CN102911872A - Scenedesmus sp. strain and application thereof - Google Patents

Abstract

The invention discloses a scenedesmus sp. strain and application thereof. The scenedesmus sp. strain has the advantages of high growth speed, high grease accumulation speed, high oil content, capability of effectively removing nitrogen containing compounds in water, and the like, contains partially beneficial pigments, and can be used in production of biodiesel and production of foods, health care products, feeds, aquatic product baits, medicines, cosmetics and pigments.

Description

Scenedesmus strain and its application

technical field

The invention relates to the field of microorganisms, in particular to Scenedesmus ENN33 (Scenedesmus sp.) and its application.

Background technique

With the gradual depletion of the world's fossil fuel reserves, the continuous increase of carbon dioxide emissions and the increasing warming of the global climate, human survival and development have faced unprecedented challenges. Therefore, countries all over the world are actively looking for renewable energy sources that can replace petroleum products. Among them, biodiesel is an important bioenergy source. If there are too many, the problem of "competing with people for food and land with food" will arise, which will lead to the embarrassing result of "the energy crisis has been solved, but the food crisis has emerged".

Microalgae is a kind of low-level plants that are widely distributed on land and in the ocean, rich in nutrients, short in growth cycle, and high in photosynthetic utilization. material resources. Under certain conditions, many microalgae can accumulate a large amount of storage triacylglycerol (microalgal oil) in their cells (similar to vegetable oil), and its maximum content can reach more than 50% of the dry weight of the cells. Compared with higher terrestrial oil crops, the oil yield per unit area of microalgae is dozens of times higher than that of oil plants. After biosmelting, it can produce biodiesel and other biofuels. It is considered to be the most likely and most potential to become an oil substitute. Biomass resources for products.

In addition to very high oil production, microalgae are also rich in protein, fat, carbohydrates, various amino acids, vitamins, antibiotics, high unsaturated fatty acids and other biologically active substances. Therefore, they are widely used in health food, medicine and feed, etc. The field also has good development prospects, for example, for food and pharmaceutical industries (nutritional supplements for humans and animals: vitamins, proteins, fatty acids, polysaccharides, etc.); extraction of chemical industries such as cosmetics, fine chemical products, etc.; as energy Produce biogas and fuel; used for bait and feed industry (bait for fish, shrimp, crustaceans and other aquatic animals, poultry feed, etc.); agriculturally used as soil conditioner, fertilizer, etc.

In addition, microalgae have very strong environmental adaptability and simple nutritional requirements, and can grow in various harsh environments. For example, microalgae can absorb nitrogen and phosphorus from inorganic salts such as nitrate, nitrite, ammonium salt, and phosphate in sewage through photosynthesis to synthesize organic substances such as amino acids, proteins, and phospholipids to form microalgae cells, thereby reducing nitrogen in water bodies. Phosphorus content, so microalgae can be used to remove nitrogen and phosphorus in sewage. Microalgae can also use CO ₂ in industrial waste gas to reduce CO ₂ emissions. Therefore, microalgae also have good application prospects in both industrial environmental protection and urban environmental protection. There are also some microalgae that can grow in water bodies with high salinity and high alkali environment, and can make full use of tidal flats, saline-alkali land, deserts and other lands that are not suitable for cultivation for large-scale cultivation, and can also use seawater, saline-alkali water, industrial wastewater, etc. Cultivation with non-agricultural water is known as a recyclable "green oil field". Therefore, the development of microalgae cultivation and related industries is also very suitable for land, especially in my country where cultivated land resources are relatively scarce.

At present, the development of microalgae, especially its application in bioenergy, has been actively carried out at home and abroad, among which the research on chlorella is mostly. CN101460609 discloses a new Chlorella genus (CHLORELLA) species and its application CN101565675 discloses a Chlorella and its cultivation method and its application in biodiesel production; CN101824386 discloses a strain of Chlorella, and Its application in producing microalgae oil and fixing _CO2 ; CN102093955 discloses a strain of chlorella and its application in the utilization of sewage cultivation and biodiesel conversion system; CN102229889 discloses a strain of chlorella and its cultivation method and In the fixation of _CO2 , the purification of waste water, the production and application of biomass such as oil, protein, pigment, starch, polysaccharide, nucleic acid, etc.

