CN106520559B - A kind of chlorella high efficiency light autotrophy cultural method - Google Patents
A kind of chlorella high efficiency light autotrophy cultural method Download PDFInfo
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Abstract
The present invention provides a kind of chlorella high efficiency light autotrophy cultural method, culture solution is added in bioreactor, chlorella is accessed, and guarantees algae cell density >=1,000,000/milliliter, using natural lighting, daytime, luminous intensity was in 10-130Klx, periodicity of illumination 12/12, blowing air amount 1.0-10.0L/min, carbon dioxide gas intake 0.05-0.5L/min, algae solution temperature is 26-33 DEG C, and pH is maintained at 7.0-9.0；The present invention realizes the high efficiency large-scale production that continuous incubation time is long, yield is high, nutritive salt utilization rate is high; entire culture period; the algae butt of harvesting is 22.131-23.181 kilograms, and frond protein content reaches 55% or more, and nutritive salt utilization rate reaches 90% or more.
Description
Technical field
The present invention relates to technical field of microalga biology, and in particular to a kind of chlorella high efficiency light autotrophy cultural method.
Background technique
Chlorella is the general natural disposition monoplast green alga of Chlorophyta Chlorella, is a kind of spherical, oval unicellular alga, directly
3~8 microns of diameter, be one of life earliest on the earth, is a kind of efficient photosynthetic plant, with light before appearing in more than 20 hundred million years
Autophyting growth breeding is closed, is distributed all over the world, is moved in smaller shallow water more, also there are marine breeds.It is individual under natural endowment
It is less, manually cultivate mass propagation.Intracellular protein, fat and carbohydrate content is all very high, and there are many dimension lifes
Element, it is edible and as bait.
Photosynthesis can simply be summarised as containing photosynthetic pigments be mainly chlorophyll plant cell and bacterium, in the sunlight
Utilize inorganic matter (CO2、H2O、H2S etc.) synthesis of organic substance (C6H12O6), and discharge oxygen (O2) or other substances (such as S) mistake
Journey.Photosynthesis is the maximum organic synthesis carried out on the earth simultaneously.The energy from the sun to the earth is about 1.5 daily
×1022KJ is converted into the chemical energy of molecular forms by photosynthesis wherein about 1% is absorbed by photosynthetic organism, and passes through food
Chain is utilized by other members in biosphere.Therefore, sunlight is the final source of energy needed for almost all creatures on the earth.
Currently, the main training method of chlorella is divided into autotrophic type and feeding type and heterotrophism according to chlorella metabolic type
Type.Autotrophy culture there are culture densities it is low, growth cycle is long the disadvantages of；Mixotrophic cultivation, which exists, needs to provide ready-made organic carbon source
The consumption problem of the energy caused by energy source as its growth, meanwhile, heterotrophism is transitioned into the autotrophy stage and be easy to cause frond
There is the disadvantages of a part of phenomena of mortality so as to cause frond in discomfort；Heterotrophic culture equally exists the ready-made organic carbon source of consumption
Situation, and nutriment such as protein, chlorophyll, vitamin by accumulating in the chlorella cells of heterotrophism etc. is generally than certainly
The related substance for supporting chlorella cylinder accumulation is low.
Summary of the invention
Above-mentioned 3 kinds of training methods there are aiming at the problem that, the present invention, which was provided in a kind of short time, reaches continuous incubation time
The high chlorella light autotrophy cultural method of length, high efficiency output, nutritive salt utilization rate, the cultural method include the following steps:
(1) nutritive salt is added in tap water sufficiently to dissolve and obtain culture solution, using ultraviolet sterilization device to culture solution into
Row sterilization processing makes content of microorganisms in sterilized rear culture solution be less than 100cfu/ milliliters；
(2) culture solution is added in bioreactor, accesses chlorella, and guarantee algae cell density >=1,000,000/milli
It rises, carries out the culture of light autotrophy；
(3) culture uses natural lighting, and daytime, luminous intensity was in 10-130Klx, periodicity of illumination 12/12, blowing air amount 1.0-
10.0L/min, carbon dioxide gas intake 0.05-0.5L/min, algae solution temperature are 26-33 DEG C, and pH is maintained at 7.0-9.0；
(4) in incubation, start to harvest when intensity of illumination decays to 50% in algae solution, harvest 50% algae solution every time, then
Fresh medium is supplemented, such operation was repeated once every 1 day, and until chlorella culture enters the decline phase, culture terminates.
