CN107746809A - The method for improving algae bio amount - Google Patents
The method for improving algae bio amount Download PDFInfo
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- CN107746809A CN107746809A CN201711324639.5A CN201711324639A CN107746809A CN 107746809 A CN107746809 A CN 107746809A CN 201711324639 A CN201711324639 A CN 201711324639A CN 107746809 A CN107746809 A CN 107746809A
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- 241000195493 Cryptophyta Species 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 28
- 241000195649 Chlorella <Chlorellales> Species 0.000 claims abstract description 54
- 239000001963 growth medium Substances 0.000 claims abstract description 33
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 9
- 239000008103 glucose Substances 0.000 claims abstract description 9
- 239000011734 sodium Substances 0.000 claims abstract description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 6
- 230000003203 everyday effect Effects 0.000 claims abstract description 6
- 239000011790 ferrous sulphate Substances 0.000 claims abstract description 6
- 235000003891 ferrous sulphate Nutrition 0.000 claims abstract description 6
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims abstract description 6
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims abstract description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 6
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- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 6
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- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims description 22
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- 241000193830 Bacillus <bacterium> Species 0.000 claims description 16
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- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims 1
- 238000005286 illumination Methods 0.000 claims 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 abstract description 10
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- 239000000843 powder Substances 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 3
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
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- 241000726221 Gemma Species 0.000 description 2
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- 150000003722 vitamin derivatives Chemical class 0.000 description 2
- 235000019786 weight gain Nutrition 0.000 description 2
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- 238000004820 blood count Methods 0.000 description 1
- YYRMJZQKEFZXMX-UHFFFAOYSA-L calcium bis(dihydrogenphosphate) Chemical compound [Ca+2].OP(O)([O-])=O.OP(O)([O-])=O YYRMJZQKEFZXMX-UHFFFAOYSA-L 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
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- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
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- 210000003743 erythrocyte Anatomy 0.000 description 1
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- -1 it by sunlight Substances 0.000 description 1
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Classifications
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- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- 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
-
- 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
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The present invention provides the method for improving algae bio amount, and its step is as follows：（1）Picking chlorella algae kind illuminance 5000lux, 26 DEG C of cultures, is shaken every day triangular flask 2~3 times into the triangular flask containing growth medium, to be grown to exponential phase, obtains seed liquor, every liter of the growth medium contains：Glucose 5g, dusty yeast 1g, ferrous sulfate 1g, Na2SO32g, sodium chloride 0.5g, potassium dihydrogen phosphate 1g, magnesium sulfate 1g, ammonium chloride 1g；（2）By step（1）The algae solution of acquisition is inoculated into according to 15% volume ratio to be expanded in culture medium；（3）Expand under conditions of culture is 20 100rpm in 20 ± 10 DEG C, 2000 ± 500LUX, shaking table vibration velocity and cultivate.The application cultural method simplicity is easily operated, and green, beneficial to large-scale production.
Description
Technical field
The invention belongs to biological technical field, and in particular to the method for improving algae bio amount.
The content of the invention
Microalgae refers to containing chlorophyll A and can carry out the general name of photosynthetic microorganism, and its individual is small, generally requires
Form could be distinguished under the microscope, and microalgae is widely distributed, land lake, maritime waters are distributed, and planktonic microalgae is to pond
The material circulation of cultivation and energy flow have the function that it is very important, it for maintenance pond ecosystem normal function,
Stable pond environment is indispensable.Chlorophyta and Cyanophyta phytoplankton are all likely to become the sociales in natural water body,
And for cultivating pool, people are more desirable to can be in water body to the beneficial Chlorophyta phytoplankton of cultivation object in cultivating pool
Advantage is occupied, excellent planktonic microalgae algae mutually during population stabilization, biomass sustainable growth, can promote nutrition in water body
The decomposition and conversion of salt, reduce and eliminate ammonia nitrogen, a variety of noxious materials such as cultured water, organic pollution；But also it can pass through
Photosynthesis produces oxygen and increases the dissolved oxygen of breeding water body, promotes the oxidation Decomposition of oxygen-consuming organic matter being rich in water body.