Scenedesmus is a common planktonic microalgae in fresh water, which likes to reproduce in nutrient-rich still water. Many species have strong tolerance to organic pollutants, can be used as indicator organisms in water quality evaluation, and play a certain role in water self-purification and sewage purification, and are the dominant species in the biophase of organic sewage oxidation ponds. It can attach to organic fragments and other aquatic plants in water at the same time as bacteria, forming a colloidal layer and adsorbing organic matter. When Scenedesmus carries out photosynthesis, on the one hand, it produces oxygen for bacteria to decompose organic matter, and on the other hand, it can directly use organic matter as carbon and nitrogen sources, so that organic matter in water can be rapidly degraded, thereby purifying the water body. Scenedesmus cells are rich in protein and vitamins, and are good feed for fish. If fish are raised in organic sewage oxidation ponds, high yields can be obtained. The prolific algae can also be used as feed for poultry. At present, there are not many developments on Scenedesmus at home and abroad. CN101565674 discloses a low-nutrient Scenedesmus and its application in sewage advanced treatment. The Scenedesmus strain can grow in a low-nutrient environment, so the secondary output Nitrogen and phosphorus in water are removed to a very low level; CN102083957 discloses a microalgae of Scenedescens var. Algae and uses thereof; CN102250773 discloses a strain of Scenedesmus and its cultivation method and in the fixation of _CO2 , the purification of waste water, the application in fields such as the production of biomass such as oil, protein, pigment, starch, polysaccharide, nucleic acid; Quarterly Xiang, Wang Jinrong et al. (Screening of a strain of Scenedesmus obliquus and optimization of growth conditions [J]. Aquatic Science and Technology Information, 2010: 37(6)) obtained a strain of Scenedesmus obliquus with relatively high oil content and good growth Algal strains.

Although microalgae have very good development prospects in energy, environmental protection and even medicine, and related research work has been gradually carried out, the currently known microalgae, especially Scenedesmus, usually have an oil content below 40% and are resistant to CO ₂ 's processing capacity, extreme environmental tolerance and other aspects of performance are generally low. Therefore, it is necessary to develop algal species with better performance and more suitable for industrial applications.

Contents of the invention

The object of the present invention is to provide a Scenedesmus that is more adaptable to the environment, has a high oil content, and has a strong ability to process CO ₂ and is more suitable for industrial applications.

For reaching this purpose, the present invention adopts following technical scheme:

In the first aspect, the present invention provides a Scenedesmus ENN33 (Scenedesmus sp.), which is preserved in the General Microorganism Center (CGMCC) of the China Microbiological Culture Collection Management Committee (CGMCC), the preservation date is July 13, 2012, and the preservation number is CGMCC No. 6347.

The content of fatty acids in the algae flour of Scenedesmus ENN33 provided by the present invention may be greater than 40% by dry weight, preferably greater than 50% by dry weight, more preferably 56% to 60% by dry weight, most preferably 58%.

Scenedesmus ENN33 provided by the invention contains astaxanthin, β-carotene, lutein, zeaxanthin and canthaxanthin.

The tolerance temperature of Scenedesmus ENN33 provided by the present invention can be from -10°C to 44°C, preferably from 6°C to 40°C, more preferably from 8°C to 30°C, still more preferably from 10°C to 20°C, most preferably 15°C.

Scenedesmus ENN33 provided by the present invention can be used at -10°C, -9°C, -8°C, -7°C, -6°C, -5°C, -4°C, -3°C, -2°C, -1°C, 0°C ℃, 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, 10℃, 11℃, 12℃, 13℃, 14℃, 15℃, 16℃, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C , 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 46, 43°C or 44°C.

