CN106754390A - The albuminiferous chlorella of one plant height and its cultural method and application - Google Patents
The albuminiferous chlorella of one plant height and its cultural method and application Download PDFInfo
- Publication number
- CN106754390A CN106754390A CN201611255690.0A CN201611255690A CN106754390A CN 106754390 A CN106754390 A CN 106754390A CN 201611255690 A CN201611255690 A CN 201611255690A CN 106754390 A CN106754390 A CN 106754390A
- Authority
- CN
- China
- Prior art keywords
- chlorella
- algae
- application
- cultural method
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
- C12N1/125—Unicellular algae isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/89—Algae ; Processes using algae
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
- C02F3/322—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae use of algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
Abstract
The invention discloses a chlorella, chlorella (Chlorella sorokiniana) BL ch1 are named as, are preserved in China typical culture collection center, address on November 8th, 2016：Wuchang, wuhan Luo Jia Shan, its deposit number is：CCTCC M 2016626.The algae strain cultural method be：Algae strain is seeded on culture medium, inoculum concentration is 5 15%, liquid amount is 400 800mL/L, initial pH is 6.0 8.0,33 DEG C of cultivation temperature 28 DEG C.The chlorella that present invention screening is obtained, its protein content is high and high to the degradation rate of total nitrogen, total phosphorus, ammonia nitrogen, can be used for the multiple fields such as protein production, feed preparation, sewage disposal.
Description
Technical field
The present invention relates to the albuminiferous chlorella of a plant height and its cultural method and application, belong to algae bio technology neck
Domain.
Background technology
Microalgae is that one kind can carry out photoautotrophic microorganism, primary producer is in the ecosystem, in whole thing
Act on huge in matter circulation, production efficiency is high, and development prospect is tempting.Microalgae can absorb a large amount of nitrogen phosphorus units in growth course
Element, therefore post-processing unit culture microalgae using sewage treatment plant carries out denitrogenation dephosphorizing deep purifying sewage, early in 1957
Year Oswald etc. is it has been suggested that microalgae to be used as a kind of biosystem of the activated sludge in substitution sewage disposal.Hereafter
The oxidation pond technology set up on artificial mode water body helotisn self-cleaning basis is widely used and develops.Separately
Outward, microalgae also have that growth is fast, harvest time is short, rich in grease, photosynthetic utilization ratio is high the features such as, can be as food and biology
One of main source of the energy, is also increasingly paid close attention to by people.
At present, about administering waste water and also relatively fewer as the research of food and bioenergy by the use of microalgae, mostly
The laboratory lab scale stage of Strain selection is in, and from the point of view of having been reported, the algae strain generally existing condition of culture for being utilized
Cause albumen to be difficult to the problems such as being utilized by organism it is required that high, protein yield is low, frustule wall is relatively thick, and be difficult to wide
General application.Therefore, excellent algae kind is screened to have great importance for solving the above problems.
The content of the invention
For above-mentioned prior art, it is an object of the invention to provide the albuminiferous chlorella of a plant height and its cultural method and
Using.
To achieve the above object, the present invention uses following technical proposals：
The first aspect of the present invention a, there is provided chlorella, is named as chlorella (Chlorella sorokiniana)
BL-ch1, is preserved in China typical culture collection center, address on November 8th, 2016：Wuchang, wuhan Luo Jia Shan, its
Deposit number is：CCTCC NO:M 2016626.
Algae strain is isolated from the dirty discharge irrigation canals and ditches of excrement pond and excrement on Taian Shandong Ningyang pig farm, algae strain be it is unicellular, it is spherical,
Cell membrane is thin, 3-6 μm of cell dia.Cell green, chromatoplast cup-shaped accounts for cell major part, with a pyrenoids.
The second aspect of the present invention, there is provided the cultural method of the albuminiferous chlorella of a plant height, step is：Algae strain is inoculated with
To culture medium, inoculum concentration is 5-15% (preferably 10%), and liquid amount is 400-800mL/L (preferably 600mL/L), initially
PH is 6.0-8.0 (preferably 7.0), 28 DEG C -33 DEG C of cultivation temperature.
In above-mentioned cultural method, the composition of the culture medium is：In No. 10 culture mediums of Zhu Shi add 1 ‰ glucose,
0.1 ‰ dipotassium hydrogen phosphates, 0.1 ‰ urea and 0.1 ‰ glycine.
The third aspect of the present invention, there is provided application of the above-mentioned chlorella in sewage disposal.
Experiment proves that, 94.31% is reached as high as to the degradation rate of total nitrogen in sewage using chlorella of the invention, to total
The degradation rate of phosphorus reaches as high as 90.07%, and the degradation rate to ammonia nitrogen reaches as high as 99.21%.To the excellent of sewage disposal.
The fourth aspect of the present invention, there is provided application of the above-mentioned chlorella in protein production.Concrete application method is：Will be small
Ball algae is cultivated by above-mentioned cultural method, obtains algae solution；Algae solution is centrifuged, is concentrated, dried, obtain final product the algae powder containing albumen.
Application of the above-mentioned chlorella in aquatic livestock open-mouthed bait and/or fish meal substitute is prepared is also guarantor of the invention
Shield scope.
