CN107287125B - Method for culturing chlorella pyrenoidosa - Google Patents
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Abstract
The invention provides a culture method beneficial to improving the growth rate of chlorella pyrenoidosa, increasing the cell density and promoting the accumulation of oil, which is characterized in that a mixed zymocyte liquid of inactivated Rhodobacter (Rhodobacter sp.) and Rhodopseudomonas (Rhodopseudomonas sp.) bacteria is added into a culture medium for culturing the chlorella pyrenoidosa. The method can lead the algae cells to grow rapidly, the cultured chlorella pyrenoidosa has high cell density, and particularly, the method is added with the mixed bacterial liquid, thereby promoting the rapid proliferation of the algae cells, increasing the concentration of the algae cells and obviously increasing the oil content, and is suitable for large-scale culture production.
Description
Technical Field
The invention belongs to the technical field of algae culture, and particularly relates to a method for culturing chlorella pyrenoidosa.
Background
Chlorella pyrenoidosa (Chlorella pyrenoidosa) belongs to Chlorophyta, Chroococcales, Chlorella, and is the only species with a pyrenoid nucleus in plants of this genus. The chlorella pyrenoidosa cells contain rich protein, polysaccharide, unsaturated fatty acid, dietary fiber, vitamins, trace elements and the like, have high nutritional value and are excellent health-care food and aquaculture bait for human beings. Has been approved as a new resource food by the Ministry of health of the people's republic of China. The chlorella pyrenoidosa serving as a renewable energy biomass can accumulate grease under photoautotrophic and heterotrophic culture conditions, can effectively utilize solar energy and organic energy to rapidly grow and accumulate the grease, can be used as renewable energy to prepare biodiesel, and has great potential in replacing fossil fuel diesel. However, the existing large-scale culture of the chlorella pyrenoidosa has the problems of slow growth, low biomass and low oil content, and is not beneficial to large-scale culture.
Disclosure of Invention
The invention aims to solve the technical problem of providing a culture method for the chlorella pyrenoidosa, which is beneficial to improving the growth rate of the chlorella pyrenoidosa, increasing the cell density and promoting the accumulation of oil, thereby improving the culture efficiency and being particularly suitable for large-scale culture.
The culture method of the chlorella pyrenoidosa provided by the invention is characterized in that a zymocyte liquid of inactivated Rhodobacter sp and Rhodopseudomonas sp bacteria is added into a culture medium for culturing the chlorella pyrenoidosa;
the Rhodobacter genus (Rhodobacter sp.), one of the examples of which is preferably Rhodobacter capsulatus (Rhodobacter capsulatus);
the Rhodopseudomonas sp, one of the embodiments of which is preferably Rhodopseudomonas palustris (Rhodopseudomonas palustris);
the culture medium of the zymocyte liquid comprises the following components: NH (NH)4Cl 1g/L、K2HPO40.5g/L、MgCl20.2g/L, 0.5g/L yeast powder, 4g/L sodium acetate, 30 deg.C, 24 hr continuous illumination, intensity of 10000Lux, 3 days fermentation time, inoculation concentration of each bacterium of 1 × 107one/mL.
The used protein chlorella culture medium comprises the following specific components: peptone 300-500mg/L, K2HPO416-21mg/L，MgSO4·7H2O 60-90mg/L，CaCl2·2H230-42mg/L of O, 8-16mg/L of ferric ammonium citrate and NaHCO310-30mg/L and 0.5-1.5ml/L of trace element solution.
The culture medium may also contain plant growth hormone and vitamin solution.
The auxin is indoleacetic acid, and the vitamin solution is vitamin B2And (3) solution.
More specifically, the culture method adopts the temperature of 30 ℃ and 24-hour continuous illumination, and the intensity is 10000 Lux.
The method can lead the algae cells to grow rapidly, the cultured chlorella pyrenoidosa has high cell density, and particularly, the method is added with the mixed bacterial liquid, thereby promoting the rapid proliferation of the algae cells, increasing the concentration of the algae cells and obviously increasing the oil content, and is suitable for large-scale culture production.
Drawings
FIG. 1 is a graph showing the effect of different medium concentrations and their flora on the oil content of Chlorella pyrenoidosa.