In a second aspect, the present invention provides a method for cultivating Scenedesmus ENN33 as described in the first aspect, the method comprising inoculating the green cells of Scenedesmus strain ENN 33 in BG11 medium, wherein the light intensity is 50-500 μmol/ m ² .s, preferably 100-450 μmol/m ² .s, more preferably 150-400 μmol/m ² .s, still more preferably 200-350 μmol/m ² .s, most preferably 250-300 μmol/m ² .s, pH The value is 7-9, preferably 7.5-8.5, more preferably 8, and the temperature is 15-35°C, preferably 20-30°C, more preferably 25°C.

In the cultivation method of Scenedesmus ENN33 of the present invention, the light intensity can be 50 μmol/m ² .s, 80 μmol/m ² .s, 100 μmol/m ² .s, 120 μmol/m ² .s, 150 μmol/m ² .s , 180μmol/m ² .s, 200μmol/m ² .s, 220μmol/m 2 .s, 250μmol/m ^{2 .s} , 280μmol/m ² .s, 300μmol/m ² .s, 320μmol/m ² .s, ^{pH _ _ _ _ _ _} Values can be 7, 7.2, 7.5, 7.8, 8, 8.2, 8.5, 8.8, or 9; temperatures can be 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C , 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C.

In a third aspect, the present invention provides the application of Scenedesmus ENN33 in fatty acid production as described in the first aspect, preferably, the fatty acid is myristic acid, palmitic acid, palmitoleic acid, stearic acid, stearic acid decenoic acid, octadecadienoic acid, stearidonic acid, alpha-linolenic acid, gamma-linolenic acid and/or eicosapentaenoic acid.

In the fourth aspect, the present invention provides the application of Scenedesmus ENN33 as described in the first aspect in the production of biofuel, preferably, the biofuel is biodiesel or bioethanol.

In a fifth aspect, the present invention provides the application of Scenedesmus ENN33 in environmental protection as described in the first aspect, preferably the environmental protection is sewage treatment or _CO2 emission reduction, and the sewage treatment includes denitrification of sewage , phosphorus removal, organic matter degradation and/or heavy metal ion adsorption.

In the sixth aspect, the present invention provides Scenedesmus ENN33 as described in the first aspect in the presence of myristic acid, palmitic acid, palmitoleic acid, stearic acid, octadecenoic acid, linoleic acid, octadecanetra enoic acid, alpha-linolenic acid, gamma-linolenic acid, arachidic acid, behenic acid, eicosapentaenoic acid, astaxanthin, beta-carotene, lutein, zeaxanthin and/or canthaxanthin Use in the preparation of medicaments.

In the seventh aspect, the present invention provides Scenedesmus ENN33 as described in the first aspect containing myristic acid, palmitic acid, palmitoleic acid, stearic acid, octadecenoic acid, linoleic acid, octadecanetetra enoic acid, alpha-linolenic acid, gamma-linolenic acid, arachidic acid, behenic acid, eicosapentaenoic acid, astaxanthin, beta-carotene, lutein, zeaxanthin and/or canthaxanthin Use in the production of food, health products, feed and/or aquatic product bait.

In the eighth aspect, the present invention provides Scenedesmus ENN33 as described in the first aspect in the presence of myristic acid, palmitic acid, palmitoleic acid, stearic acid, octadecenoic acid, linoleic acid, octadecanetra enoic acid, alpha-linolenic acid, gamma-linolenic acid, arachidic acid, behenic acid, eicosapentaenoic acid, astaxanthin, beta-carotene, lutein, zeaxanthin and/or canthaxanthin Application in cosmetic production.

In the present invention, BG11 medium is the most commonly used algae medium in the field, and its specific components are described below.

The beneficial technical effect of the present invention is:

1. This algae grows fast, has a high _CO2 utilization rate, and has good cell sedimentation, which is easy to collect, suitable for high-density cultivation, and suitable for industrialization promotion

2. The algal oil accumulation is fast and the oil content is high, which is suitable for the production of biodiesel

3. This algae species can effectively remove nitrogen in water and is suitable for wastewater treatment

4. This species of algae has a wide range of temperature tolerance and is suitable for breeding throughout the year.

Description of drawings

Fig. 1 is a microscopic examination picture of Scenedesmus ENN33 of the present invention under a microscope.