Chlorella (Chlorella) is the general natural disposition monoplast green alga of Chlorophyta Chlorella, is a kind of spherical unicellular light
Algae class, 3~8 microns of diameter, is one of life earliest on the earth, is a kind of efficient photosynthetic before appearing in more than 20 hundred million years
Plant, it is wide with photoautotrophy growth and breeding, distributed pole.Chlorella is lived in fresh water, it by sunlight, water and carbon dioxide,
To divide the vigorous fertility of 4 cells every 20 hours, ceaselessly solar energy is converted into contain a variety of nutrition
The frond of composition, and substantial amounts of oxygen is discharged in propagation；And its photosynthetic capacity is higher than other plant more than 10 times.Bead
Algae acts not only as the outstanding natural bait of aquatic economic animal, while can also absorb the elements such as the nitrogen in water, phosphorus, reduces
The eutrophication of water body, purifies water.Moreover, chlorella is rich in grease, can be used for producing biodiesel；Also contain
There are some hydrocarbons, gasoline can be processed into after extraction, diesel oil uses.If with sewage mass propgation chlorella and make it have height
Fat content and hydrocarbon content, it can not only slow down water quality deterioration, and the generation mass-energy that can make a living provides a large amount of quality raw materials.
Instantly, the growth of microalgae belongs to popular research subject, and the optimization of microdisk electrode condition and oil and fat accumulation amount turn into
The emphasis of research.At present, the large-scale culture of microalgae mainly has two approach --- autotrophy culture and heterotrophic fermentation, autotrophy culture
GHG carbon dioxide can be fixed and discharge oxygen, it is environmentally friendly, but due to the mutual masking between microalgae cell
Effect, the utilization of luminous energy suffer from great limitation.Cell concentration is higher, and this shadowing effect embodies more obvious, seriously
The growth and Fatty synthesis, this High Density Cultivation for making to realize oil-containing micro-algae in photo-biological reactor for influenceing cell become ten
Divide difficulty.For Heterotrophic culture, the growth of cell relies primarily on absorption of the cell to organic carbon source, due to its not light
Limitation, therefore can realize that high density fermentation efficiently synthesizes with fatty by stream plus organic carbon.Although Heterotrophic culture has
The advantages that growth rate is fast, cultivation cycle is short, incubation is easy to control, but produce the main of biodiesel using heterotrophic microalgae and ask
It is raw material that topic, which is that this method relies on organic carbon source (such as glucose, starch), adds production cost.Microalgae in the prior art
Production be to utilize photoproduction reaction device mostly, their research emphasis by general production technology research turn to its deep processing with
The research of particular matter extraction, the technology of China Taiwan production chlorella is also quite ripe, the products of some chlorellas by
Broad masses are received, although the 1960s has been carried out research to chlorella on ground in China, due to can't
Being mass produced reduces cost, so, the production primary product stage is only rested on, causes input-output ratio extremely uneven
Weighing apparatus, while also constrain and the process of developmental research is carried out to it, the extensive open type cultivation pattern biomass in ground reaches in China
10000000/ml or so, by input, high production is low is limited, larger with foreign countries cultivation level disparities, in addition, prior art
There is the method that biodiesel is prepared using amylorrhexis culture heterotrophism algae fast pyrogenation.The patent is using low-quality foodstuff starch as original
Material, nutrient solution is prepared using enzymatic starch D/W, then heterophytic chlorella is obtained by heterotrophism transformation technology；Then
With the heterotrophism frustule fast pyrogenation of high fat content, the biodiesel of acquisition high yield and high quality.This method selects low-quality
Glucose sugar is provided after foodstuff starch hydrolysis and is used as organic carbon source, cost is higher, it is difficult to realizes large-scale production.
In recent years, aquaculture industry of China is able to fast development, and breeding wastewater is because of having containing substantial amounts of nitrogen, phosphorus and high concentration
Machine thing, is considered as high-concentration sewage, and directly discharge can cause body eutrophication, polluted source.In addition, breeding wastewater carries greatly
The pathogen of amount, extremely dense stink is distributed, increasingly environment is caused seriously to pollute.Preserve the ecological environment very urgent.