Detailed Description
The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention.
The following description of the materials and processes involved in the present invention is as follows:
various nutrient salts (except plant growth hormone and vitamin solution) used by the invention can be prepared into mother liquor with higher concentration, the mother liquor is prepared into 1000 times of concentrated mother liquor in general, and medicines are respectively prepared and stored when the mother liquor (1) - (7) is prepared; (8) the medicines in the trace elements are mixed and stored after being prepared according to the amount. After the mother liquid is prepared, the mother liquid is respectively stored by covering with a brown glass bottle, labeled, marked with the name of the stored liquid, the preparation times, the date and the like, and stored in a refrigerating chamber of a refrigerator. When working solution culture medium is prepared, the stock solution is gently shaken, and if sediment, suspended matters or microorganism pollution is found, the stock solution needs to be prepared again. Finally, according to the volume of the prepared culture medium, the required dosage is respectively taken out from various storage solutions by using a pipette, water is continuously added and stirred in the process of adding the nutrient salt, the various nutrient salts are added according to the sequence provided in the formula so as to avoid chemical reaction, and distilled water is added to dilute the nutrient salts to the required concentration. Adjusting the prepared culture medium to 7.0 with pH meter, packaging, and sterilizing with high pressure steam at 121 deg.C under 0.103MPa for 20 min.
Oil extraction and content determination: weighing dry algae powder M1, and adding into a mixture with a certain volume ratio of 1: 2 chloroform: oscillating and mixing the methanol solution uniformly, and extracting for 20-30min by using an ultrasonic crusher; centrifuging at 3000r/min for 5min, collecting chloroform layer solution, repeatedly extracting oil for 3 times in a glass centrifuge tube with pre-weighed M2, blow-drying the collected chloroform layer in nitrogen gas, drying in a vacuum drier, weighing to constant weight M3, and weighing
The total lipid content was calculated as follows: total lipid (%) ═ 100 × (M3-M2)/M1.
Nile Red dyeing method: taking a certain volume of the late stage of logarithmic growthCentrifuging the algae solution at 8000 r/min for 5min, removing supernatant, washing the algae cell precipitate with phosphate buffer twice, and resuspending the algae cells with 20% volume of dimethyl sulfoxide aqueous solution to make the algae solution OD540And (3) carrying out water bath at 40 ℃ for 20min, adding 15 mu L of nile red dye (the mass concentration is 0.1mg/mL of acetone solution) into 1mL of algae liquid, uniformly mixing and dyeing for 5min, exciting the wavelength to be 480nm, and measuring the fluorescence intensity of the algae liquid at the wavelength of 575nm to screen the total lipid content of the algae.
The present invention will be described in detail with reference to specific examples.
Example 1: screening of chlorella pyrenoidosa and mixed flora symbiotic system and community structure identification
And (3) putting a water sample extracted from the activated sludge in the biochemical pool of the Harbin municipal sewage plant into an aseptic sampling bag, taking the water sample back to a laboratory, observing the water sample by using a microscope to find that the sample has more microorganisms and algae cells, and enriching, separating and screening corresponding microalgae and microalgae associated flora. Putting the raw water sample into a simulated biochemical pool reactor for continuous culture, placing the reactor under natural illumination, uniformly mixing the reactor with the prepared simulated urban sewage, carrying out aeration treatment for a long time, keeping the oxygen content at 6-8mg/L, culturing at 32 ℃, keeping the pH at 7.5COD at about 400mg/L, TN at about 50mg/L and TP at about 10 mg/L. Municipal sewage was simulated by a modified BG11 medium, which simulated sewage had the following composition (mg/L): peptone 400, potassium dihydrogen phosphate 18.5, magnesium sulfate heptahydrate 75.0, calcium chloride dihydrate 36, ferric ammonium citrate 12, sodium bicarbonate 20, and finally 1mL of trace elements are added. Wherein the formula of the trace elements is (mg/L): h3BO31660，MnCl2·4H2O 1860，ZnSO4·7H2O 220，Na2MoO4·2H2O 21，CuSO4·5H2O 80，Co(NO3)2·6H2O 50，NiCl250，KI 30。