Fig. 2 shows the results of BLAST alignment of the ITS and 5.8S partial nucleotide sequences of Scenedesmus ENN33 of the present invention in the GenBank database.

Fig. 3 is the phylogenetic tree of Scenedesmus ENN33 constructed by the Neighbor-joining algorithm.

Fig. 4 is the indoor cultivation growth curve of Scenedesmus ENN33 of the present invention.

Fig. 5 is a growth curve of Scenedesmus ENN33 of the present invention under high temperature conditions.

Figure 6 is the growth curve (1) of Scenedesmus ENN33 of the present invention under low temperature conditions.

Fig. 7 is the growth curve (2) of Scenedesmus ENN33 of the present invention under low temperature conditions.

Fig. 8 is the growth curve of waste water and waste gas cultivation of Scenedesmus ENN33 of the present invention.

Fig. 9 is a graph showing the pigment percentage content of Scenedesmus ENN33 of the present invention.

Detailed ways

The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation methods.

Embodiment 1 screening oil-producing Scenedesmus strains

Take the soil retrieved from Wulanchabu after diluting and dissolving with sterile water. After observing under a 400-fold microscope, the approximate cell concentration is 800-1200 cells/ml. Take about 1 μl of algae liquid with a capillary tube and inoculate it in 48 wells In the culture plate with BG11 medium, culture at 25°C and light intensity of 50 μmol/m2.s. When a certain cell concentration is reached, observe with a microscope, select a well with only a single algae strain, and dilute to a cell concentration of 1000 Plate after -3000/ml to obtain a single algae strain. This process is repeated until a purely cultured algal strain is obtained, which is numbered "ENN33" in the present invention.

Example 2 Identification of algae species and their classification on the phylogenetic tree.

Identification of algae strains takes a combination of morphological identification and molecular identification.

1. Shape identification (see Figure 1): Observe the above-mentioned isolated algae strain ENN33 under a 1000-fold microscope. The algal body is a single cell or a stereotyped group. The stereotyped group consists of 2 or 4 cells, and the group cells are juxtaposed Straight lines are arranged in a row or slightly alternately; the cells are spindle-shaped, the upper and lower ends are gradually tapered, and the cell walls are smooth. The population of 4 cells is 12-34 μm wide, and the cells are 10-20 μm long and 3-9 μm wide. Chromosomes are perinatal, flaky, with a protein nucleus. Asexual reproduction produces spore-like spores. Retrieval of "Freshwater Algae in China--System, Classification and Ecology", found that the algae in the same morphological classification are Chlorophyta, Chlorophyceae, Chlorococcales, Scenedesceae ( Scenedesmaceae), Scenedesmus (Scenedesmus).

2. Molecular identification:

A, A, Amplify its ITS and 5.8S partial nucleotide sequence from the isolated Chlamydomonas

Genomic DNA of ENN33 algae cultured for 4 days was extracted by CTAB method. Take a certain amount of cells, add an appropriate amount of CTAB buffer after grinding, homogenate, add an equal volume of phenol:chloroform for extraction, an equal volume of isopropanol for precipitation, wash with 75% ethanol and dissolve in a certain volume of sterilized double-distilled water. UV spectrophotometer to detect its concentration and purity.

ITS sequence amplification uses eukaryotic ITS amplification general primers (primers are synthesized by Shanghai Sangon Bioengineering Co., Ltd.).

Primer 1 5'GGAAGTAAAAGTCGTAACAAGG 3'

Primer 2 5'GCATATCAATAAGCGGAGGA 3'

Take 1 μl of total DNA as template. The amplification conditions are as follows: denaturation at 94°C for 5 minutes, followed by 30 cycles of 94°C for 40s, 56°C for 40s, 72°C for 2min, and finally 72°C for 10min. The amplified product was detected by 1.0% agarose gel electrophoresis, and a fragment of about 750bp was obtained by PCR amplification. The sequence is as follows (the underlined part is the primer sequence):

GGAAGTAAAAATCGTAACAAGGTCCTCCGCTTATTGATATGC .

