CN104232559A - Microalgae culturing method and grease producing method - Google Patents
Microalgae culturing method and grease producing method Download PDFInfo
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Abstract
The invention relates to a microalgae culturing method and a grease producing method. The culturing method includes: adding glucose and an alcohol into a culture medium of microalgae, and culturing under photoautotrophy conditions, wherein the alcohol is methanol and/or ethanol. The culturing method and the grease producing method can be used for large-scale low-cost efficient culturing of the microalgae, guarantee rapid growth of the microalgae and allow the microalgae to have a high grease content.
Description
Technical field
The present invention relates to the method for cultivating microalgae and produce the method for grease.
Background technology
Micro-algae is grow in water of a great variety of a class and distribution lower plant extremely widely, and it is the cell factory driven by sunlight, by the efficient photosynthesis of microalgae cell, absorbs CO
2, be the chemical energy of the carbohydrate such as fat or starch by light energy conversion, and release O
2.Utilize micro-algae production bioenergy and chemical to reach " to substitute fossil energy, reduce CO simultaneously
2discharge, purifying exhaust air and sewage " three objects.The advantage of " Microalgae biotechnology " is the following aspects: 1. micro-algae is the primordial plant that photosynthetic efficiency is the highest, and compared with farm crop, the productive rate of unit surface exceeds decades of times.Micro-algae is also an occurring in nature growth kind of plant the most rapidly, and usually in 24h, contained by micro-algae, biomass can be double, and within its " exponential phase of growth ", its biomass doubling time can shorten to 3.5h.2. micro-algae can grow in the water body of high salt, high alkali environment, beach can be made full use of, saltings, desert carry out large scale culturing, also the non-agricultural water such as seawater, saline-alkali water, trade effluent can be utilized to cultivate, and therefore micro-algae different crops can strive ground, strives water.3. oil yield rate is high, and the oleaginousness of micro-algae stem cell can up to 70%, and micro-algae does not have the cytodifferentiation such as the root, stem and leaf of higher plant, and under the conditions such as nitrogen stress, some unicellular micro-algae can accumulate grease in a large number, is the most promising oil-producing organism.4. the cultivation of micro-algae needs the CO that utilizes in industrial gaseous waste
2, alleviate the discharge of greenhouse gases, also can absorb the NOx in industrial gaseous waste, reduce the pollution of environment.5. while production microalgae biodiesel, a considerable amount of microalgae biomass can also be produced, also can obtain the high-value products such as protein, polysaccharide, lipid acid further.
Micro-algae can be divided into protokaryon algae and Eukaryotic Algae, and protokaryon algae is based on blue-green algae, and containing chlorophyll a, do not form organoid, can carry out photosynthesis, in cell, protein content is high, and can reach 70% of dry weight, lipid content is low, is about 5%; Eukaryotic Algae kind is many, is the source of main biofuel algae kind.Common micro-algae is mainly attributed to following eight classes: Bacillariophyta (Bacillariophyta), Chlorophyta (Chlorophyta), Chrysophyta (Chrysophyta), Cyanophyta (Cyanophyta), Pyrrophyta (Pyrroptata), rhodophyta (Rhodophyta), Cryptophyta (Cryptophyta) and Xanthophyta (Xanthophyta).Wherein, Bacillariophyta, Chlorophyta and Chrysophyta are the biofuel algae kind sources of most potentiality.
The efficient large-scale cultivation technology of micro-algae, new installation and novel process namely by researching and developing micro-algae large-scale cultivation come efficiently, at low cost to obtain micro algae biomass, are one of cores of Microalgae biotechnology.The factor affecting micro algae growth is a lot, mainly contains light, nutritive salt, CO
2, pH, temperature and O
2deng, the impact that these factors produce in microalgae mass culturing process is particularly outstanding.Research shows, the growth of micro-algae and the culture condition such as the content of lipid material and illumination, nitrogen, phosphorus and temperature closely related.The culture condition of different algal species growth and accumulation lipid material is not quite similar, and according to different algae kind determination optimal culture conditions, should improve growth velocity and fat content.The cultivation of micro-algae needs sufficient sunlight, CO
2, water and minerals, temperature will control usually at 20 ~ 30 DEG C, and cultivation medium must can provide and form the inorganic elements of microalgae cell, as nitrogen, P, K, Si, Fe etc.