According to the results of the detection, when COD in the culture system was reduced to 300mg/L, 100mg/L glucose was added as a supplementary carbon source. A circulating device is arranged in the aeration tank, 1.5L/min and 5h of conversion can circulate a water sample to facilitate water body backflow and uniform mixing, and a living body is obtained after the continuous aeration tank is cultured for more than 20 daysHas stable properties, can tolerate active bacteria-algae symbiotic system (described below) with certain organic concentration, and has viable bacteria number maintained at 109The number of microalgae is maintained at 10 per mL7one/mL. In the process of treating the wastewater, the initial COD of a water sample is 100mg/L, and the biochemical treatment adopts a method for gradually increasing the proportion of the wastewater, so that the functional flora is gradually adapted to the high-concentration municipal sewage environment. The first generation acclimatization system is added with 1/4 simulated wastewater, supplemented with 3/4 distilled water, inoculated with flora, inoculum size of 20% (v/v), shaking table at 30 ℃ and shaking culture at 160 rpm. After 2 days, the COD degradation rate is 40.25%, and the COD is kept stable and does not decrease any more after the culture is continued. Transferring the culture medium into a second generation domestication culture medium to improve the proportion of wastewater. 50% of the simulated wastewater volume is added with 50% of distilled water volume, the inoculum size is 20%, and the shaking culture is carried out at 30 ℃. After 2d, COD is detected to be reduced from the initial 210.5mg/L to 68.75mg/L, the degradation rate is 67.34%, the steps are repeated until the fourth generation domestication system, all culture media simulate municipal sewage, and the biochemical treatment effect of the active microalgae symbiotic flora on the wastewater is shown in the table 1. COD in the sewage is reduced to about 37.53mg/L and the activity is kept stable, the removal rate of the COD is 91.00 percent, and the removal rate of the COD is similar to the quality of the effluent of the biochemical pool of the original sewage plant, so that a group of stable bacteria-algae symbiotic systems is obtained, and the COD in the system is remarkably reduced.
TABLE 1 acclimatization and sewage treatment effect table for symbiotic system of bacteria and algae
Screening, identifying and separating the enriched mixed microalgae flora by adopting a thallus partial reflux method, and carrying out community structure analysis on the microalgae symbiotic system by utilizing morphological and molecular means to find that the following members mainly exist in the bacterial flora: rhodobacter sp, Rhodopseudomonas sp, Paenibacillus sp, Enterobacter sp, halobacter chromogenes sp, chrysophallum sp, in total up to 80% or more. Among them, Rhodobacter sp and Rhodopseudomonas sp have the highest ratio, which is 21% and 19%, respectively, and secondly, Paenibacillus sp and Enterobacter sp have 11% ratios, and Chromohalobacter sp and chrysophallum sp have 10% and 9%, respectively. Among the active bacterial microbial communities, Rhodobacter (Rhodobacter sp.), Rhodopseudomonas (Rhodopseudomonas sp.) and Paenibacillus (Paenibacillus sp.) widely exist in the nature and biochemical ponds of various large sewage treatment plants, can grow under autotrophic or heterotrophic conditions, and participate in the conversion and circulation of substances such as carbon, nitrogen and phosphorus elements. Enterobacter (Enterobacter) and Chromohalobacter (Chromohalobacter) have certain halophilicity and a certain function of degrading organic matters, and are microorganisms widely existing in various sewage. Chryseobacterium exists in functional flora for municipal and chemical wastewater treatment, and can degrade organic matters such as aromatic hydrocarbons, benzene and the like.
Through the method and the means, the main species of the microalgae are found to comprise two types of microalgae: chlorella sp and Scenedesmus sp, wherein Chlorella sp accounts for 90% of the total. The oil content of the two microalgae is measured by a Nile red dyeing method, and the oil content of the chlorella is found to reach 21.25%, the total lipid yield reaches 113.8 mg/(L.d), the oil content of scenedesmus reaches 18.76%, and the total lipid yield reaches 87.8 mg/(L.d). The invention is identified by molecular biology means such as 16S rRNA and the like, and the oil-rich microalgae Chlorella (Chlorella sp.) in the method is determined to be Chlorella pyrenoidosa (Chlorella pyrenoidosa).
Example 2: compound flora and activity verification
The formulation of the photosynthetic bacterial microorganism Rhodobacter (Rhodobacter sp.) and Rhodopseudomonas sp (Rhodopseudomonas sp.) in example 1 was simulated and was compounded with Chlorella pyrenoidosa (Chlorella pyrenoidosa). Wherein the Rhodobacter (Rhodobacter sp.) is selected from Rhodobacter capsulatus (Rhodobacter capsulatus) and Rhodopseudomonas (Rhodopseudomonas sp.) is selected from Rhodopseudomonas palustris (Rhodopseudomonas palustris). Wherein, the single bacterium (algae) is respectively activated by LB culture medium, the cell concentration is adjusted after activation, and the adding concentration of the chlorella pyrenoidosa is more than two numbers of the two photosynthetic bacteria concentrationAnd step one, analyzing the influence of a compound bacterium group on the oil content of the chlorella pyrenoidosa according to the oil content detection method of the specific embodiment by the microbial algae system obtained by compounding, wherein the oil content of the chlorella pyrenoidosa after the culture is finished is 35.43%, the chlorella pyrenoidosa grows stably, and the cell density of the algae reaches 5 × 107The total lipid yield reaches 133.9 mg/(L.d). The results show that the prepared composite flora verifies the promotion effect of the functional flora on the oil production of the chlorella pyrenoidosa in the example 1, and simultaneously shows that the influence of the flora obtained by compounding two single bacteria on the algae growth density, the oil content and the sewage COD removal efficiency is better than that of the bacterial-algae symbiotic system screened in the example 1, and is respectively improved by 400%, 66.73% and 7.79%.
Example 3: optimization of growth and oil content of chlorella pyrenoidosa by different culture medium concentrations and flora concentrations
The culture medium after the optimization of the chlorella pyrenoidosa with the following formula is prepared by sterile water: peptone 300mg/L, KH2PO416mg/L，MgSO4·7H2O 60mg/L，CaCl2·2H2O30 mg/L, ferric ammonium citrate 8mg/L, NaHCO310mg/L, and 0.5ml/L of trace element solution; also comprises auxin indoleacetic acid 1.0mg/L and vitamin B2The solution was 0.5 mg/L. Comprises 10mL of mixed flora fermentation liquor of Rhodobacter capsulatus and Rhodopseudomonas palustris, wherein the inoculation ratio of the two strains is 1: 1. The mixed flora fermentation liquid comprises the following components: NH (NH)4Cl1g/L、K2HPO40.5g/L、MgCl20.2g/L, 0.5g/L of yeast powder, 4g/L of sodium acetate, 30 ℃ of fermentation temperature, 24 hours of continuous illumination, 10000Lux of intensity, 3 days of fermentation time, 1:1 of inoculation ratio, and 1 × 10 of inoculation concentration of each bacterium7one/mL. Inoculating chlorella pyrenoidosa in logarithmic growth phase into the culture medium for culture, continuously illuminating for 24 hours, controlling the intensity to be 10000Lux, controlling the culture temperature to be about 30 ℃, manually shaking twice a day to prevent the sediment from adhering to the wall, and regularly recording the cell concentration of the chlorella pyrenoidosa in the culture medium. BG-11 medium was used as a control medium without addition of mixed flora broth to the mediumAnd special nutrient substances as a control experiment group. The algae density values of each experimental group were measured at the late stage of the logarithmic growth phase and are detailed in table 2; meanwhile, the oil content of the chlorella pyrenoidosa is measured, and the detailed description is shown in figure 1.
TABLE 2 comparison table of the effect of different flora mixture concentrations and nutrient concentrations on the growth of Chlorella pyrenoidosa
Example 4: optimization of growth and oil content of chlorella pyrenoidosa by different culture medium concentrations and flora concentrations
The culture medium after the optimization of the chlorella pyrenoidosa with the following formula is prepared by sterile water: peptone 300mg/L, KH2PO416mg/L，MgSO4·7H2O 60mg/L，CaCl2·2H2O30 mg/L, ferric ammonium citrate 8mg/L, NaHCO310mg/L, and 0.5ml/L of trace element solution; also comprises auxin indoleacetic acid 1.0mg/L and vitamin B2The solution was 0.5 mg/L. Comprises 30mL of mixed flora fermentation liquor of Rhodobacter sp and Rhodopseudomonas sp, and the composition, fermentation conditions and inoculation ratio of the mixed flora fermentation liquor are the same as those in example 3. Inoculating chlorella pyrenoidosa in logarithmic growth phase into the culture medium for culture, continuously illuminating for 24 hours, controlling the intensity to be 10000Lux, controlling the culture temperature to be about 30 ℃, manually shaking twice a day to prevent the sediment from adhering to the wall, and regularly recording the cell concentration of the chlorella pyrenoidosa in the culture medium. In addition, BG-11 medium was used as a control medium, and mixed flora fermentation broth and special nutrients were not added as control experimental groups. The algae density values of each experimental group were measured at the late stage of the logarithmic growth phase and are detailed in table 3; meanwhile, the oil content of the chlorella pyrenoidosa is measured, and the detailed description is shown in figure 1.
TABLE 3 comparison table of the effect of different flora mixture concentrations and nutrient concentrations on the growth of Chlorella pyrenoidosa
Example 5: optimization of growth and oil content of chlorella pyrenoidosa by different culture medium concentrations and flora concentrations
The culture medium after the optimization of the chlorella pyrenoidosa with the following formula is prepared by sterile water: peptone 500mg/L, KH2PO421mg/L，MgSO4·7H2O 90mg/L，CaCl2·2H2O42 mg/L, ferric ammonium citrate 16mg/L, NaHCO330mg/L, and 1.5ml/L of trace element solution; also comprises auxin indoleacetic acid 5.0mg/L and vitamin B2The solution was 1.0 mg/L. Comprises 10mL of mixed flora fermentation liquor of Rhodobacter sp and Rhodopseudomonas sp, and the composition, fermentation conditions and inoculation ratio of the mixed flora fermentation liquor are the same as those in example 3. Inoculating chlorella pyrenoidosa in logarithmic growth phase into the culture medium for culture, continuously illuminating for 24 hours, controlling the intensity to be 10000Lux, controlling the culture temperature to be about 30 ℃, manually shaking twice a day to prevent the sediment from adhering to the wall, and regularly recording the cell concentration of the chlorella pyrenoidosa in the culture medium. BG-11 medium was used as control medium without addition of mixed flora fermentation broth and special nutrients. The algae density values of each experimental group were measured at the late stage of the logarithmic growth phase and are detailed in table 4; meanwhile, the oil content of the chlorella pyrenoidosa is measured, and the detailed description is shown in figure 1.
TABLE 4 comparison table of the effect of different flora mixture concentrations and nutrient concentrations on the growth of Chlorella pyrenoidosa
Example 6: optimization of growth and oil content of chlorella pyrenoidosa by different culture medium concentrations and flora concentrations
The culture medium after the optimization of the chlorella pyrenoidosa with the following formula is prepared by sterile water: peptone 500mg/L, KH2PO421mg/L，MgSO4·7H2O 90mg/L，CaCl2·2H2O42 mg/L, ferric ammonium citrate 16mg/L, NaHCO330mg/L, and 1.5ml/L of trace element solution; also comprises auxin indoleacetic acid 5.0mg/L and vitamin B2The solution was 1.0 mg/L. Comprises 30mL of mixed flora fermentation liquor of Rhodobacter sp and Rhodopseudomonas sp, and the composition, fermentation conditions and inoculation ratio of the mixed flora fermentation liquor are the same as those in example 3. Inoculating chlorella pyrenoidosa in logarithmic growth phase into the culture medium for culture, continuously illuminating for 24 hours, controlling the intensity to be 10000Lux, controlling the culture temperature to be about 30 ℃, manually shaking twice a day to prevent the sediment from adhering to the wall, and regularly recording the cell concentration of the chlorella pyrenoidosa in the culture medium. BG-11 medium was used as control medium without addition of mixed flora fermentation broth and special nutrients. The algae density values of each experimental group were measured at the late stage of the logarithmic growth phase and are detailed in table 5; meanwhile, the oil content of the chlorella pyrenoidosa is measured, and the detailed description is shown in figure 1.