B, Homology search of the cloned nucleotide sequence

The obtained ITS sequence and 5.8S partial sequence of ENN33 were registered in the GenBank database for BLAST comparison, and the results showed that it was compatible with the internal transcriptional spacer 1, 5.8S ribosomal RNA gene and internal transcriptional spacer of Scenedesmus.sp with ACCESSION number AM419228.1 The sequence of region 2 has a high similarity of 93% (Fig. 2).

C, Make a phylogenetic tree based on the above sequence.

Using the BLAST tool in the NCBI database, the sequence similarity analysis was carried out on the ITS partial sequence of algal strain ENN33. Selected partial homologous sequences and partial ITS sequences of the algae strains were compared using the clustaLx software package to form a multiple sequence matching alignment matrix. Then, using the Mega4.0 software, using the neighbor joining algorithm, the bootstraps value of the bootstrap data set is 1000 to construct a phylogenetic evolutionary tree. See Figure 3.

According to the results of morphology and ITS blast, ENN33 was defined as Scenedesmus.

The inventor has preserved the algae strain in the General Microorganism Center of China Microbiological Culture Collection Management Committee (No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, referred to as CGMCC) on July 13, 2012, and its preservation number is CGMCC No. 6347.

Indoor culture and oil accumulation of embodiment 3 ENN 33 algae

The green swimming cells of Scenedesmus strain ENN 33 in the logarithmic growth phase were inoculated in the prepared BG11 medium (see Table 1 for the formula), so that the cell density reached an OD750 of 0.2-0.8. During the cultivation process, the light intensity is controlled at 50-500μmol/m2.s. During the cultivation period, the pH value of the medium is adjusted between 7-9 by introducing a mixture of 1.5-2% carbon dioxide and air into the culture medium. During the period, the temperature is controlled within the range of 15-35°C. The reactor used for the cultivation was a column reactor with an inner diameter of 40 mm and a length of 600 mm. During the cultivation process, samples were taken regularly to measure the dry weight, and the results are shown in Figure 4. On the 16th day of cultivation, when the color of the algae liquid turns yellow, the algae liquid is collected, and the algae mud is obtained by centrifugation or natural sedimentation, and the algae mud is vacuum freeze-dried.

Table 1BG11 medium formula

_NaNO3 1.5g/l K ₂ HPO ₄ 3H ₂ O 0.04g/l

MgSO ₄ 7H ₂ O 0.075g/l CaCl ₂ 2H ₂ O 0.036g/l citric acid 0.006g/l FeCl ₃ 6H ₂ O 0.00315g/l Na ₂ EDTA·2H ₂ O 0.00436g/l Na ₂ CO ₃ 0.02g/l A5 trace elements* 1ml

*A composition of ₅ trace elements

Add to 1000ml deionized water

H ₃ BO ₃ 2.86g MnCl ₂ ·H ₂ O 1.81g ZnSO ₄ 7H ₂ O 0.222g CuSO ₄ 5H ₂ O 0.079g Na ₂ MoO ₄ 2H ₂ O 0.390g Co(NO ₃ ) ₂ 6H ₂ O 0.0494g

After drying, measure its oil component content as shown in Table 2, and the analysis method is as follows:

1) Fatty acid extraction:

Take 50mg or 100mg of freeze-dried algal powder and place it in a small glass bottle with a volume of 15-20ml with a Telfnon screw cap, then place a small magnetic bar, add 2-4ml of 10% DMSO-methanol solution, and place it in a sand bath at 40°C (Place the beaker containing sand on a constant temperature heating magnetic stirrer) for 5 minutes; then extract with magnetic stirring at 4°C for 30 minutes, centrifuge at 3500 rpm, and transfer the supernatant to another vial. Add 1:1 diethyl ether and 4-8ml of n-hexane to the remaining algae residue, extract with magnetic stirring at 4°C for 1 hour, centrifuge at 3500 rpm, and transfer the supernatant to the above-mentioned small bottle. The above process can be repeated until the algae residue turns white. Add pure water to the above combined extracts so that the volume ratio of the four (water, DMSO-Methanol, ether, n-hexane) is 1:1:1:1, vibrate and separate the phases, transfer the organic phase to another small glass In the bottle, blow it with nitrogen in a fume hood until it becomes a concentrated solution, then transfer it to a previously weighed 1.5ml plastic centrifuge tube, and dry it with nitrogen to constant weight.