Beneficial effects of the present invention：
(1) chlorella that obtains of present invention screening, its protein content can be up to more than 71%, and, in high yield albumen
While, chlorella of the invention is high to the degradation rate of total nitrogen in sewage, total phosphorus, ammonia nitrogen, can be used for protein production, feed system
The multiple fields such as standby, sewage disposal.
(2) chlorella protein content is of a relatively high, can as single cell protein important sources, but, mostly
The miniature green alga of number all has one layer of tough and tensile cell membrane compared with thick fiber element, so as to have impact on profit of the organism to its protein
With digestibility is also decreased.And the present invention screens the chlorella for obtaining, its cell membrane is thin, and the content of protein is high, non-
Often be conducive to absorption and utilization of the organism to chlorella protein.
Brief description of the drawings
Fig. 1：The cellular morphology microphoto of algae strain of the present invention；
Fig. 2：Based on maximum brief (Maximum Parsimony) tree that 18S rDNA fragment sequences build；(inframe is marked
Be testing sample sequence)；
Fig. 3：The experimental rig figure of application of the chlorella in excrement sewage of degrading.
Specific embodiment
Involved instrument, reagent, material etc. in following embodiments, unless otherwise noted, are existing in the prior art
Conventional instrument, reagent, material etc., can be obtained by regular commercial sources.Involved experimental technique, inspection in following embodiments
Survey method etc., unless otherwise noted, is existing normal experiment method, detection method etc. in the prior art.
Embodiment 1：Separation, screening and the identification of algae strain
1 sample collection
Chlorella separates sample collection place：The dirty discharge irrigation canals and ditches of the excrement pond and excrement on Taian Shandong Ningyang pig farm.
The separation of 2 algaes strain, screening
2.1 test materials：It is No. 10 culture mediums of Zhu Shi that the present embodiment uses culture medium, and 1% is added in its solid medium
The agar of (mass percent), without agar in its fluid nutrient medium.Culture medium constituent is shown in Table 1.
Table No. 10 culture medium constituents of 1 Zhu Shi
2.2 test methods
2.2.1 the sample of collection is placed in 30 DEG C of illumination boxs and is cultivated 5-7 days, after to appear turn green, with Zhu Shi 10
Number fluid nutrient medium carries out 10,50,100,500 and 1000 times of gradient dilutions, and the μ L of sample 100 are uniformly coated on Zhu Shi after taking dilution
On No. 10 solid mediums, it is placed in 30 DEG C of illumination boxs and cultivates；Picking list when observation has single algae to be born long in incubation
One algae falls to accessing equipped with No. 10 test tubes of fluid nutrient medium of Zhu Shi；Wait turn it is green after take algae solution microscopy, choosing purebred algae strain is carried out
Expand numerous.
2.2.2 diluted excrement sewage sample 300mL is taken, loaded in 1L conical flasks, 121 DEG C of autoclaving 30min are accessed
Algae solution 100mL, in 30 DEG C, 180r/min continuously cultivates 5d, every 24h samplings once, determines chlorophyll, total phosphorus, total nitrogen, ammonia
The indexs such as nitrogen, nitrite.
2.3 index determinings
Determine chlorophyll in purebred algae strain culture, total phosphorus, total nitrogen, ammonia nitrogen, nitrite and protein content.
The assay method of These parameters is as follows：
(1) total phosphorus：Ammonium molybdate spectrophotometric method；
(2) total nitrogen：Potassium persulfate oxidation method；
(3) ammonia nitrogen：Reagent colorimetric method；
(4) nitrite：Alpha-naphthylamine colorimetric method；
(5) protein content：Kjeldahl's method；
(6) chlorophyll：Heat ethanol methodses.
2.4 result of the tests
2.4.1 algae strain isolate and purify
By isolating and purifying, 7 plants of single algae strains are obtained, numbering is 1-1,2-1,3-1,4-1,5-1,6-1,7-1.In optics
Under 100 times of microscope, the cell of 7 plants of algaes is spherical, Dan Sheng, and can see it is intracellular containing more obvious chromatoplast,
Possesses the morphological feature of Chlorophyta, this 7 plants of algaes of Preliminary Identification are green alga.
2.4.2 the screening of algae strain
(1) measure of total nitrogen content and its degradation rate
The results are shown in Table 2.
The total nitrogen content of table 2 (mg/L) and degradation rate are determined
As can be seen from Table 2：In addition to 5-1,7-1, each algae strain total nitrogen degradation rate is up to more than 75%.
(2) measure of total phosphorus content and its degradation rate
The results are shown in Table 3.
The total phosphorus content of table 3 (mg/L) and degradation rate are determined
As can be seen from Table 3：In addition to 4-1, each algae strain total phosphorus degradation rate is up to more than 78%.
(3) measure of ammonia-nitrogen content and its degradation rate
The results are shown in Table 4.
The ammonia-nitrogen content of table 4 (mg/L) and degradation rate are determined
Each algae strain ammonia nitrogen degradation rate is higher as can be seen from Table 4.
(4) content of nitrite is determined
The results are shown in Table 5.