Microalgae itself oil content is high, can use various aquifer cultivations, has and is not take up the advantages such as arable land, utilizes breeding wastewater
Microalgae is cultivated, the problem of environmental pollution of direct discharging of waste water is not only solved, also achieves the recycling of breeding wastewater, it is right
The energy and the aspect of environment two generate active influence.
The content of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide improve algae bio amount.The present invention is using such as
What lower technical scheme was realized：
A kind of method for improving algae bio amount, it is characterised in that step is as follows：
（1）Picking chlorella algae kind is into the triangular flask for filling 100mL growth mediums, illuminance 5000lux, 26 DEG C of cultures,
It is shaken every day triangular flask 2~3 times.The gradual greening of nutrient solution color can be observed after 3~4d.It is to be grown to exponential phase, obtain
To seed liquor (algae solution), every liter of the growth medium contains：Glucose 5g, dusty yeast 1g, ferrous sulfate 1g, Na2SO32g, chlorine
Change sodium 0.5g, potassium dihydrogen phosphate 1g, magnesium sulfate 1g, ammonium chloride 1g.
The chlorella is chlorella.Sp ATCC30412
（2）By step（1）The algae solution of acquisition according to 15% volume ratio be inoculated into expand culture medium in, it is described expansion culture medium into
It is divided into：Bacteria residue hydrolyzate：Growth medium：Bacillus nutrient solution is according to volume ratio=5-6:1-2:1-2 is mixed；
The bacteria residue hydrolyzate is：Propylhomoserin mother liquor is obtained after amino acid fermentation is terminated mycoprotein is collected by centrifugation, adjust thalline
The solid content of albumen is 8%, adds 5-10wt% 5mol/L NaOH, water at normal temperature solution 5-8d, obtains bacteria residue hydrolyzate.
The bacillus nutrient solution is：Take bacillus amyloliquefaciens（Bacillus amyloliquefaciens）ATCC
23843, first activation culture, shaken cultivation in triangular flask is then seeded into, it is 1 × 10 to be cultivated to concentration8Individual/ml bacterium
Liquid；
（3）Expand under conditions of culture is 20-100 rpm in 20 ± 10 DEG C, 2000 ± 500LUX, shaking table vibration velocity and cultivate；
Traditional bead algae culture medium contains more than 20 kindizations such as macroelement, trace element, vitamin and some growth conditioning agent
Medicine is learned, troublesome in poeration, workload is big, particularly some micro constitutents and element, and dosage is few, and error is big, and inconvenience transport
With commercialization, market operation.Growth medium disclosed by the invention, the nutrient solution comprise only 8 kinds of nutrients, user
Just, workload is small, storage, convenient transportation, is easy to the batch production of nutrient solution, standardization, the marketization, commercialization, Virtual production,
Micro constitutent and element are quantitatively accurate, and error is small.And it can also realize preferable chlorella culture effect；
The present invention adds cheap carbon source as Heterotrophic culture process after microalgae uses medium culture during expanding culture
Growth carbon source, the cheap carbon source is a kind of treated bacteria residue, and the carbon source is than simple addition glucose or Starch Hydrolysis
The heterotrophism such as liquid carbon source is more according to growth vigor, while the bacteria residue in standard biologic fermenting and producing is realized that circulation is sharp again by the inventive method
With solving the pollution problem of bacteria residue, moreover it is possible to turn waste into wealth, because bacteria residue is substantially solid matter, in order to more preferable quilt
Chlorella absorbs, and the application is more conducive to the absorption of nutriment by the way of basic hydrolysis；
Strain of the present invention can be from American Type Culture collection warehousing（ATCC）It is commercially available etc. commercial sources.
Strain of the present invention can obtain the bacterium solution of required concentration by the cultural method of routine, as space is limited, and
Do not repeat one by one.
Embodiment
Embodiment 1
A kind of method for improving algae bio amount, it is characterised in that step is as follows：
（1）Picking chlorella algae kind is into the triangular flask for filling 100mL growth mediums, illuminance 5000lux, 26 DEG C of cultures,
It is shaken every day triangular flask 2~3 times.The gradual greening of nutrient solution color can be observed after 3~4d.It is to be grown to exponential phase, obtain
To seed liquor (algae solution), every liter of the growth medium contains：Glucose 5g, dusty yeast 1g, ferrous sulfate 1g, Na2SO32g, chlorine
Change sodium 0.5g, potassium dihydrogen phosphate 1g, magnesium sulfate 1g, ammonium chloride 1g.
The chlorella is chlorella.Sp ATCC30412
（2）By step（1）The algae solution of acquisition according to 15% volume ratio be inoculated into expand culture medium in, it is described expansion culture medium into
It is divided into：Bacteria residue hydrolyzate：Growth medium：Bacillus nutrient solution is according to volume ratio=5:1:1 mixing；
The bacteria residue hydrolyzate is：Propylhomoserin mother liquor is obtained after amino acid fermentation is terminated mycoprotein is collected by centrifugation, adjust thalline
The solid content of albumen is 8%, adds 5wt% 5mol/L NaOH, water at normal temperature solution 5d, obtains bacteria residue hydrolyzate.
The bacillus nutrient solution is：Take bacillus amyloliquefaciens（Bacillus amyloliquefaciens）ATCC
23843, first activation culture, shaken cultivation in triangular flask is then seeded into, it is 1 × 10 to be cultivated to concentration8Individual/ml bacterium
Liquid；
（3）Expand under conditions of culture is 20-100 rpm in 20 ± 10 DEG C, 2000 ± 500LUX, shaking table vibration velocity and cultivate；
Embodiment 2
A kind of method for improving algae bio amount, it is characterised in that step is as follows：
（1）Picking chlorella algae kind is into the triangular flask for filling 100mL growth mediums, illuminance 5000lux, 26 DEG C of cultures,
It is shaken every day triangular flask 2~3 times.The gradual greening of nutrient solution color can be observed after 3~4d.It is to be grown to exponential phase, obtain
To seed liquor (algae solution), every liter of the growth medium contains：Glucose 5g, dusty yeast 1g, ferrous sulfate 1g, Na2SO32g, chlorine
Change sodium 0.5g, potassium dihydrogen phosphate 1g, magnesium sulfate 1g, ammonium chloride 1g.
The chlorella is chlorella.Sp ATCC30412
（2）By step（1）The algae solution of acquisition according to 15% volume ratio be inoculated into expand culture medium in, it is described expansion culture medium into
It is divided into：Bacteria residue hydrolyzate：Growth medium：Bacillus nutrient solution is according to volume ratio=3:1:1 mixing；
The bacteria residue hydrolyzate is：Propylhomoserin mother liquor is obtained after amino acid fermentation is terminated mycoprotein is collected by centrifugation, adjust thalline
The solid content of albumen is 8%, adds 10wt% 5mol/L NaOH, water at normal temperature solution 8d, obtains bacteria residue hydrolyzate.
The bacillus nutrient solution is：Take bacillus amyloliquefaciens（Bacillus amyloliquefaciens）ATCC
23843, first activation culture, shaken cultivation in triangular flask is then seeded into, it is 1 × 10 to be cultivated to concentration8Individual/ml bacterium
Liquid；
（3）Expand under conditions of culture is 20-100 rpm in 20 ± 10 DEG C, 2000 ± 500LUX, shaking table vibration velocity and cultivate；
Embodiment 3
The symbiosis experiment of chlorella and bacillus and the scale processing of bacteria residue：Using bacillus amyloliquefaciens and chlorella
Symbiosis culture chlorella more promotes the growth of chlorella, adds the system of chlorella and bacillus amyloliquefaciens, solves starch gemma
Collaboration facilitation is played in growth of the growth of bacillus to chlorella, it may be possible to because in growth course, frustule can be outside
Environment secretion in boundary is advantageous to the material of bacterial growth, and bacterium produces carbon dioxide and inorganic simultaneously using materials of these secretions
Salt promotes the growth of chlorella simultaneously again.
Fat content determines：Algae solution 3000r/min in stabilizer is centrifuged, algal gel cleaned with deionized water, so
65 degree of drying obtain algae powder in electric heating air blower drying box afterwards, will be placed in plug rub oral examination tube, add after algae powder fracturing cell walls
Absolute ethyl alcohol, mix, room temperature extraction 3h, during which suitably mix, supernatant moves into centrifuge tube, adds activated decoloration, and centrifugation is received
Collect supernatant, supernatant is boiled off into absolute ethyl alcohol in electric drying oven with forced convection, weighed, calculate fat content, fat content=grease contains
Amount/algae silty amount %；
Experimental group：According to the operation of step embodiment 1；
Contrast 1 group：Without using bacillus cogeneration system, that is, expand and bacillus amyloliquefaciens nutrient solution is deleted in culture medium, its
Remaining all same；
Contrast 2 groups：Expand culture medium and do not contain bacteria residue hydrolyzate, remaining all same；
Contrast 3 groups：Expand culture medium and only contain bacteria residue hydrolyzate and bacillus amyloliquefaciens nutrient solution, remaining all same；
4 groups are contrasted, expands series bacillus disclosed in culture medium selection addition CN2015100638727, remaining all same.
The above-mentioned start recording color after blake bottle culture, 2 days, microscope and red blood cell count are used every 24h
Plate counts to microalgae cell in each bottle.Carry out counting statistics by count results and draw growth curve to be compareed.As a result
It is shown in Table 1：
The symbiosis of table 1 is tested
| Blake bottle color | Chlorella cells concentration | Fat content wt% | |
| Experimental group | Blake bottle color virescence, color are gradually deepened after 2 days | 5.3×1010Individual/mL | 44.2 |
| Contrast 1 | 4th day blake bottle colors green | 1.3×109Individual/mL | 32.1 |
| Contrast 2 | Blake bottle color virescence, color are gradually deepened after 2 days | 4.7×109Individual/mL | 41.8 |
| Contrast 3 | 3rd day colors green | 1.8×109Individual/mL | 40.4 |
| Contrast 4 | 3rd day color virescence | 1.7×1010Individual/mL | 38.2 |
By above-mentioned experiment, bacillus and chlorella cogeneration system can produce bigger bead concentration of algae, 2 after inoculation
Its blake bottle color greening, and deeper green is shown than contrast experiment's 1-3 groups, the group, show the number of chlorella cells
Amount increases significantly than the latter, because the symbiosis of bacillus amyloliquefaciens and chlorella promotes relation, solves starch gemma bar
The presence of bacterium improves the growth microenvironment of chlorella so that the biomass of chlorella is more, and fat content shows compared with control group
Write.1 group is compareed, due to eliminating the symbiosis conditions of bacillus amyloliquefaciens so that the growth of chlorella is with respect to retardation, logarithmic phase
Biomass is remarkably decreased than experimental group, and the chlorella fat content produced is relatively low.The application experimental group is than right
Than 2 groups, bacteria residue nutrient solution is used in culture is expanded, has not only realized twice laid, and obtain than conventional medium
Similar result is achieved in sheet, than growth medium effect phase on fat content and the speed of growth and biomass
When it makes use of cheap carbon source, greatlys save the energy.3 groups are contrasted than experimental group, bacterium has been directly entered after algae solution is obtained
Cultivated in slag hydrolyzate and bacillus amyloliquefaciens nutrient solution, without the presence of any nutrient solution composition before, due to lacking
Certain domestication environment, causes chlorella growth slow than experimental group, thus final biomass is also affected；Contrast 4 groups
Than experimental group, the bacillus of symbiosis replaces with series bacillus by the bacillus amyloliquefaciens of the application, is obtained in biomass
Obtain and improved on fat content than without using helotism system, but in biomass and caused chlorella
On fat content, the application obtains more excellent effect, and ball frustule biomass improves 2.12 times, and fat content improves
15.7%, the chlorella and bacillus amyloliquefaciens for showing the application obtain more excellent synergy.
Embodiment 4
Growth medium screening test
BG-11 is the conventional culture medium of usually algae culture, however, its contain macroelement, trace element, vitamin and
Some growth conditioning agent etc., complex operation, workload is big, and chlorella has 15-20 kinds in the nutrient needed for growth course,
Most elements will not turn into it is restricted therefore, wherein C, N, P be growth essential element, growth and accumulation to chlorella have
Considerable influence, the application applicant delete choosing by orthogonal optimization test and obtain simple chlorella medium component glucose 5g, ferment
Female powder 1g, ferrous sulfate 1g, Na2SO32g, sodium chloride 0.5g, potassium dihydrogen phosphate 1g, magnesium sulfate 1g, ammonium chloride 1g, than existing
There is technology, easy to use, preparation workload is small, and can also realize preferable chlorella culture effect.
Experimental group：Picking chlorella algae kind is into the triangular flask for filling 100mL growth mediums, illuminance 5000lux,
26 DEG C of cultures, are shaken every day triangular flask 2~3 times.It is to be grown to exponential phase, obtain seed liquor (algae solution)；
Compare 1 group：Growth medium is replaced with into BG-11, remaining same experimental group；
Compare 2 groups：Growth medium culture is prepared as nutrient source using the ammonium hydrogen carbonate of conventional Tu Chi cultures and calcium superphosphate, its
Remaining same experimental group.
It the results are shown in Table 2
The growth medium screening test of table 2
| The speed of growth（After 5d） | Growth conditions | |
| Experimental group | 0.338d-1 | Greening after 3d, leaf green content significantly increase |
| Compare 1 group | 0.347 d-1 | Greening after 3d, leaf green content significantly increase |
| Compare 2 groups | 0.102 d-1 | Surface forms algae film, and attached wall is serious, and microscopy cell bonds, and cell space has a certain proportion of decomposition dead |
It can be seen that the application culture medium achieves the result substantially similar than BG-11, medium component is enormously simplify, and reduce
Cost.
Embodiment 5 feeds experiment
Experimental group：The chlorella and bacillus amyloliquefaciens mixture that embodiment 2 is cultivated add powder according to 30% mass ratio
Crucian carp feed raw material after broken sieving, pellet is made using granulator after mixing, feeds crucian after drying, day feeding volume is
The 3%-5% of fish body weight.
It is in 500L aquariums that experiment crucian prelarva is put in a suitable place to breed in volume respectively, puts 20 tail fishes in each aquarium in a suitable place to breed.
Control group：During chlorella is cultivated, bacillus amyloliquefaciens are not added, remaining same experimental group.
Every kind of feed sets three repetitions, and after cultivating 8 weeks, the fasting of experiment fish is weighed in after one day, and muscle sample is taken after dissection
Product carry out conventional nutrients analysis, take liver specimens to carry out salivary lysozyme and Antioxidant Indexes measure, as a result see
Table 3.
Table 3 feeds experiment
| Rate of body weight gain | Feed coefficient | Survival rate | Moisture（Nutritive composition in muscle） | |
| Experimental group | 147.34±6.21 | 2.03±0.17 | 95% | 76.44±1.23 |
| Control group | 102.15±5.27 | 2.92±0.13 | 90% | 78.31±1.27 |
Understood through above-mentioned experiment, chlorella prepared by the application feeds experiment in crucian and obtains significant effect, than only cultivating
Chlorella, crucian are improved in rate of body weight gain, survival rate, and feed coefficient declines, and bait utilization is improved, and the crucian carp fed
Fish moisture also decreases, and strengthens protein quality.
The phycomycete system sewage disposal of embodiment 6 is tested
Example 1 expands the phycomycete system of culture, and 10%v/v is added in artificial sewage, the pH of artificial sewage is 7.0-8.0,
COD contents are the mg/L of 100 mg/L, TP content of 500mg/L, TN content 5.3；Simulated flue gas is exposed into reactor, simulates cigarette
Road gas composition 5-20% CO2 and 50-100ppm NO, other compositions N2, after reactor is run one day, reactor outlet gas
Composition mean concentration is stablized in 2.5%CO2,33ppmNO (nitrogen balance), and after cultivating 3 days, microalgae is collected using the method for centrifugation,
Water after discharge processing.COD, TN, TP of sewage clearance are respectively 87.0%, 90%, 97% after processing, and water outlet reaches《Cities and towns are dirty
Water treatment plant's pollutant emission standard》(GB8978-2002) secondary discharge standard.
Control group, bacillus amyloliquefaciens are not added, remaining is with embodiment 1, and by the chlorella system of acquisition, 10%v/v adds
Enter in artificial sewage, the pH of artificial sewage is that 7.0-8.0, COD content are mg/L, TP content 5.3 of 500mg/L, TN content 100
mg/L；Simulated flue gas, simulated flue gas composition 5-20% CO2 and 50-100ppm NO are exposed into reactor, other compositions are
N2, after cultivating 3 days, microalgae, water after discharge processing are collected using the method for centrifugation.COD, TN, TP of sewage removal after processing
Rate is respectively 53%, 65%, 73%, than experimental group, significant difference.
The syntaxial system of the application chlorella and bacillus amyloliquefaciens is more than single small to the obvious processing effect of sewage
Ball frond system.
Although above detailed explanation is made to this case with generality explanation and embodiment, in the present invention
On the basis of, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore, not
Deviate the modification or improvement made on the basis of present invention spirit, belong to the scope of protection of present invention.
Claims (10)
1. improve the method for algae bio amount, it is characterised in that bacillus amyloliquefaciens are added during algae culture and are total to
Culture.
2. according to the method for claim 1, it is characterised in that bacillus amyloliquefaciens are（Bacillus amyloliquefaciens）ATCC 23843.
3. according to the method for claim 1, it is characterised in that comprise the following steps that：
（1）Picking chlorella algae kind is into the triangular flask containing growth medium, intensity of illumination 5000lux, 26 DEG C of cultures,
It is shaken every day triangular flask 2~3 times, it is to be grown to exponential phase, obtain algae solution；Every liter of the growth medium contains with the following group
Point：Glucose 5g, dusty yeast 1g, ferrous sulfate 1g, Na2SO32g, sodium chloride 0.5g, potassium dihydrogen phosphate 1g, magnesium sulfate 1g, chlorine
Change ammonium 1g；
（2）By step（1）The algae solution of acquisition is inoculated into expand in culture medium according to 15% volume ratio is enlarged culture；
（3）Expand under conditions of culture is 20-100 rpm in 20 ± 10 DEG C, 2000 ± 500 lux, shaking table vibration velocity and cultivate.
4. according to the method described in claim 1-3, it is characterised in that the step（2）Middle expansion culture medium contains bacteria residue water
Solve liquid.
5. according to the cultural method described in claim 1-4, it is characterised in that it is described expansion medium component be：Bacteria residue hydrolyzes
Liquid：Growth medium：Bacillus nutrient solution is according to volume ratio=5-6:1-2:1-2 is mixed.
6. according to the cultural method described in claim 1-5, it is characterised in that
The bacteria residue hydrolyzate is prepared according to following technique：After amino acid fermentation terminates, mycoprotein is collected by centrifugation, adjusts
The solid content of mycoprotein is 8wt%, adds 5-10wt% 5mol/L NaOH, water at normal temperature solution 5-8d, obtains bacteria residue hydrolysis
Liquid.
7. according to the cultural method described in claim 1-6, it is characterised in that the bacillus nutrient solution is：Take solution starch
Bacillus, first activation culture, shaken cultivation in triangular flask is then seeded into, it is 1 × 10 to be cultivated to concentration8Individual/ml's
Bacterium solution, produce.
8. according to the cultural method described in claim 1-7, it is characterised in that the chlorella is chlorella.Sp
ATCC30412。
9. claim 1-7 methods describeds obtain the purposes that product is used for sewage disposal.
10. claim 1-7 methods describeds obtain the purposes that product is used to feed feed.
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