TABLE 5 comparison table of the effect of different flora mixture concentrations and nutrient concentrations on the growth of Chlorella pyrenoidosa
From the above results, it was found that the culture method and the culture medium provided by the present invention can rapidly proliferate chlorella pyrenoidosa, advance the time of logarithmic growth of chlorella pyrenoidosa, and shorten the culture time, compared with the case of using BG-11 medium alone. As can be seen from the figure 1, the oil content of the chlorella pyrenoidosa under the optimal culture conditions of each example is remarkably improved compared with that of a control culture medium BG-11, so that the method for culturing the chlorella pyrenoidosa provided by the invention has obvious culture advantages, is more beneficial to the rapid growth of algae cells, and simultaneously promotes the improvement of the oil content.
The above description is only an exemplary embodiment of the present invention, and is not intended to limit the scope of the present invention. Any equivalent changes and modifications that can be made by one skilled in the art without departing from the spirit and principles of the invention should fall within the protection scope of the invention.
Claims (7)
1. A method for culturing Chlorella pyrenoidosa, characterized in that the culture medium for culturing Chlorella pyrenoidosa is supplemented with inactivated Rhodobacteria (genus: (A) (B))Rhodobacter sp.) And Rhodopseudomonas (R.), (Rhodopseudomonas sp.) A zymogen solution of bacteria;
the genus rhodobacter (a)Rhodobacter sp.) The bacteria are rhodobacter capsulatus (Rhodobacter capsulatus)；
The Rhodopseudomonas sp is Rhodopseudomonas palustris.
2. The method according to claim 1, wherein the ratio of the culture medium used for fermentation of the fermented bacterial liquid is as follows: NH (NH)4Cl 1 g/L、K2HPO40.5 g/L、MgCl20.2g/L, 0.5g/L yeast powder and 4g/L sodium acetate.
3. The method of claim 1, wherein the culture medium is prepared from the following components: peptone 300-500mg/L, K2HPO416-21 mg/L，MgSO4·7H2O 60-90 mg/L，CaCl2·2H230-42mg/L of O, 8-16mg/L of ferric ammonium citrate and NaHCO310-30mg/L and 0.5-1.5ml/L of trace element solution.
4. A method according to claim 2 or 3, wherein the nutrient medium is supplemented with an auxin and/or vitamin solution.
5. The method of claim 4, wherein the auxin is indoleacetic acid and the vitamin solution is vitamin B2And (3) solution.
6. The method according to claim 1, wherein the cultivation is carried out at a temperature of 30 ℃ and a continuous illumination for 24 hours at an intensity of 10000 Lux.
7. A culture medium for culturing chlorella pyrenoidosa, which is characterized by comprising the following components: peptone 300-500mg/L, K2HPO416-21 mg/L，MgSO4·7H2O 60-90 mg/L，CaCl2·2H230-42mg/L of O, 8-16mg/L of ferric ammonium citrate and NaHCO310-30mg/L, 0.5-1.5ml/L of trace element solution; inactivated rhodobacter genus (Rhodobacter sp.) And Rhodopseudomonas (R.), (Rhodopseudomonas sp.) A zymogen solution of bacteria; auxin and vitamin solutions;
the inactivated rhodobacter genus (Rhodobacter sp.) And Rhodopseudomonas (R.), (Rhodopseudomonas sp.) A zymogen solution of bacteria; the proportion of the culture medium used in the fermentation is as follows: NH (NH)4Cl 1 g/L、K2HPO40.5 g/L、MgCl20.2g/L, 0.5g/L yeast powder and 4g/L sodium acetate;
the genus rhodobacter (a)Rhodobacter sp.) The bacteria are rhodobacter capsulatus (Rhodobacter capsulatus)；
Said Rhodopseudomonas genus (A), (B), (CRhodopseudomonas sp.) The bacteria are Rhodopseudomonas palustris (A)Rhodopseudomonas palustris)。
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