At present for the micro-algae of major part especially green alga, the main cultural method improving its cell grease content is that condition is coerced, especially nitrogen stress is coerced, be inoculated in by frustule in the culture system of nitrogen stress environment to change the approach of internal metabolism, synthetic fat and nonprotein or carbohydrate more, thus reach the object improving lipid content.But restriction nitrogenous source can affect proper splitting and the growth of cell, cell viability is declined, causes biomass sharply to decline, finally total fat production does not promote.Existing method is, first Rapid Accumulation micro algae biomass under the condition of nitrogenous source abundance, and then is separated from the substratum of rich nitrogen by micro-algae and put into nitrogen stress substratum again, and the condition of carrying out coerces to improve lipid content.This method is not suitable for large-scale commercial production, puts into that nitrogen stress substratum process is loaded down with trivial details again and energy consumption is high because separated from the substratum of rich nitrogen by micro-algae.
Light autotrophy refers under light illumination, with CO
2for carbon source cultivates micro-algae.Heterotrophism refers to cultivates micro-algae with organic carbon source in dark conditions.A lot of micro-algae can carry out mixotrophic cultivation, and therefore a lot of scholar has carried out micro-algae, as the technology of supporting of holding concurrently of chlorella, to improve the fat content of its cultivation efficiency and micro-algae.Such as Chinese patent 200810112998.9 discloses a kind of method of two steps cultivation chlorella production biofuel from autotrophy to heterotrophism, cultivate from the autotrophy of chlorella, concentrated autotrophy algae proceeds to fermentor tank and carry out heterotrophic growth, Rapid Accumulation grease by the technique such as cell concentration, fermentation Environmental capacity.Heterotrophic culture needs to add organic carbon source, and as glucose, although Heterotrophic culture can improve rapidly algae cell density and the speed of growth, this process needs the strict pollution controlling miscellaneous bacteria, otherwise can cause cultivating unsuccessfully, and the chlorella quality of Heterotrophic culture can decline.In addition, the obvious process of the method is comparatively loaded down with trivial details, cost is higher.Chinese patent 200980128146.1,201010545871.3 and 201110029154.x disclose similar both culturing microalgae method, the Heterotrophic culture step that these methods comprise micro-algae algae kind and the frustule that obtains using Heterotrophic culture are as the light autotrophy culturing step of seed, these methods can improve the cultivation efficiency of micro-algae, but separate two steps obviously make operation more complicated.
How high-level efficiency, at low cost microalgae are the difficult problems in Microalgae biotechnology always, and major cause is: micro-phycobiont small (being usually less than 20 microns) and algae liquid concentration very low (under concentration of gathering, usually less than 3 ‰ of algae liquid quality).Existing microalgae recovery method mainly flocculence, filtration method, centrifuging and settling process.Wherein, the technique of flocculence, filtration method and centrifuging is more complicated, cost is higher.Settling process have easy and simple to handle, save the advantage such as the energy, as CN101748068 discloses a kind of microalgae harvesting method, the method utilizes barodynamics principle, not exclusively gathers in the crops for high-density cultured continuously photosynthetic organism reactive system, is greater than 10 for concentration
7the algae liquid of individual every milliliter (is equivalent to OD
680=1.0), rate of descent generally can reach more than 60%, and after sedimentation, isolated supernatant liquid back light reactive system continues to utilize, for the micro-algae being not easy sedimentation, need use pH adjusting agent by algae liquid pH regulator to strong basicity, make micro-algae produce autoflocculation to accelerate sedimentation.The method still has the following disadvantages: 1. regulate pH strong basicity to make micro-algae autoflocculation, can cause the increase of cost and affect the vigor of micro-algae, being unfavorable for recycle algae kind; 2. in some cases, slight disturbance will make micro-algae again disperse, and is unfavorable for the clear liquid after separate and subside.