The content of nitrite of table 5 determines (mg/L)
Each algae strain as can be seen from Table 5 does not have obvious effect to nitrite.
(5) measuring chlorophyll content
The results are shown in Table 6.
The measuring chlorophyll content of table 6 (μ g/L)
Each algae strain chlorophyll content persistently increases as can be seen from Table 6, illustrates each algae strain well-grown.
(6) protein content and biomass estimation
The results are shown in Table 7.
The protein content of table 7 and biomass
6-1 algaes strain protein content is up to 67.9% as can be seen from Table 7.
By the above results as can be seen that algae strain 6-1 protein content highests, and the degradation rate of total nitrogen, total phosphorus, ammonia nitrogen is reachable
More than 75%.Lay eggs white so as to obtain a plant height, and to the strain of the algae of good waste water treatment effect, be named as BL-ch1.
The identification of 3 algaes strain
3.1 morphologic observations
Algae strain 6-1 Main Biological be：Algae strain is unicellular, and spherical, cell membrane is thin, 3-6 μm of cell dia.Carefully
Born of the same parents' green, chromatoplast cup-shaped accounts for cell most of.With a pyrenoids.Microphoto such as Fig. 1 institutes of algae strain cellular morphology
Show.
3.2 Molecular Identifications
3.2.1 method
3.2.1.1 algal gene group is extracted using CTAB (Cetyltrimethylammonium bromide) method, and
With reference to《Molecular systematics》, specific method is as follows：
10mL algae solutions are taken, 8000rpm centrifugation 10min add 100 μ L TE, vibrate suspension cell, add 800 μ L cracking
Liquid and 0.8 μ L beta -mercaptoethanols, rapid fully vibration are mixed, and are placed in 65 DEG C of warm bath 30min, are overturned for several times back and forth therebetween, then add
Enter 700 μ L chloroforms, fully mix, be transferred to supernatant in new centrifuge tube after centrifugation by 12000rpm centrifugation 5min, adds 700 μ
L combination buffers, are sufficiently mixed uniformly, are then added in centrifugal adsorbing column, place 1min, 12000rpm centrifugation 1min,
Fall the waste liquid in collecting pipe, add 700 μ L lavation buffer solutions, 12000rpm centrifugation 1min, repeated washing 3 times, last time
12000rpm is centrifuged 2min, fully removes lavation buffer solution, takes out centrifugal adsorbing column, be inserted in a clean 1.5mL from
Heart pipe, and 50 μ L elution buffers are added, after placing 1min, 1min is centrifuged in 12000rpm, centrifugal adsorbing column is taken out, take 10 μ L
Electrophoresis.
3.2.1.2 sequence amplification and measure
PCR is expanded and is determined core encoding ribosomal 18S rDNA fragment sequences.
50 μ L PCR reaction systems include：10 × PCR buffer, 0.2mM dNTP, 2.5mM Mg2+, 1U Taq enzymes, mould
Plate DNA, forward and reverse primer, ddH2O.Response procedures are：95 DEG C of denaturation 5min, then by 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min
34 circulations of program, last 72 DEG C of extensions 10min.PCR primer electrophoresis in 1.5% agarose gel.After PCR primer is purified
Directly it is sequenced by Huada gene company.
3.2.1.3 data analysis
Using Blast functions, resulting sample 18S rDNA fragment sequences are compared with GenBank databases.
The sequence of similar algae on GenBank is downloaded, gene order is compared using Clustal X, be aided with SEAVIEW craft school
Just, morphological development tree is built using MEGA5, analyzes the systematic growth position of sample.
3.2.2 interpretation of result
3.2.2.1 sequence analysis
Through sequencing, as shown in SEQ ID NO.1, clip size is sample 18S rDNA fragment sequences to be identified
1693bp.Data in this sequence and NCBI Genbank databases are carried out into sequence homology to compare, find algae strain with
Recently, sequence homology can reach 100% to Chlorella sorokiniana affiliations.
3.2.2.2 Phylogenetic Analysis
(the i.e. 6-1 algaes of phylogenetic tree (Fig. 2) display testing sample Contig 1 built based on 18S rDNA fragment sequences
Strain) with Chlorella sorokiniana affiliations closely, on same evolutionary branching.
According to microexamination and molecular sequences sequencing result comprehensive analysis, show that testing sample is a kind of chlorella
Chlorella sorokiniana, are under the jurisdiction of Chlorophyta (Chlorophyta), Chlorophyceae (Chlorophyceae), Chlorococcum
Mesh (Chlorococcales), chlorella section (Chlorellaceae), Chlorella (Chlorella).
The sequence analysis result of biological property analysis and 18s rDNA based on bacterial strain, algae strain 6-1 is accredited as bead
Algae, is named as chlorella (Chlorella sorokiniana) BL-ch1, is preserved in Chinese Typical Representative on November 8th, 2016
Culture collection, address：Wuchang, wuhan Luo Jia Shan, its deposit number is：CCTCC M 2016626.