The method of the supplement culture solution is added using at the uniform velocity mode, and primary addition was finished using time and algae solution circulation one
It is identical the time required to all.
The nutritive salt composition includes: urea 0.38-0.47g/L, MgSO4·7H2O 0.25-0.5g/L, KH2PO4
0.095-0.12g/L, ferrous sulfate 0.005-0.006g/L, CaCl2·2H2O 0.02-0.03g/L, A5 liquid microelement 0.8-
1mL/L；Wherein A5 microelement formula of liquid is H3BO3 2.86g/L、MnCl2·4H2O 1.86g/L、ZnSO4·7H2O
0.22g/L、Na2MoO4·2H2O 0.39g/L、CuSO4·5H2O 0.08g/L and Co (NO3)2·6H2O 0.05g/L。
The chlorella of harvesting is centrifuged, is cleaned, dries acquisition dry algae powder, measures its algae butt；Frond internal protein
Assay uses " measurement of Protein in Food " (GB 5009.5-2010) Kjeldahl nitrogen determination；It cultivates total in water body
Phosphorus measurement is using the national standard method (GB/T that potassium peroxydisulfate (or nitric acid-perchloric acid) oxidation is added in neutral conditions
11893-89)；The determination of total nitrogen content national standard quantitative in 120-124 DEG C of resolution, uv-spectrophotometric using alkaline chitinase
Method (GB/T11894-1989).
Compared with prior art, the present invention has the following advantages: (1) chlorella culture water passes through sterilization treatment, matter
Amount meets the relevant regulations of existing National Standard of the People's Republic of China " standards for drinking water quality "；(2) bead is improved
Algal nutrient formula, the optimal proportion of nutritive salt needed for making it meet chlorella physiologically；(3) in light autotrophy incubation,
Harvest is a part of when intensity of illumination decays to 50% in algae solution, adds fresh medium, single-cell algae again in remaining algae solution
Life continued, ensure that the vigor of frustule, improve frustule growth rate, extend and continue cultivation cycle.This culture
Method realizes high efficiency large-scale production, and in 5.5 tons of bioreactors, chlorella can continuously be cultivated 20 days, from the 6th
It rises, and harvesting in every 2 days is primary, harvests 50% algae solution every time, is centrifuged, is cleaned to the algae solution of harvesting, drying acquisition dry algae powder,
Algae butt and frond internal protein content, entire culture period are measured, the algae butt of harvesting is 25.203-25.226 kilograms, frond
Protein content reaches 69% or more, and nutritive salt utilization rate reaches 90% or more.
Specific embodiment
By specific embodiment, invention is further explained.
Embodiment 1
The following table 1 component nutritive salt is added first in tap water after completely dissolution to each component to be added to through ultraviolet sterilization
In 5 tons of bioreactor, it is inoculated with chlorella, and guarantees that algae cell density is 1,000,000/milliliter, starts chlorella light autotrophy；
Autotrophy condition of culture are as follows: use natural lighting, daytime, luminous intensity was in 10-130Klx, periodicity of illumination 12/12, blowing air amount 1.0L/
Min, carbon dioxide gas intake 0.05L/min, algae solution temperature are 26-33 DEG C, and pH is maintained at 7.0-9.0；In culture the 6th day
It rises, primary every harvesting in 1 day, the algae solution of harvesting 50%, is supplemented fresh medium and continues to cultivate every time, until the 20th day, training
Supporting terminates.The chlorella of harvesting is centrifuged, is cleaned, dries acquisition dry algae powder, measures its algae butt and frond internal protein
Content, entire culture period, the algae butt of harvesting are 25.226 kilograms (being shown in Table 4), and frond internal protein is 69.2%-70.9%
(being shown in Table 5)；The total nitrogen total phosphorus consumption rate situation of detection culture water body, total nitrogen total phosphorus utilization rate respectively reach 90.05%-
91.54% and 90.12%-91.56% (is shown in Table 6).