Summary of the invention
An object of the present invention is to provide a kind of method of cultivating microalgae, can ensure the quick growth of micro-algae, can significantly improve its fat content again.Two of object of the present invention is to provide a kind of method of producing grease, and the method, on the basis realizing object one, solves Problems existing in micro-algae sedimentation recovery process further, thus can obtain grease with lower cost.
A method for cultivating microalgae, comprising: add glucose and alcohol in the substratum of Xiang Weizao, cultivates under the condition of light autotrophy; Described alcohol is methyl alcohol and/or ethanol.
The addition of glucose is 0.1 ~ 50g/L.
The addition of alcohol is 0.1 ~ 10mL/L, is preferably 1 ~ 10mL.
Described nitrogenous fertilizer can be selected from one or more in urea, ammonium salt, nitrate and amino acid; Be preferably urea, its dosage is 1 ~ 5g/L.
Described micro-algae is to carry out the micro-algae supported of holding concurrently, as chlorella, grid algae or single needle algae, and preferred chlorella.
According to method of the present invention, also need to maintain other prerequisite required for micro-algae normal growth in the process of cultivating micro-algae, as provided suitable illumination, temperature, and the necessary nutrition of other micro algae growth, the CO in regulation and control algae liquid
2, dissolved oxygen, water, inorganic salt, necessary nutrient matter, pH value etc. in suitable scope, make the quick growth and reproduction of its suitable micro-algae.These technology are well-known to those skilled in the art.As substratum can select BG-11 substratum; The pH value of algae liquid controls between 6 ~ 10, preferably controls between 7 ~ 9; Culture temperature is 15 ~ 40 DEG C, is preferably 25 ~ 35 DEG C; Light intensity is 2000 ~ 200000 luxs, is preferably 5000 ~ 150000 luxs.
In the present invention, the mode that the cultivation of micro-algae can adopt classification to expand, such as algae kind is through 1L, 5L, 50L, 500L ... amplification culture step by step.It is in the scope of 0.2 ~ 1 that the initial concentration of inoculation generally can control in algae liquid optical density value (OD value).Culturing time is usually more than 10 days, and preferably more than 15 days, for the cultivation of oil-producing microalgae, culturing time can obtain better effect in more than 20 days.
Produce a method for grease, comprise the separation and extraction of both culturing microalgae, microalgae recovery, microalgae grease, both culturing microalgae wherein adopts above-mentioned method.
In preferred situation, microalgae recovery step comprises: as the optical density value OD of algae liquid
680>1.6 and rely on micro-algae own growth when making algae liquid pH value>=7, under the condition of temperature≤40 DEG C, sedimentation more than 24 hours; Then at 20 DEG C ~ 40 DEG C, the algae liquid of results lower floor sedimentation, upper strata OD
680the algae liquid of≤0.6 returns both culturing microalgae step cycle and utilizes.
Test finds, although the algae liquid concentration very low (less than 3 ‰ of algae liquid quality) when gathering, algae liquid concentration still has obvious impact, as the optical density value OD of algae liquid to micro-algae sedimentation
680during < 1.6, micro-algae is extremely difficult to sedimentation.According to method of the present invention, need by the optical density value OD of algae liquid
680control at >1.6, preferred > 2, more preferably > 3.5, further preferred > 7.
Test finds, when algae liquid is in time acid, micro-algae is extremely difficult to sedimentation.According to method of the present invention, need the pH value of algae liquid to control >=7.The present invention not to need algae liquid pH regulator to strong basicity, only needs to rely on algae liquid pH value that micro-algae own growth causes and rises and make it be in nonacid state.Under preferred condition, rely on micro-algae own growth when making algae liquid pH value be 7 ~ 9, then carry out sedimentation.
In preferred situation, as the optical density value OD of algae liquid
680>3.5 and rely on micro-algae own growth when making algae liquid pH value be 7 ~ 9, then gather; In preferred situation, as the optical density value OD of algae liquid
680>7 and rely on micro-algae own growth when making algae liquid pH value be 7 ~ 9, then gather.
The sedimentation degree of depth can be determined according to concrete micro-algae and prior art, is generally 50mm ~ 1000mm, and preferably the degree of depth is 50mm ~ 120mm.
Test finds, as temperature <20 DEG C, slight algae liquid disturbance, will make micro-algae of sedimentation again disperse, and therefore needs results temperature to control at 20 DEG C ~ 40 DEG C.
In preferred situation, return the OD of the algae liquid of both culturing microalgae step
680be 0.4 ~ 0.6.
When without prejudice to the present invention object and not conflicting, each technical characteristic in the present invention can arbitrary combination, and it belongs to content disclosed by the invention equally.
Those skilled in the art know, and adopt nitrogen technology of hungering and thirst can improve the fat content of micro-algae, but the method are not suitable for large-scale commercial production, put into that nitrogen stress substratum process is loaded down with trivial details again and energy consumption is high because separated from the substratum of rich nitrogen by micro-algae.In addition, the restriction due to nitrogenous source makes the division of cell, growth is restricted, and cell viability declines and causes biomass sharply to decline, and finally total fat production does not promote.Prior art adopts the segmentation cultural method of autotrophy+heterotrophism, can improve the cultivation efficiency of micro-algae, but separate two steps obviously makes operation more complicated.In addition, be that carbon source carries out heterotrophism or hold concurrently supporting with glucose, usually need sterile environment, otherwise cultivation is easy to suffer infecting of miscellaneous bacteria and failure, and gnotobasis obviously causes more complicated operation and the raising of cost.The present invention has larger advantage in breeding scale, owing to need not adopt the pattern of subsection filter, breeding process is greatly simplified.What is more important, the present invention can ensure the quick growth of micro-algae, it can be made again to accumulate grease in a large number, can also avoid the living contaminants caused due to organotrophic adding.
Accompanying drawing explanation
Accompanying drawing 1 is the growth curve of micro-algae.
Embodiment
Describe the present invention in detail with embodiment below, but be not therefore construed as limiting the invention.
algae liquid optical density value (OD
680
value) measure
Optical density value spectrophotometric determination, compares with distilled water, measures the light absorption value of algae liquid at wavelength 680nm place, as the index of both culturing microalgae concentration.
oil content of microalgae measures
Employing apparatus,Soxhlet's measures, first by centrifugal, dry for appropriate algae liquid, carefully grind to form fine powder one and guarantee the effective broken wall of micro-algae, weigh, put into extractor with filter paper parcel, add methyl alcohol and trichloromethane, extracting 6 hours at 90 DEG C, dried by two kinds of mixed solvents, constant weight takes left material, records the formula of oleaginousness: heavy (G) 100% of heavy (the G)/dry powder of oil-containing %=extract.
the preparation of micro-algae culture medium:
By the table 1 formulated aqueous solution (the micro-A5 in table 1 presses table 2 formulated), during use, be diluted to the substratum mother liquor of 1000 times, cool for subsequent use after 121 DEG C of high pressure, high-temperature sterilization 20min.
Table 1
Table 2
the mensuration of algae liquid settling property
The change of algae liquid optical density value before and after sedimentation is designated as rate of descent, and as the index of the settling property of measurement algae liquid, measuring position is 4cm under liquid level.Optical density value before note sedimentation is OD
1, the optical density value after sedimentation is OD
2.
Rate of descent=(OD
2-OD
1)/OD
2× 100%
Embodiment 1
The cultivation of micro-algae is carried out in 5L Erlenmeyer flask, (substratum adopts aforesaid method preparation to load 3 ~ 4L substratum, the tap water of cultivation hydromining filter-sterilized), be 0.3 ~ 0.6 access chlorella algae kind by final algae liquid OD value, and add glucose by 20g/L, 3g/L adds urea, and 3ml/L adds ethanol.Take natural light as light source, control light intensity in the scope of 2000 ~ 20000 luxs, incubated at room temperature.Pass into 2%(v/v) CO
2/ air Mixture carries out aerated culture, gas mixture membrane filtration purification after filtration.Micro algae growth curve is shown in Fig. 1.