Embodiment 2：Application in chlorella production albumen of the invention
The determination of algae powder amount needed for 1 determining the protein quantity
1.1 test materials
Algae strain：The embodiment of the present invention 1 screens isolated chlorella (Chlorella sorokiniana) BL-ch1；
Culture medium：Zhu Shi 10, formula：Sodium nitrate 0.1g, dipotassium hydrogen phosphate 0.01g, magnesium sulfate 0.25g, sodium carbonate
0.02g, sodium metasilicate 0.025g, iron chloride 0.008g, water 1L.
1.2 test methods
Biomass is detected --- measuring cup method；
Protein content is detected --- Kjeldahl's method.
1.3 test procedures
No. 10 culture mediums of Zhu Shi are prepared, in inoculation algae strain BL-ch1 to culture medium, 33 DEG C are cultivated 5 days, by algae solution 5000r/
Min is centrifuged 5min, concentration gained algae mud, and with pure washing three times, during algae mud all then moved into measuring cup, 70 DEG C dry
Algae powder, weighs and calculates biomass, and algae powder surveys protein content.
1.4 result of the tests
The results are shown in Table 8.
The algae silty amount of table 8 and protein content
As can be seen from Table 8, chlorella BL-ch1 protein contents measure bead for 67% or so, 0.13g-0.24g algae powder
Algae BL-ch1 protein content no significant differences.
The optimization of 2 production albumen used mediums
2.1 test methods
On the basis of No. 10 culture mediums of Zhu Shi, glucose, dipotassium hydrogen phosphate, urea, potassium nitrate and sweet are further added
Propylhomoserin, by orthogonal design, the prescription to culture medium is optimized, and the formula composition of orthogonal design is as shown in table 9.
The orthogonal design formula of table 9 is constituted
2.2 result of the tests
The embodiment of the present invention 1 is screened into isolated chlorella (Chlorella sorokiniana) BL-ch1 inoculations
To the culture medium of different numberings, 33 DEG C are cultivated 5 days, and algae solution 5000r/min is centrifuged into 5min, concentration gained algae mud, with pure
Washing three times, during algae mud all then moved into measuring cup, 70 DEG C dry to obtain algae powder, weigh and calculate biomass, and algae powder is surveyed albumen and contained
Amount, the results are shown in Table 10.
The culture medium prescription of table 10 optimizes
As can be seen from Table 10, it is formula 9 that culture medium is preferably formulated, and formula composition is：No. 10 culture mediums+1 ‰ of Zhu Shi
The glycine of+0.1 ‰ urea of ‰ dipotassium hydrogen phosphate of glucose+0.1+0.1 ‰.Algae strain is carried out using the culture medium prescription after optimization
Culture, is cultivated relative to No. 10 culture mediums of Zhu Shi, and its biomass can improve 47.54%, and protein content improves 2.98%.
The optimization of 3 high yield protein pellet algae condition of culture
3.1 test materials
Algae strain：The embodiment of the present invention 1 screens isolated chlorella (Chlorella sorokiniana) BL-ch1；
Culture medium：Optimization wild Oryza species formula：Sodium nitrate 0.1g, dipotassium hydrogen phosphate 0.11g, magnesium sulfate 0.25g, sodium carbonate
0.02g, sodium metasilicate 0.025g, iron chloride 0.008g, 1g glucose, 0.1g urea, 0.1g glycine water 1L.
3.2 test methods
Chlorophyll is detected --- ethanol extraction method；
Biomass is detected --- measuring cup method；
Protein content is detected --- Kjeldahl's method.
Different vaccination amount, the influence of liquid amount, initial pH, cultivation temperature to chlorella protein yield are investigated respectively.
3.3 result of the tests
3.3.1 the influence (continuously cultivating 5d) of different vaccination amount, liquid amount to chlorophyll
The different vaccination amount of table 11, influence of the liquid amount to chlorophyll (μ g/L)
As can be seen from Table 11, the inoculum concentration of algae strain is preferably 5%-10%, and liquid amount is advisable with 600mL/L.
3.3.2 different vaccination amount, liquid amount are to biomass, the influence (continuously cultivating 5d) of protein content
The different vaccination amount of table 12, liquid amount are to biomass, the influence of protein content
As can be seen from Table 12, different vaccination amount and liquid amount do not make significant difference to algae strain biomass and protein content.
3.3.3 change (continuously cultivate 6d) of the different initial pH values to pH value in growth course
Change of the different initial pH values of table 13 to pH value in growth course
As can be seen from Table 13, in incubation pH value into continuous ascendant trend, chlorella BL-ch1 is in growth course
In its pH value up to more than 9.
3.3.4 influence (continuously cultivate 6d) of the different initial pH values to algae strain chlorophyll
Influence of the different initial pH values of table 14 to algae strain chlorophyll (μ g/L)
As can be seen from Table 14, chlorella BL-ch1 chlorophyll is fallen after rising, and most suitable initial pH value is 6.00.
3.3.5 different initial pH values are to biomass, the influence of protein content
The different initial pH values of table 15 are to biomass, the influence of protein content
As can be seen from Table 15, different initial pH values influence not significantly, with initial pH on chlorella BL-ch1 protein contents
Be worth for 7 when it is slightly excellent.