Table 1
| Urea | 0.38g/L |
| MgSO4·7H2O | 0.25g/L |
| KH2PO4 | 0.095g/L |
| Ferrous sulfate | 0.005g/L |
| CaCl2·2H2O | 0.02g/L |
| A5 liquid microelement | 0.8mL |
Remarks: growth analysis calculates average daily biological yield, and formula is as follows: K=(InN2–InN0)/(T2–T0),
Middle K is that biological production goes out efficiency, as one of harvesting judgment basis；T0、T2For corresponding incubation time；N1And N2Respectively T0、T2
When biomass；T0For the inoculation same day.
Embodiment 2
The following table 2 component nutritive salt is added first in tap water after completely dissolution to each component to be added to through ultraviolet sterilization
In 5 tons of bioreactor, it is inoculated with chlorella, and guarantees that algae cell density is 1,200,000/milliliter, starts chlorella light autotrophy；
Autotrophy condition of culture are as follows: use natural lighting, daytime, luminous intensity was in 10-130Klx, periodicity of illumination 12/12, blowing air amount
10.0L/min, carbon dioxide gas intake 0.5L/min, algae solution temperature are 26-33 DEG C, and pH is maintained at 7.0-9.0；It is cultivating
From 6th day, primary every harvesting in 1 day, the algae solution of harvesting 50%, is supplemented fresh medium and continues to cultivate, until the 20th every time
It, culture terminates.The chlorella of harvesting is centrifuged, is cleaned, dries acquisition dry algae powder, measures egg in its algae butt and frond
White matter content, entire culture period, the algae butt of harvesting are 25.203 kilograms (being shown in Table 4), and frond internal protein is 69.2%-
70.9% (being shown in Table 5)；The total nitrogen total phosphorus consumption rate situation of detection culture water body, total nitrogen total phosphorus utilization rate respectively reach 90.12%-
91.9% and 90.22%-91.89% (is shown in Table 6).
Table 2
Embodiment 3
The following table 3 component nutritive salt is added first in tap water after completely dissolution to each component to be added to through ultraviolet sterilization
In 5 tons of bioreactor, it is inoculated with chlorella, and guarantees that algae cell density is 1,400,000/milliliter, starts chlorella light autotrophy；
Autotrophy condition of culture are as follows: use natural lighting, daytime, luminous intensity was in 10-130Klx, periodicity of illumination 12/12, blowing air amount 5.0L/
Min, carbon dioxide gas intake 0.2L/min, algae solution temperature are 26-33 DEG C, and pH is maintained at 7.0-9.0；In culture the 6th day
It rises, primary every harvesting in 1 day, the algae solution of harvesting 50%, is supplemented fresh medium and continues to cultivate every time, until the 20th day, training
Supporting terminates.The chlorella of harvesting is centrifuged, is cleaned, dries acquisition dry algae powder, measures its algae butt and frond internal protein
Content, entire culture period, the algae butt of harvesting are 25.216 kilograms (being shown in Table 4), and frond internal protein is 69.2%-70.9%
(being shown in Table 5)；The total nitrogen total phosphorus consumption rate situation of detection culture water body, total nitrogen total phosphorus utilization rate respectively reach 91.21%-
92.01% and 91.24%-92.09% (is shown in Table 6).
Table 3
| Urea | 0.40g/L |
| MgSO4·7H2O | 0.50g/L |
| KH2PO4 | 0.10g/L |
| Ferrous sulfate | 0.005g/L |
| CaCl2·2H2O | 0.025g/L |
| A5 liquid microelement | 0.9mL |
Table 4 harvests algae butt situation
Table 5 detects frond protein content after harvesting
The total nitrogen total phosphorus situation of the culture water body of table 6
Claims (3)
1. a kind of chlorella high efficiency light autotrophy cultural method, which is characterized in that this method comprises the following steps:
(1) nutritive salt is added in tap water sufficiently to dissolve and obtain culture solution, culture solution is killed using ultraviolet sterilization device
Bacterium processing makes content of microorganisms in sterilized rear culture solution be less than 100cfu/ milliliters；The nutritive salt composition includes: urea
0.38-0.47g/L, MgSO4·7H2O 0.25-0.5g/L, KH2PO40.095-0.12g/L, ferrous sulfate 0.005-
0.006g/L, CaCl2·2H2O 0.02-0.03g/L, A5 liquid microelement 0.8-1mL/L；The A5 microelement formula of liquid is
H3BO3 2.86g/L、MnCl2·4H2O 1.86g/L、ZnSO4·7H2O 0.22g/L、Na2MoO4·2H2O 0.39g/L、
CuSO4·5H2O 0.08g/L and Co (NO3)2·6H2O 0.05g/L；
(2) culture solution is added in bioreactor, accesses chlorella, and guarantee algae cell density >=1,000,000/milliliter, into
Row light autotrophy culture；
(3) culture uses natural lighting, and daytime, luminous intensity was in 10-130Klx, periodicity of illumination 12/12, blowing air amount 1.0-
10.0L/min, carbon dioxide gas intake 0.05-0.5L/min, algae solution temperature are 26-33 DEG C, and pH is maintained at 7.0-9.0；
(4) in incubation, start to harvest when intensity of illumination decays to 50% in algae solution, harvest 50% algae solution every time, be supplemented
Fresh medium, such operation were repeated once every 1 day, and until chlorella culture enters the decline phase, culture terminates.
2. a kind of chlorella high efficiency light autotrophy cultural method as described in claim 1, which is characterized in that the supplement culture
The method of liquid is added using at the uniform velocity mode, and primary addition finishes identical as algae solution one week required time of circulation using the time.
3. a kind of chlorella high efficiency light autotrophy cultural method as described in claim 1, which is characterized in that the nutritive salt group
At including: urea 0.4g/L, MgSO4·7H2O 0.5g/L, KH2PO40.1g/L, ferrous sulfate 0.005g/L, CaCl2·2H2O
0.025g/L, A5 liquid microelement 0.9mL/L.
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| CN102021208A (en) * | 2010-11-16 | 2011-04-20 | 华东理工大学 | Method for rapidly accumulating micro-algae intracellular grease |
| CN103805514A (en) * | 2014-02-25 | 2014-05-21 | 中国科学院水生生物研究所 | Microalga photosynthetic aerobic high-density fermentation culture method utilizing inorganic nitrogen source and application |
| CN105985989A (en) * | 2015-01-30 | 2016-10-05 | 中国石油天然气股份有限公司 | Method for producing biodiesel from chlorella |
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| CN102021208A (en) * | 2010-11-16 | 2011-04-20 | 华东理工大学 | Method for rapidly accumulating micro-algae intracellular grease |
| CN103805514A (en) * | 2014-02-25 | 2014-05-21 | 中国科学院水生生物研究所 | Microalga photosynthetic aerobic high-density fermentation culture method utilizing inorganic nitrogen source and application |
| CN105985989A (en) * | 2015-01-30 | 2016-10-05 | 中国石油天然气股份有限公司 | Method for producing biodiesel from chlorella |
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