glucose as the BG-11 basal medium Heterotrophic culture single needle algae of carbon source at 25 DEG C
It is long to collect frustule to logarithmic growth phase later period (biomass reaches 5 g/L), it is dilute with the BG-11 culture medium containing 20 g/L NaCl
Resuspension frustule is released to 0.9g/L as induction algae solution；
(2) the induction algae solution of step (1) is placed in temperature is 25 DEG C, intensity of illumination is 30 μm of ol m−2 s−1Under the conditions of, cold light lamp
Continuous light Fiber differentiation, is collected by centrifugation frustule daily；
(3) grease in organic solvent extraction step (2) frustule is utilized；Wherein organic solvent is chloroform-methanol, chlorine
The imitative volume ratio with methanol of imitation-carbinol Chlorine in Solution is 1:2；The method that organic solvent extracts the grease in frustule are as follows: will train
Nutrient solution is through 5000 r/min centrifugal enrichment, 5 min, and dry algae powder is made in freeze-drying after washing 3 times repeatedly with distilled water, weighs；It is added dry
2 times of quartz sand of algae silty amount is simultaneously ground, and is added chloroform-methanol and is repeated to extract to frond and whiten, centrifugation is received
Collect organic phase, organic phase is drying to obtain grease；Under 20 g/L salt stress, 1) single needle algae fat content is shown in Table up to 42.93%(, raw
Object amount is 0.96 g/L, and micro algae biomass reduces 1.03% compared with comparative example 1 under salt stress, and frustule fat content and grease
Comparative example 1 increases 1.18 and 1.16 times to yield respectively.
Embodiment 1: this is as follows using the method that epiphysin joint salt stress improves heterotrophic microalgae lipid-producing:
(1) raw to single needle algae using 10g/L glucose as the BG-11 basal medium Heterotrophic culture single needle algae of carbon source at 25 DEG C
Length to the logarithmic growth phase later period collects frustule, extremely with the BG-11 culture medium dilution resuspension frustule containing 20 g/L NaCl
0.9g/L is as induction algae solution；
(2) epiphysin mother liquor, is added to the induction algae of step (1) by the epiphysin mother liquor that 100 μm of ol/L are prepared with dehydrated alcohol
In liquid dilute epiphysin concentration be 1 μm of ol/L, be placed in 25 DEG C, intensity of illumination be 29 μm of ol m−2 s−1Under the conditions of, cold light lamp is held
Continuous light induction culture, is collected by centrifugation frustule daily；
(3) grease in organic solvent extraction step (2) frustule is utilized；Wherein organic solvent is chloroform-methanol, chlorine
The imitative volume ratio with methanol of imitation-carbinol Chlorine in Solution is 1:2；The method that organic solvent extracts the grease in frustule are as follows: will train
Nutrient solution is through 5000 r/min centrifugal enrichment, 5 min, and dry algae powder is made in freeze-drying after washing 3 times repeatedly with distilled water, weighs；It is added dry
2 times of quartz sands of algae silty amount are simultaneously ground, and are added chloroform-methanol and are repeated to extract and whiten to frond, are collected by centrifugation
Organic phase is dried in vacuo up to grease；Under 1 μm of ol/L MT joint salt stress, microalgae grease content is up to that 49.18%(is shown in Table
1), biomass is 0.95 g/L；As known from Table 1, biomass respectively reaches 97.93% and of comparative example 1, comparative example 2
98.96%；Frustule fat content and lipid-producing have increased separately 1.35 and 1.32 times compared with comparative example 1, increase compared with comparative example 2
1.15 and 1.14 times.
Embodiment 2: this is as follows using the method that epiphysin joint salt stress improves heterotrophic microalgae lipid-producing:
(1) at 24 DEG C, using 10 g/L glucose as the BG-11 basal medium Heterotrophic culture single needle algae of carbon source, to single needle algae
Logarithmic growth phase later period collection frustule is grown to, extremely with the BG-11 culture medium dilution resuspension frustule containing 20 g/L NaCl
0.9g/L is as induction algae solution；
(2) epiphysin mother liquor, is added to the induction algae of step (1) by the epiphysin mother liquor that 100 μm of ol/L are prepared with dehydrated alcohol
In liquid dilute epiphysin concentration be 10 μm of ol/L, be placed in 24 DEG C, intensity of illumination be 30 μm of ol m−2 s−1Under the conditions of, cold light lamp
Continuous light Fiber differentiation, is collected by centrifugation frustule daily；
(3) grease in organic solvent extraction step (2) frustule is utilized；Wherein organic solvent is chloroform-methanol, chlorine
The imitative volume ratio with methanol of imitation-carbinol Chlorine in Solution is 1:2；The method that organic solvent extracts the grease in frustule are as follows: will train
Nutrient solution is through 5000 r/min centrifugal enrichment, 5 min, and dry algae powder is made in freeze-drying after washing 3 times repeatedly with distilled water, weighs；It is added dry
2 times of quartz sands of algae silty amount are simultaneously ground, and are added chloroform-methanol and are repeated to extract and whiten to frond, are collected by centrifugation
Organic phase is dried in vacuo up to grease；Under 10 μm of ol/L MT joint salt stress, microalgae grease content is up to 51.74 %, raw
Object amount is 1) 0.96g/L(is shown in Table.Its biomass reaches the 98.97% of comparative example 1, with maintaining an equal level for comparative example 2；Frustule grease contains
Amount and lipid-producing have increased separately 1.42 and 1.41 times compared with comparative example 1, increase 1.21 times compared with comparative example 2.
Embodiment 3: this is as follows using the method that epiphysin joint salt stress improves heterotrophic microalgae lipid-producing:
(1) at 24 DEG C, using 10 g/L glucose as the BG-11 basal medium Heterotrophic culture single needle algae of carbon source, to single needle algae
Logarithmic growth phase later period collection frustule is grown to, extremely with the BG-11 culture medium dilution resuspension frustule containing 20 g/L NaCl
0.9 g/L is as induction algae solution；
(2) epiphysin mother liquor, is added to the induction of step (1) by the epiphysin mother liquor that 100 μm of ol/L are prepared with dehydrated alcohol
In algae solution dilute epiphysin concentration be 100 μm of ol/L, be placed in 26 DEG C, intensity of illumination be 31 μm of ol m−2 s−1Under the conditions of, it is cold
Light lamp continuous light Fiber differentiation, is collected by centrifugation frustule daily；
(3) grease in organic solvent extraction step (2) frustule is utilized；Wherein organic solvent is chloroform-methanol, chlorine
The imitative volume ratio with methanol of imitation-carbinol Chlorine in Solution is 1:2；The method that organic solvent extracts the grease in frustule are as follows: will train
Nutrient solution is through 5000 r/min centrifugal enrichment, 5 min, and dry algae powder is made in freeze-drying after washing 3 times repeatedly with distilled water, weighs；It is added dry
2 times of quartz sands of algae silty amount are simultaneously ground, and are added chloroform-methanol and are repeated to extract and whiten to frond, are collected by centrifugation
Organic phase, organic phase are dried in vacuo up to grease；Under 100 μm of ol/L MT joint salt stress, microalgae grease content is up to
47.81%, biomass is 1) 0.99 g/L(is shown in Table.Its biomass has increased separately 2.06% He compared with comparative example 1 and comparative example 2
3.12%；Frustule fat content and lipid-producing have increased separately 1.31 and 1.33 times compared with comparative example 1, increase respectively compared with comparative example 2
1.11 and 1.15 times are added.
Biomass, Biomass yield, fat content and the lipid-producing of single needle algae under the conditions of 1 different disposal of table
；
Heterotrophic culture microalgae can quickly increase micro algae biomass, and the frustule after heterotrophism is induced using light autotrophy accumulates grease, hair
Existing salt stress can promote the accumulation of grease in frustule, but the growth of frustule receives certain inhibition；Add appropriate concentration
Epiphysin joint salt stress can further promote the accumulation of grease, and maintain the growth of frustule, to improve significantly
The lipid-producing of microalgae；By the method for this induction microalgae, quick, a large amount of accumulation of grease in frustule had not only been realized, but also had been protected
The stable growth of microalgae is demonstrate,proved.
Claims (4)
1. a kind of method for improving heterotrophic microalgae lipid-producing using epiphysin joint salt stress, which is characterized in that specific steps
It is as follows:
(1) at 24 ~ 26 DEG C, using 8 ~ 12g/L glucose as the BG-11 basal medium Heterotrophic culture single needle algae of carbon source, to list
Needle algae grows to the logarithmic growth phase later period, collects frustule, is resuspended with the BG-11 culture medium dilution of the NaCl Han 15 ~ 25g/L single
Needle frustule is to 0.9 ~ 1.2g/L as induction algae solution；
(2) prepare epiphysin mother liquor with dehydrated alcohol, epiphysin mother liquor is added in the induction algae solution of step (1) dilution take off it is black
Plain concentration is 1 μm of ol/L ~ 100 μm ol/L, is placed in 24 ~ 26 DEG C, Fiber differentiation under illumination condition；
(3) grease in frustule is extracted using organic solvent after cultivating.
2. the method according to claim 1 for improving heterotrophic microalgae lipid-producing using epiphysin joint salt stress, special
Sign is: single needle algae is the strain of single needle phycomyceteMonoraphidium sp. QLY-1。
3. the method according to claim 1 for improving heterotrophic microalgae lipid-producing using epiphysin joint salt stress, special
Sign is: step (2) intensity of illumination is 29 ~ 31 μm of ol m−2 s−1。
4. the method according to claim 1 for improving heterotrophic microalgae lipid-producing using epiphysin joint salt stress, special
Sign is: organic solvent is chloroform-methanol in step (3), and the volume ratio of chloroform and methanol is 1 in chloroform-methanol:
2；The method that organic solvent extracts the grease in frustule is that medium centrifugal is enriched with to and is collected frond, is washed with distilled water 3
Dry algae powder is made in freeze-drying after secondary；Quartz sand is added in dry algae powder and is ground, adds chloroform-methanol and repeats to extract
Whiten to frond, organic phase is collected by centrifugation, is dried in vacuo up to grease, wherein quartz sand additive amount is 2 times of dry algae powder quality.
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| CN110106210A (en) * | 2019-04-22 | 2019-08-09 | 昆明理工大学 | Epiphysin is promoting the application in Haematococcus pluvialis production γ-aminobutyric acid |
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