3.3.6 the influence (continuously cultivating 6d) of different temperatures and condition of culture to chlorophyll
The influence of the different temperatures of table 16 and condition of culture to chlorophyll (μ g/L)
As can be seen from Table 16, growth tendency is optimal at 33 DEG C for chlorella BL-ch1.
3.3.7 different temperatures and condition of culture are to biomass, the influence of protein content
The different temperatures of table 17 and condition of culture are to biomass, the influence of protein content
As can be seen from Table 17, chlorella BL-ch1 biomass and protein content highest in 28 DEG C of illumination.
Thereby determine that, high yield protein pellet algae BL-ch1 optimal condition of culture is：Inoculum concentration 10%, liquid amount 600mL/
L, initial pH value 7.0,28 DEG C -33 DEG C of temperature.
The measure of 4 high yield protein pellet algae growth tendencies and protein content
The chlorella BL-ch1 growth tendencies of table 18 and protein content
As can be seen from Table 18, high after pH value is first low, biomass is incremented by always, reaches 1.5660g/L, chlorophyll within the 8th day
Changes of contents trend is identical with biomass, and protein content gradually rises, and 70% or so is substantially remained in after five days, reaches as high as
71%.
5 high yield protein pellet algae analysis of amino acids
5.1 test materials
(algae solution distills water washing three to chlorella BL-ch1 algaes powder prepared by the present invention through 5000 turns/min, 5min centrifugations
It is secondary, 65 C overnights drying gained)
5.2 assay methods
Being posted after sample preparation success to Shandong University carries out Contents of Amino Acids, and instrument is Hitachi's L-8900 types
High speed automatic amino acid analyzer.
5.3 amino acid analysises are reported
Amino acid content in the chlorella BL-ch1 algae powder of table 19
Result display gained chlorella contains amino acid classes up to 17 kinds.
5.4 chlorella BL-ch1 algaes powder and amino acid content contrast in fish meal
The chlorella BL-ch1 algaes powder of table 20 and amino acid content contrast in fish meal
As can be seen from Table 20, compare with fish meal, the algae strain valine of BL-ch1, leucine are higher than fish meal, it is threonine, different
Leucine, phenylalanine are similar to fish meal, and methionine, histidine are less than fish meal.
To sum up, the chlorella BL-ch1 of present invention screening can carry out Heterotrophic culture, and contain by albumen after formulation optimization
Amount reaches as high as more than 69%, and containing 17 kinds of amino acid, two of which amino acid is higher than amino acid content, three kinds of ammonia in fish meal
Base acid content is substantially close with amino acid content in fish meal.Therefore, the chlorella powder of present invention screening can be as aquatic livestock
Open-mouthed bait and part substitute fish meal.
Embodiment 3：Application of the chlorella of the invention in excrement sewage of degrading
1 experimental design
Experimental design is as shown in table 21.
The chlorella of table 21 degraded excrement sewage experimental design
The cultural method of algae solution：Algae strain is the algae strain BL-ch1 of the screening of the embodiment of the present invention 1, and inoculum concentration is 10% (volume
Fraction), liquid amount 600mL/L, initial pH value 7.0,28 DEG C -33 DEG C of temperature.
This experiment considers that excrement dirt smell and sampling are convenient, employs box with cover, and it is poor to do illuminance with 3L triangular flasks
Different contrast, as shown in Figure 3.
2 experimental results
2.1 pairs of degradeds (total nitrogen content mg/L) of total nitrogen
Result is as shown in table 22.
Degradation results of the table 22 to total nitrogen
As can be seen from Table 22：Treatment 4,5,6, i.e. algae are dirty than being 1：1、4：1、8：The degradation rate of 1 pair of total nitrogen is reachable
More than 80%.
2.2 pairs of degradeds of total phosphorus (total phosphorus content content mg/L)
Result is as shown in table 23.
Degradation results of the table 23 to total phosphorus
As can be seen from Table 23：4,5,6 pairs of degradeds of total phosphorus for the treatment of are up to more than 40%, wherein 5 pairs of total phosphorus for the treatment of turn
Change up to 90%.
2.3 pairs of degradeds of ammonia nitrogen (ammonia-nitrogen content content mg/L)
Result is as shown in table 24.
Degradation results of the table 24 to ammonia nitrogen
As can be seen from Table 24：Treatment 4,5,6 i.e. algae is dirty than being 1：1、4：1、8：1 pair of degradation rate of ammonia nitrogen up to 90% with
On.
The measure (chlorophyll content μ g/L) of 2.4 chlorophyll
Result is as shown in Table 25.
The measurement result of the chlorophyll of table 25
As can be seen from Table 25：Although slightly fluctuation still can embody becoming for growth on 4,5,6 chlorophyll measurings for the treatment of
Gesture, with reference to culture color change, illustrates algae strain well-grown.
Consider result above, in phosphorus decomposing, drop nitrogen, total nitrogen initial value should be for chlorella BL-ch1 of the invention
400mg/L or so is more suitable.
SEQUENCE LISTING
<110>Shandong Baolai-leelai Bio-engineering Co., Ltd.