Embodiment 2
The cultivation of micro-algae is carried out in 5L Erlenmeyer flask, (substratum adopts aforesaid method preparation to load 3 ~ 4L substratum, the tap water of cultivation hydromining filter-sterilized), be 0.3 ~ 0.6 access chlorella algae kind by final algae liquid OD value, and add glucose by 10g/L, 2g/L adds urea, and 5ml/L adds ethanol.Take natural light as light source, control light intensity in the scope of 2000 ~ 20000 luxs, incubated at room temperature.Pass into 2%(v/v) CO
2/ air Mixture carries out aerated culture, gas mixture membrane filtration purification after filtration.Micro algae growth curve is shown in Fig. 1.
Embodiment 3
The cultivation of micro-algae is carried out in 5L Erlenmeyer flask, (substratum adopts aforesaid method preparation to load 3 ~ 4L substratum, the tap water of cultivation hydromining filter-sterilized), be 0.3 ~ 0.6 access chlorella algae kind by final algae liquid OD value, and add glucose by 15g/L, 1.5g/L adds urea, and 6ml/L adds ethanol.Take natural light as light source, control light intensity in the scope of 2000 ~ 20000 luxs, incubated at room temperature.Pass into 2%(v/v) CO
2/ air Mixture carries out aerated culture, gas mixture membrane filtration purification after filtration.Micro algae growth curve is shown in Fig. 1.
Comparative example 1
The cultivation of micro-algae is carried out in 5L Erlenmeyer flask, (substratum adopts aforesaid method preparation to load 3 ~ 4L substratum, the tap water of cultivation hydromining filter-sterilized), be 0.3 ~ 0.6 access chlorella algae kind by final algae liquid OD value, and add glucose by 20g/L, 1.5g/L adds urea, does not add ethanol.Take natural light as light source, control light intensity in the scope of 2000 ~ 20000 luxs, incubated at room temperature.Pass into 2%(v/v) CO
2/ air Mixture carries out aerated culture, gas mixture membrane filtration purification after filtration.Micro algae growth curve is shown in Fig. 1.
Comparative example 2
The cultivation of micro-algae is carried out in 5L Erlenmeyer flask, (substratum adopts aforesaid method preparation to load 3 ~ 4L substratum, the tap water of cultivation hydromining filter-sterilized), be 0.3 ~ 0.6 access chlorella algae kind by final algae liquid OD value, and add glucose by 20g/L, 0.5g/L adds urea, does not add ethanol.Take natural light as light source, control light intensity in the scope of 2000 ~ 20000 luxs, incubated at room temperature.Pass into 2%(v/v) CO
2/ air Mixture carries out aerated culture, gas mixture membrane filtration purification after filtration.
Table 3
Table 3 result shows, adopts method of the present invention, and the fat content of chlorella significantly improves.
Embodiment 4
Chlorella algae liquid is placed in beaker, algae liquid OD
680be 2.367, pH=7, envrionment temperature is 25 DEG C, lucifuge sedimentation 24h, supernatant liquor OD
680be 0.250, after rate of descent reaches 89%, 48h, rate of descent reaches 90%.
Embodiment 5
Chlorella algae liquid is placed in beaker, algae liquid OD
680be 7.290, pH=8, envrionment temperature is 30 DEG C, lucifuge sedimentation 24h, supernatant liquor OD
680be 0.507, after rate of descent reaches 93%, 48h, rate of descent reaches 93.4%.Collect supernatant liquor, it is mixed with 1:1 ratio with BG11 substratum and is put in cultivating system
Continue to cultivate, OD after 14 days
680be 6.521.
Embodiment 6
Chlorella algae liquid is placed in beaker, algae liquid OD
680be 3.521, pH=9, envrionment temperature is 35 DEG C, lucifuge sedimentation 24h, supernatant liquor OD
680be 0.247, rate of descent reaches 93%.
Comparative example 3
Chlorella algae liquid is placed in beaker, algae liquid OD
680be 1.575, pH=8, envrionment temperature is 25 DEG C, lucifuge sedimentation 24h, supernatant liquor OD
680be 1.114, after rate of descent only reaches 29%, 48h, rate of descent only reaches 66%.
Comparative example 4
Chlorella algae liquid is placed in beaker, algae liquid OD
680be 3.928, pH=6, envrionment temperature is 25 DEG C, lucifuge sedimentation 48h, supernatant liquor OD
680be 1.288, rate of descent is 67%.
Comparative example 5
Chlorella algae liquid is placed in screw thread reagent bottle, algae liquid OD
680be 9.2, pH=8,45 DEG C of constant temperature water bath 2h, lucifuge sedimentation 24h, supernatant liquor OD
680be 0.94, clear liquid is reclaimed, add BG11 substratum with the ratio of 1:1 and continue to cultivate, OD after 24h
680be be 0.1 after 0.4,48h.
Claims (10)
1. a method for cultivating microalgae, comprising: add glucose and alcohol in the substratum of Xiang Weizao, cultivates under the condition of light autotrophy; Described alcohol is methyl alcohol and/or ethanol.
2. in accordance with the method for claim 1, it is characterized in that, the addition of glucose is 0.1 ~ 50g/L, and the addition of alcohol is 0.1 ~ 10mL/L.
3. in accordance with the method for claim 1, described nitrogenous fertilizer is selected from one or more in urea, ammonium salt, nitrate and amino acid.
4. in accordance with the method for claim 1, it is characterized in that, described nitrogenous fertilizer is urea, and its dosage is 1 ~ 5g/L.
5. in accordance with the method for claim 1, described micro-algae is chlorella, grid algae or single needle algae.
6. in accordance with the method for claim 1, it is characterized in that: culture temperature is 15 ~ 40 DEG C, light intensity 2000 ~ 200000 lux, the pH value of algae liquid controls between 6 ~ 10.
7. in accordance with the method for claim 6, it is characterized in that, culture temperature is 25 ~ 35 DEG C, and light intensity 5000 ~ 150000 lux, the pH value of algae liquid controls between 7 ~ 9.
8. in accordance with the method for claim 1, it is characterized in that, culturing time is more than 10 days.
9. produce a method for grease, comprise the separation and extraction of both culturing microalgae, microalgae recovery, microalgae grease, both culturing microalgae wherein adopts method arbitrary in claim 1 ~ 8.
10. in accordance with the method for claim 9, it is characterized in that, microalgae recovery step comprises: as the optical density value OD of algae liquid
680>1.6 and rely on micro-algae own growth when making algae liquid pH value>=7, under the condition of temperature≤40 DEG C, sedimentation more than 24 hours; Then at 20 DEG C ~ 40 DEG C, the algae liquid of results lower floor sedimentation, upper strata OD
680the algae liquid of≤0.6 returns both culturing microalgae step cycle and utilizes.
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| CN106635805A (en) * | 2015-07-23 | 2017-05-10 | 中国石油化工股份有限公司 | A culture method for monoraphidium |
| CN109609382A (en) * | 2018-12-07 | 2019-04-12 | 中国石油大学(华东) | A kind of method that phycomycete co-cultures promotion chlorella growth and oil and fat accumulation |
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| CN101280328B (en) * | 2008-05-27 | 2011-06-29 | 清华大学 | Method for producing biodiesel by autotrophic culture and heterotrophic culture of chlorella |
| CN102443562B (en) * | 2010-10-12 | 2016-04-13 | 中国石油化工股份有限公司 | A kind of substratum and cultural method promoting proliferation of microalgae papidly |
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| CN106399105A (en) * | 2015-07-23 | 2017-02-15 | 中国石油化工股份有限公司 | Monoraphidium breeding method |
| CN106635805A (en) * | 2015-07-23 | 2017-05-10 | 中国石油化工股份有限公司 | A culture method for monoraphidium |
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| CN109609382A (en) * | 2018-12-07 | 2019-04-12 | 中国石油大学(华东) | A kind of method that phycomycete co-cultures promotion chlorella growth and oil and fat accumulation |
| CN109609382B (en) * | 2018-12-07 | 2022-03-22 | 中国石油大学(华东) | Method for promoting growth of chlorella and oil accumulation by algal-bacteria co-culture |
| CN117625397A (en) * | 2023-11-30 | 2024-03-01 | 山东省农业科学院 | Vermicelli wastewater treatment process based on microalgae |
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