2) Fatty acid analysis:

After extraction according to the above method, dissolve with n-hexane, and use Agilent 6820 gas chromatograph for gas chromatographic analysis (chromatographic conditions are carrier gas: nitrogen flow rate 1ml/min, hydrogen flow rate 30ml/min, air flow rate 300ml/min, inlet Temperature: 280°C, detector temperature: 280°C, detector type: FID, chromatographic column: DB-5 capillary column (30m×0.25mm, 0.25μm), split ratio: 4:1. Analysis method: internal standard method (Gas chromatography uses nitrogen as a carrier gas, which is equivalent to the mobile phase of liquid chromatography).

Table 2 The sample total lipid composition table determined by gas chromatography

The Scenedesmus strain ENN33 has undergone multiple growth cultures and fatty acid analysis experiments to determine that its fatty acid content can reach 56% to 60% dry weight.

Embodiment 4ENN 33 high temperature resistance

The green motile cells of Scenedesmus strain ENN33 in the logarithmic growth phase were inoculated in the prepared BG11 medium (see Table 1 for the formula), so that the cell density reached an OD ₇₅₀ of 1.2-2.0. During the cultivation process, the light intensity is controlled at 400-1200μmol/m ² .s. During the cultivation period, the pH value of the medium is adjusted to 7-9 by introducing a mixture of 1.5-2% carbon dioxide and air into the culture medium. between. The reactor used for cultivation is a 600*5cm column reactor. On the 4th day of cultivation, the temperature rise treatment was carried out. The first day of treatment was 40-43°C for 2h, and then 40-44°C for 6h, and the rest of the time was kept at a constant temperature of about 25°C. During the cultivation process, samples were taken regularly to measure the dry weight, and the results are shown in Fig. 5 .

ENN33 can maintain a certain growth rate under high temperature conditions, and finally the dry weight reaches about 3.7g/L in 15 days, and the high temperature of 40-44℃ for 6 hours has little effect on the growth of ENN33. However, the high temperature of 40°C is a temperature that may cause death for other algae strains.

Embodiment 5 ENN33 low temperature tolerance

(1) Inoculate the green motile cells of Scenedesmus strain ENN33 in the logarithmic growth phase in the prepared BG11 medium (see Table 1 for the formula), so that the cell density reaches an OD ₇₅₀ of 1.2-2.0. During the cultivation process, the light intensity is controlled at 600-1500μmol/m2/s. During the cultivation period, the pH value of the medium is adjusted between 6.5-8 by introducing a mixture of 1.5-2% carbon dioxide and air into the culture medium. between. The reactor used for cultivation is a 0.5*0.03*0.5m plate reactor. Two experimental groups were set up: the first group was cultured at room temperature (the temperature range of the algae liquid ranged from 15-30°C), and the second group was cultured outdoors at low temperature during the day (the temperature range of the outdoor algae liquid ranged from 6-20°C, which was maintained most of the time. Between 10-15°C, the outdoor ambient temperature is mostly between -10°C and 8°C), and they are moved indoors at night for breeding. During the cultivation process, samples were taken regularly to measure the dry weight, and the results are shown in Figure 6.

Although there is a certain gap between the growth of ENN33 low temperature group and the normal temperature group, it can also maintain a certain growth rate under low temperature conditions, and the dry weight is basically the same as that of the normal temperature group in the later stage of cultivation, and finally the dry weight reaches about 3.8g/L in 20 days .