<120>The albuminiferous chlorella of one plant height and its cultural method and application
<130> 2016
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1693
<212> DNA
<213>Algae strain 6-1
<400> 1
tgtctaagta taaactgctt tatactgtga aactgcgaat ggctcattaa atcagttata 60
gtttatttga tggtacctac tactcggata cccgtagtaa atctagagct aatacgtgcg 120
taaatcccga cttctggaag ggacgtattt attagataaa aggccgaccg ggctctgccc 180
gactcgcggt gaatcatgat aacttcacga atcgcatggc cttgcgccgg cgatgtttca 240
ttcaaatttc tgccctatca actttcgatg gtaggataga ggcctaccat ggtggtaacg 300
ggtgacggag gattagggtt cgattccgga gagggagcct gagaaacggc taccacatcc 360
aaggaaggca gcaggcgcgc aaattaccca atcctgacac agggaggtag tgacaataaa 420
taacaatact gggccttttc aggtctggta attggaatga gtacaatcta aaccccttaa 480
cgaggatcaa ttggagggca agtctggtgc cagcagccgc ggtaattcca gctccaatag 540
cgtatattta agttgctgca gttaaaaagc tcgtagttgg atttcgggtg gggcctgccg 600
gtccgccgtt tcggtgtgca ctggtagggc ccaccttgtt gccggggacg ggctcctggg 660
cttcactgtc cgggactcgg agtcggcgct gttactttga gtaaattaga gtgttcaaag 720
caggcctacg ctctgaatac attagcatgg aataacacga taggactctg gcctatcctg 780
ttggtctgta ggaccggagt aatgattaag agggacagtc gggggcattc gtatttcatt 840
gtcagaggtg aaattcttgg atttatgaaa gacgaactac tgcgaaagca tttgccaagg 900
atgttttcat taatcaagaa cgaaagttgg gggctcgaag acgattagat accgtcctag 960
tctcaaccat aaacgatgcc gactagggat cggcggatgt ttcttcgatg actccgccgg 1020
caccttatga gaaatcaaag tttttgggtt ccggggggag tatggtcgca aggctgaaac 1080
ttaaaggaat tgacggaagg gcaccaccag gcgtggagcc tgcggcttaa tttgactcaa 1140
cacgggaaaa cttaccaggt ccagacatag tgaggattga cagattgaga gctctttctt 1200
gattctatgg gtggtggtgc atggccgttc ttagttggtg ggttgccttg tcaggttgat 1260
tccggtaacg aacgagacct cagcctgcta aatagtcacg gttggttcgc cagccggcgg 1320
acttcttaga gggactattg gcgactagcc aatggaagca tgaggcaata acaggtctgt 1380
gatgccctta gatgttctgg gccgcacgcg cgctacactg atgcattcaa cgagcctagc 1440
cttggccgag aggcccgggt aatctttgaa actgcatcgt gatggggata gattattgca 1500
attattaatc ttcaacgagg aatgcctagt aagcgcaagt catcagcttg cgttgattac 1560
gtccctgccc tttgtacaca ccgcccgtcg ctcctaccga ttgggtgtgc tggtgaagtg 1620
ttcggattgg cgaccggggg cggtctccgc tctcggccgc cgagaagttc attaaaccct 1680
cccacctaga gga 1693
Claims (10)
1. a chlorella, is named as chlorella (Chlorella sorokiniana) BL-ch1, on November 8th, 2016
It is preserved in China typical culture collection center, address：Wuchang, wuhan Luo Jia Shan, its deposit number is：CCTCC M
2016626。
2. the cultural method of the chlorella described in claim 1, it is characterised in that step is：Algae strain is seeded on culture medium,
Inoculum concentration is 5-15%, and liquid amount is 400-800mL/L, and initial pH is 6.0-8.0,28 DEG C -33 DEG C of cultivation temperature.
3. cultural method as claimed in claim 2, it is characterised in that the composition of the culture medium is：In No. 10 cultures of Zhu Shi
1 ‰ glucose, 0.1 ‰ dipotassium hydrogen phosphates, 0.1 ‰ urea and 0.1 ‰ glycine are added in base.
4. cultural method as claimed in claim 2, it is characterised in that inoculum concentration is 10%, and liquid amount is 600mL/L, initially
PH is 7.0.
5. application of the chlorella described in claim 1 in sewage disposal.
6. application as claimed in claim 5, it is characterised in that the sewage disposal includes total nitrogen, the total phosphorus in degraded sewage
And ammonia nitrogen.
7. application of the chlorella described in claim 1 in protein production.
8. application as claimed in claim 7, it is characterised in that application process is：By the chlorella described in claim 1 by power
Profit requires that the cultural method described in 2 is cultivated, and obtains algae solution；Algae solution is centrifuged, is concentrated, dried, obtain final product the algae containing albumen
Powder.
9. application of the chlorella described in claim 1 in aquatic livestock open-mouthed bait and/or fish meal substitute is prepared.
10. it is a kind of to give birth to albuminiferous method, it is characterised in that by the chlorella described in claim 1 as described in claim 2
Cultural method carries out culture 4-6 days, obtains algae solution；Algae solution is centrifuged, is concentrated, dried.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611255690.0A CN106754390B (en) | 2016-12-30 | 2016-12-30 | The albuminiferous chlorella of one plant height and its cultural method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611255690.0A CN106754390B (en) | 2016-12-30 | 2016-12-30 | The albuminiferous chlorella of one plant height and its cultural method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106754390A true CN106754390A (en) | 2017-05-31 |
| CN106754390B CN106754390B (en) | 2019-07-26 |
Family
ID=58953410
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611255690.0A Active CN106754390B (en) | 2016-12-30 | 2016-12-30 | The albuminiferous chlorella of one plant height and its cultural method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106754390B (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107345208A (en) * | 2017-06-19 | 2017-11-14 | 南京师范大学 | A kind of low-temperature epitaxy green alga and its application for removing nitrogen and phosphorus in sewage |
| CN107541466A (en) * | 2017-08-02 | 2018-01-05 | 海南大学 | A kind of chlorella of resistance to strong light of salt tolerant high temperature resistant for discharging aroma type smell and its application |
| CN107916226A (en) * | 2017-10-13 | 2018-04-17 | 浙江省农业科学院 | The screening of one strain capable of high-efficiency processing pig farm livestock and poultry sewage and the microalgae of recycling |
| CN108753623A (en) * | 2018-06-12 | 2018-11-06 | 兰溪市沉默生物科技有限公司 | A kind of chlorella store method |
| JP2020174579A (en) * | 2019-04-18 | 2020-10-29 | 三菱重工機械システム株式会社 | Microalga culture method and microalga culture device |
| CN112538431A (en) * | 2020-12-16 | 2021-03-23 | 范秀娟 | Chlorella pyrenoidosa and biological environment restoration liquid prepared from chlorella pyrenoidosa and application of chlorella pyrenoidosa |
| CN113481102A (en) * | 2021-08-19 | 2021-10-08 | 山西农业大学 | Chlorella sorokiniana strain as well as culture method and application thereof |
| CN113717854A (en) * | 2021-09-01 | 2021-11-30 | 东北农业大学 | Oil-producing chlorella ZM-5 and application thereof |
| CN114854596A (en) * | 2022-05-26 | 2022-08-05 | 珠海元育生物科技有限公司 | Chlorella protothecoides mutant strain with high protein yield and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1282511A (en) * | 1999-08-03 | 2001-02-07 | 中国科学院水生生物研究所 | Method for fixating shifting sand with algal eremophyte |
| CN101705190A (en) * | 2009-11-29 | 2010-05-12 | 中南大学 | Chlorella sorokiniana CS-01 and culture method thereof for producing grease |
| CN102943045A (en) * | 2012-10-12 | 2013-02-27 | 广东省微生物研究所 | Medium promoting growth of chlorella vulgaris and method for culturing chlorella culture therewith |
| CN103555588A (en) * | 2013-11-10 | 2014-02-05 | 湘潭大学 | Culture method of chlorella cells with high density and high oil content |
| CN103881923A (en) * | 2012-12-21 | 2014-06-25 | 中国中化股份有限公司 | Method for culturing microalgae by using coking wastewater |
| CN104152357A (en) * | 2014-08-06 | 2014-11-19 | 华南理工大学 | High-density culture method for improving chlorophyll and protein content of chlorella at same time |
| CN104387171A (en) * | 2014-11-07 | 2015-03-04 | 南京农业大学 | Method for producing organic seaweed fertilizer employing algae processing waste and prepared fertilizer |
-
2016
- 2016-12-30 CN CN201611255690.0A patent/CN106754390B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1282511A (en) * | 1999-08-03 | 2001-02-07 | 中国科学院水生生物研究所 | Method for fixating shifting sand with algal eremophyte |
| CN101705190A (en) * | 2009-11-29 | 2010-05-12 | 中南大学 | Chlorella sorokiniana CS-01 and culture method thereof for producing grease |
| CN102943045A (en) * | 2012-10-12 | 2013-02-27 | 广东省微生物研究所 | Medium promoting growth of chlorella vulgaris and method for culturing chlorella culture therewith |
| CN103881923A (en) * | 2012-12-21 | 2014-06-25 | 中国中化股份有限公司 | Method for culturing microalgae by using coking wastewater |
| CN103555588A (en) * | 2013-11-10 | 2014-02-05 | 湘潭大学 | Culture method of chlorella cells with high density and high oil content |
| CN104152357A (en) * | 2014-08-06 | 2014-11-19 | 华南理工大学 | High-density culture method for improving chlorophyll and protein content of chlorella at same time |
| CN104387171A (en) * | 2014-11-07 | 2015-03-04 | 南京农业大学 | Method for producing organic seaweed fertilizer employing algae processing waste and prepared fertilizer |
Non-Patent Citations (2)
| Title |
|---|
| 王晓晨: "小球藻油脂积累的影响条件及油脂提取方法的研究", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 * |
| 陈曦: "利用膜式光生物反应器培养微藻处理沼液氮和磷", 《厦门大学硕士学位论文》 * |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107345208B (en) * | 2017-06-19 | 2020-05-05 | 南京师范大学 | Low-temperature growth green algae and application thereof in removing nitrogen and phosphorus in sewage |