(2) Inoculate the green motile cells of Scenedesmus strain ENN33 in the logarithmic growth phase in the prepared BG11 medium (see Table 1 for the formula), so that the cell density reaches an OD ₇₅₀ of 1.2-2.0. During the cultivation process, the light intensity is controlled at 600-1500μmol/m2/s. During the cultivation period, the pH value of the medium is adjusted between 6.5-8 by introducing a mixture of 1.5-2% carbon dioxide and air into the culture medium. between. The reactor used for cultivation is a 0.5*0.03*0.5m plate reactor. Two experimental groups were set up: the first group was cultured at room temperature for 24 hours (the temperature range of the algae liquid ranged from 15-30°C), and the second group was cultured at low temperature outdoors (the temperature range of the algae liquid ranged from 6-20°C during the daytime) for 24 hours. Maintain between 10-15 ℃, freezing at night) for breeding. During the cultivation process, samples were taken regularly to measure the dry weight, and the results are shown in FIG. 7 .

The growth of the ENN33 low temperature group was not much different from that of the normal temperature group, and the dry weight of the ENN33 low temperature group was basically the same as that of the normal temperature group during the whole culture process, and the dry weight reached about 3.8g/L at the end of 19 days.

The settleability of embodiment 6 ENN33

The ENN33 cultivated for 19 days in Example 5 was allowed to stand overnight, and observed on the second day, the supernatant was relatively clear, and almost all algal cells had settled to the bottom of the reactor, indicating that ENN33 had settled overnight and had a good collection effect.

Example 7 ENN 33 waste water and waste gas cultivation

Inoculate the algal species in the logarithmic growth phase in a 50*10*50cm plate reactor, and set up two experimental groups: the first group uses tap water to prepare BG11 medium according to Table 1 for cultivation, and the second group uses chemical plant wastewater Add 1.5g/L NaNO ₃ , 0.04g/L K ₂ HPO ₄ ·3H ₂ O to prepare culture medium for cultivation. See Table 3 for the ion concentrations of the two groups of medium.

The ion concentration measurement result of two kinds of culture media of table 3

Note: The ion components in chemical plant wastewater are the results of ion chromatography measurements, and the concentrations of the components in BG11 medium are theoretically calculated values

Use natural conditions for growth and cultivation. The natural conditions during the cultivation period are as follows: the cultivation period is 11 days, the temperature range from 9:00 to 17:00 is mainly between 20-45°C, and the duration of high temperature during the day is 3-5h. During the day, the total light on both sides can reach more than 10,000 μmol/m ² /s in sunny days, and less than 5,000 μmol/m ² /s in cloudy days, and there are only two cloudy days in the whole cultivation process. A certain proportion of CO ₂ (CO ₂ comes from a chemical plant with a purity of more than 95%) is introduced, and the pH value is controlled between 7.5-8.5. Sampling was taken intermittently during the culture process to determine the dry weight (results shown in Figure 8).

The algae strains appeared certain death until the 5th day after cultivation, but then all of them grew up. Compared with tap water aquaculture, wastewater aquaculture has a slight decline in the middle stage of cultivation, but it grows in the later stage, which is not much different from tap water aquaculture. After 11 days of culture, the dry weight reached about 2g/L.

Embodiment 8 ENN 33 pigment content determination

1. Extraction of pigments in algae cells: Take a certain amount of algae cells collected by centrifugation, immerse them in methanol for extraction several times, combine the methanol extracts, remove the methanol with a rotary vacuum evaporator, and then dissolve the concentrate with CCl4 to be tested.

2. Preparation of pigment standard solution: Weigh a certain amount of standard products of astaxanthin, lutein, zeaxanthin, canthaxanthin and β-carotene, and prepare standard working solutions with different mass concentrations with methanol.

3. Perform liquid chromatography to draw the chromatogram and standard curve of the pigment

4. Quantitatively calculate the content of the pigment with the pigment standard by the external standard method (see Table 4, Figure 9)

The sample pigment component table that table 4 liquid chromatography measures

Pigment name Percentage of algae flour Astaxanthin 0.0039% Lutein 0.0021% Zeaxanthin 0.0366% Canthaxanthin 0.0106% β-carotene 0.0548%

The chemical name of astaxanthin is 3,3′-dihydroxy-4,4′-diketone-β,β′-carotene, which can be combined with protein in the body to turn green or blue. Anti-oxidation, anti-aging, anti-tumor, prevention of cardiovascular and cerebrovascular diseases;

Lutein, also known as "plant lutein", is the main pigment that constitutes the macular area of the human eye. It has the functions of protecting eyes, delaying eye aging, preventing disease and antioxidation;

Zeaxanthin is an isomer of lutein, which plays a particularly important role in maintaining eye health;

Canthaxanthin (International Food Additive Number: INS161g) is mainly used as an animal feed additive, and can also be used in foods such as jams, candies, syrups, sauces, and beverages containing carbon dioxide as a coloring agent.

B-carotene is an antioxidant with detoxification effect. It is an indispensable nutrient for maintaining human health. It has significant functions in anti-cancer, prevention of cardiovascular disease, cataract and anti-oxidation, and then prevents aging and aging-induced diseases. Various degenerative diseases.

The applicant declares that the present invention illustrates the detailed structural features and methods of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed structural features and methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed structural features and methods. implement. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of selected components in the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (10)

1. a grid algae ENN33 (Scenedesmus sp.) is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), and preservation date is on 07 13rd, 2012, and deposit number is CGMCC No.6347.

2. grid algae ENN33 according to claim 1 is characterized in that, the content of lipid acid is greater than 40% dry weight among the described grid algae ENN33, is preferably more than 50% dry weight, is 56% ~ 60% dry weight more preferably, is most preferably 58%.

3. grid algae ENN33 according to claim 1 is characterized in that, the tolerable temperature of described grid algae ENN33 is-10 ℃ to 44 ℃, preferably 6 ℃ to 40 ℃, and more preferably 8 ℃ to 30 ℃, also more preferably 10 ℃ to 20 ℃, most preferably 15 ℃.

4. grid algae ENN33 according to claim 1 is characterized in that, described grid algae ENN33 contains astaxanthin, β-carotene, xenthophylls, zeaxanthin and canthaxanthin.

5. such as the cultural method of each described grid algae ENN33 in the claim 1 to 4, described method comprises grid algae algae strain ENN33 green cell is seeded in the BG11 substratum, wherein intensity of illumination 50-500 μ mol/m ².s, preferred 100-450 μ mol/m ².s, more preferably 150-400 μ mol/m ².s, more preferably 200-350 μ mol/m also ².s, 250-300 μ mol/m most preferably ².s, pH value 7-9, preferred 7.5-8.5, more preferably 8, temperature 15-35 ℃, preferred 20-30 ℃, more preferably 25 ℃.

6. such as the application of each described grid algae ENN33 in the claim 1 to 4 in lipid acid is produced, preferably, described lipid acid is tetradecanoic acid, palmitinic acid, Zoomeric acid, stearic acid, octadecenoic acid, linolic acid, therapic acid, alpha-linolenic acid, gamma-linolenic acid, eicosanoic acid, behenic acid and/or timnodonic acid.

7. such as the application of each described grid algae ENN33 in the claim 1 to 4 in biofuel is produced, preferably, described biofuel is biofuel or bio-ethanol.

8. such as each described grid algae ENN33 application in environment protection in the claim 1 to 4, preferably described environment protection is sewage disposal or CO ₂Reduce discharging, described sewage disposal comprises denitrogenation, dephosphorization, the organic matter degradation and/or heavy metal ion adsorbed of sewage.

9. comprise application in the medicine of tetradecanoic acid, palmitinic acid, Zoomeric acid, stearic acid, octadecenoic acid, linolic acid, therapic acid, alpha-linolenic acid, gamma-linolenic acid, eicosanoic acid, behenic acid, timnodonic acid, astaxanthin, β-carotene, xenthophylls, zeaxanthin and/or canthaxanthin such as each described grid algae ENN33 in the claim 1 to 4 in preparation.

10. such as the purposes of each described grid algae ENN33 in the food that comprises tetradecanoic acid, palmitinic acid, Zoomeric acid, stearic acid, octadecenoic acid, linolic acid, therapic acid, alpha-linolenic acid, gamma-linolenic acid, eicosanoic acid, behenic acid, timnodonic acid, astaxanthin, β-carotene, xenthophylls, zeaxanthin and/or canthaxanthin, healthcare products, feed, fishery products bait, makeup and/or pigment production in the claim 1 to 4.

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