| CN107345208A (en) * | 2017-06-19 | 2017-11-14 | 南京师范大学 | A kind of low-temperature epitaxy green alga and its application for removing nitrogen and phosphorus in sewage |
| CN107541466A (en) * | 2017-08-02 | 2018-01-05 | 海南大学 | A kind of chlorella of resistance to strong light of salt tolerant high temperature resistant for discharging aroma type smell and its application |
| CN107541466B (en) * | 2017-08-02 | 2019-07-05 | 海南大学 | It is a kind of discharge aroma type smell the resistance to strong light of salt tolerant high temperature resistant chlorella and its application |
| CN107916226B (en) * | 2017-10-13 | 2020-11-27 | 浙江省农业科学院 | Screening of microalgae capable of efficiently treating livestock and poultry sewage in pig farm and realizing resource utilization |
| CN107916226A (en) * | 2017-10-13 | 2018-04-17 | 浙江省农业科学院 | The screening of one strain capable of high-efficiency processing pig farm livestock and poultry sewage and the microalgae of recycling |
| CN108753623A (en) * | 2018-06-12 | 2018-11-06 | 兰溪市沉默生物科技有限公司 | A kind of chlorella store method |
| JP2020174579A (en) * | 2019-04-18 | 2020-10-29 | 三菱重工機械システム株式会社 | Microalga culture method and microalga culture device |
| CN112538431A (en) * | 2020-12-16 | 2021-03-23 | 范秀娟 | Chlorella pyrenoidosa and biological environment restoration liquid prepared from chlorella pyrenoidosa and application of chlorella pyrenoidosa |
| CN112538431B (en) * | 2020-12-16 | 2023-08-11 | 范秀娟 | Chlorella pyrenoidosa, preparation method of biological environment restoration liquid and application thereof |
| CN113481102A (en) * | 2021-08-19 | 2021-10-08 | 山西农业大学 | Chlorella sorokiniana strain as well as culture method and application thereof |
| CN113717854A (en) * | 2021-09-01 | 2021-11-30 | 东北农业大学 | Oil-producing chlorella ZM-5 and application thereof |
| CN113717854B (en) * | 2021-09-01 | 2023-05-02 | 东北农业大学 | Chlorella oleaginous ZM-5 and application thereof |
| CN114854596A (en) * | 2022-05-26 | 2022-08-05 | 珠海元育生物科技有限公司 | Chlorella protothecoides mutant strain with high protein yield and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106754390B (en) | 2019-07-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106754390B (en) | The albuminiferous chlorella of one plant height and its cultural method and application | |
| Shen et al. | Microalgae mass production methods | |
| Cheng et al. | Screening of the dominant Chlorella pyrenoidosa for biofilm attached culture and feed production while treating swine wastewater | |
| Economou et al. | Lipid production by the filamentous cyanobacterium Limnothrix sp. growing in synthetic wastewater in suspended-and attached-growth photobioreactor systems | |
| CN109234167B (en) | Chlorella and application thereof in biogas slurry purification | |
| CN105331552B (en) | One plant of efficient denitrification acinetobacter calcoaceticus novel species and its application | |
| Pandey et al. | Bioprospecting microalgae from natural algal bloom for sustainable biomass and biodiesel production | |
| Huang et al. | A rapid sampling technique for isolating highly productive lipid-rich algae strains from environmental samples. | |
| Davis et al. | Characterization of a novel strain of Tribonema minus demonstrating high biomass productivity in outdoor raceway ponds | |
| Selvan et al. | Eco-friendly approach for tannery effluent treatment and CO2 sequestration using unicellular green oleaginous microalga Tetradesmus obliquus TS03 | |
| Hariz et al. | Growth and biomass production of native microalgae Chlorella sp., chlamydomonas sp. and Scenedesmus sp. cultivated in Palm Oil Mill Effluent (POME) at different Cultivation conditions | |
| CN107384800B (en) | Clermann dipteridium krameri (Chlamydododium SP.) and its use | |
| Camarena-Bernard et al. | Native microalgae from eutrophic water: potential for wastewater treatment, low-cost biomass, and lipid production | |
| CN113234608B (en) | Abnormal yeast Weikehan for degrading nitrite | |
| CN105349447B (en) | Vibrio strains and uses thereof | |
| Patil et al. | Isolation of microalgae for biomass and lipid enhancement through Plackett-Burmann design | |
| CN103571755B (en) | A kind of acquisition of chain band algae NMX451 and the method for genetic transformation | |
| Alshareef et al. | Identification and growth characterization of native microalgae isolated from different environments of Saudi Arabia | |
| Bruno et al. | Biomass and lipid productivity by four fresh water microalgae in photoreactor | |
| Sweiss | Microalgae for wastewater treatment and biomass production from bioprospecting to biotechnology | |
| CN105543099B (en) | One plant of oily ball algae Graesiella sp.WBG-1 and separating screening method and application | |
| CN111943360B (en) | Prevention and treatment method for algal bloom disasters of eutrophic water body | |
| CN105199961B (en) | The sharp top Fusariumsp of one kind and its application | |
| Divakaran et al. | Algae Cultivation Strategies: An Overview | |
| CN113583906B (en) | Application of pseudomonas B5 in algae removal |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |