CN110628643A - Method for screening optimal green algae combination mixed culture - Google Patents

Abstract

The invention discloses a method for screening the best green algae combination and mixed culture. In particular to a method for screening the crescent moon algaeSelenastrumcapricornutum) And Chlorella pyrenoidosa (C. pyrenoidosa) ((C. pyrenoidosa))Chlorellapyrenoidosa) The optimal combination of the two green algae is mixed culture method. The method selects a proper proportion for mixed culture under proper conditions through simulation experiments according to the biological characteristics of two kinds of algae, and selects the green algae combination with high total cell density, high chlorophyll a content, large light absorption value and large biomass to be the optimal combination from a mixed culture system. The culture method is ecological and environment-friendly, solves the problems of poor light energy utilization rate, low cell density, little biomass and the like which are usually encountered in the culture process of the green algae, and realizes the high-density large-scale culture industrialization of efficiently culturing the green algae.

Description

Method for screening optimal green algae combination mixed culture

Technical Field

The invention belongs to the technical field of algae biology, and particularly relates to a method for screening optimal green algae combination and mixed culture. The method involves the use of Hibiscus aegillus (crescent moon algae)Selenastrum capricornutum) And Chlorella pyrenoidosa (C. pyrenoidosa) ((C. pyrenoidosa))Chlorella pyrenoidosa) Two kinds of algae.

Background

The green algae is an autotrophic plant with various varieties, wide distribution, rich nutrition, various cell forms and high photosynthetic availability, and polysaccharides, proteins, pigments and the like generated by cell metabolism have good development prospects in the fields of food, medicine, genetic engineering, liquid fuel and the like. The green algae plays an extremely important role in the balance and stability of the water body as a primary producer of the aquatic ecosystem, and is an important index for reflecting the environmental quality of the water body. Crescent moon of sheep horn (crescent moon)Selenastrum capricornutum) Chlorella pyrenoidosa (C. pyrenoidosa) ((C. pyrenoidosa))Chlorella pyrenoidosa) The algae is a common algae species in biotoxicity tests in the current aquatic ecotoxicology research due to the characteristics of small individual, fast propagation, high sensitivity and the like. Meanwhile, high-concentration nitrogen and phosphorus in the sewage are essential elements for the growth of green algae, the urban domestic sewage is treated by using the green algae, the content of pollutants can be reduced, and the feed containing the green algae single cells is harvested at the same time, so that the method has the characteristics of low cost, good economic effect and the like.

However, in the prior art, the cultivation of algae is mainly based on a single algae species cultivation mode, and even if the algae cultivation is carried out by searching the optimal conditions suitable for the growth and propagation of algae, such as temperature, illumination, nutritive salt, cultivation mode and other factors, the high-density cultivation effect can not be achieved, which directly influences the success rate and the use effect of continuous propagation; meanwhile, the culture of single algae species is not suitable for a sewage system, and the production efficiency of the single algae species can be reduced even the culture system is broken down by the mixed algae, the protozoa and the like in the sewage system. The expected culture effect can be achieved by selecting proper green algae for mixed culture. At present, much research on microalgae mixed culture focuses on the growth inhibition of water bloom microalgae, and few researches on obtaining a high-density green algae mixed culture mode are reported. Therefore, the optimal combined screening method for mixed culture of multiple algae species, which is easy to operate, strong in practicability, ecological and environment-friendly, is provided, and the high-efficiency and high-density green algae culture effect is realized.

Disclosure of Invention

The invention aims to provide a method for screening the optimal green algae combination mixed culture. So that the effect of high-density green algae culture can be realized.

The method comprises the following specific steps:

(1) the algae seed pure culture includes purchasing algae seed, preparing corresponding culture medium, transferring 5 ~ 10mL algae seed directly to 250mL conical flask under aseptic operation after receiving algae seed, adding prepared fresh culture medium to 100mL, sealing the flask, culturing in light culture box, diluting and transferring algae seed in 1:1 every 2 ~ 3 days, transferring to 3 generations, and observing the growth condition of algae seed under microscope during each inoculation.

(2) And (3) algal strain mixed culture design: the two algae species combination experiments are provided with 3 treatment groups and 2 control groups in total, each algae species in the experiments is provided with 3 parallel treatments, and the initial total density of the cells of pure culture and mixed culture is set in a two-algae species mixed system.

(3) And (3) algae seed mixed culture: selecting two types of experimental algae in the logarithmic growth phase under the pure culture condition obtained in the step (1), calculating the cell concentration of the algae by a blood counting method, diluting with a culture medium to ensure the same initial inoculation cell concentration of the two types of algae, and uniformly mixing and culturing according to a proportion.

(4) And (3) measuring the related indexes of the mixed culture algae: the day after inoculation is the first day, and 4 indexes of algae cell density, chlorophyll a, light absorption value and biomass are measured at the same time point in each measurement.

(5) Screening an optimal combination: and (4) screening out the optimal combination of high cell density, high chlorophyll a content, large light absorption value and large biomass of the algae according to the related index result determined in the step (4) and compared with a control group.

The algae in the step (1) specifically refer to crescent moon algae and chlorella pyrenoidosa, and the culture temperature of the illumination incubator is 25 ℃, the illumination condition is 1000 ~ 2000lux, and the time is 12Hr day and 12Hr night.

The 3 treatment groups in the step (2) are set according to 3 different proportions, and the 2 comparison groups are respectively pure cultured crescent moon algae and pure cultured chlorella pyrenoidosa under the same conditions.

The initial inoculation algae cell concentration of the two kinds of algae in the step (3) is 5 multiplied by 10⁵one/mL.

Measuring the cell density of the green algae in the step (4) by using a blood counting chamber; the chlorophyll a is determined by an ethanol-ultrasonic method; the light absorption value is measured by an ultraviolet visible spectrophotometer UV 9000S; biomass was determined by dry weight method.

Compared with the prior art, the invention has the advantages that:

(1) the invention provides a method for screening the best green algae combination mixed culture, which is simple to operate, strong in practicability and ecological and environment-friendly; the method has the advantages of high culture speed and good culture stability.

(2) The invention can realize the high-density and low-cost culture of the green algae, and the cultured green algae has activity and can be propagated in large quantities.

Drawings

FIG. 1 is a graph showing the cell density of each component in Sc-Cp mixed culture at different ratios in examples of the present invention.

FIG. 2 is a graph showing chlorophyll-a contents of components in Sc-Cp mixed culture at different ratios in the example of the present invention.

FIG. 3 is a graph showing absorbance values of components in a Sc-Cp mixed culture at different ratios in examples of the present invention.

FIG. 4 is a graph showing the biological amounts of the components in the Sc-Cp mixed culture at different ratios in the examples of the present invention.

Detailed Description

The above-described scheme is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes and are not intended to limit the scope of the present invention. The conditions used in the examples may be further adjusted according to the conditions of the particular manufacturer, and the conditions not specified are generally the conditions in routine experiments.

Reference will now be made by way of example, if not explicitly described, to the usual laboratory manuals and manufacturer's specifications for the equipment used. Wherein the strain is purchased from freshwater algae seed bank (FACHB) of the typical culture preservation Committee of Chinese academy of sciences, and numbers of crescent moon algae and Chlorella pyrenoidosa are respectively [ FACHB-271 ] and [ FACHB-5 ].

Example (b):

1.1 preparation of culture medium: the pure culture and mixed culture of two green algae of the crescent moon algae (Sc) and the chlorella pyrenoidosa (Cp) adopt BG11 culture medium, and the formula is shown in Table 1. The formula of BG11 culture medium is prepared according to the formula of Table 1, and the preparation method comprises the following steps: preparing the components in the table 1 into corresponding mother liquor concentration, sequentially transferring the corresponding mother liquor dosage to a 1000mL volumetric flask according to the indicated sequence, sealing and labeling, fixing the volume to 1000mL by using distilled water, sterilizing at 121 ℃ and 101KPa for 20min, placing in a refrigerator at 4 ℃ after sterilizing and cooling, and storing for 2 months.

TABLE 1 BG11 Medium formulation

Note: the pH was adjusted to 7.1 using 1M NaOH or HCl.

1.2 pure culture of algae, namely purchasing algae and receiving the algae, shaking the algae liquid in a test tube uniformly, directly transferring each algae (5 ~ 10 mL) into a 250mL conical flask under aseptic operation, adding prepared fresh culture medium to a constant volume of 100mL, sealing the bottle mouth, placing the conical flask in a light incubator for culture, setting the culture temperature of the light incubator to be 25 ℃, the light condition to be 1000 ~ 2000lux, the time to be 12Hr day and night, diluting and transferring the algae seeds according to a ratio of 1:1 at intervals of 2 ~ 3d, repeatedly inoculating the algae seeds for 2 ~ 3 times at the most vigorous algae cell metabolism period (about 10M to 10 ~ 11M), shaking the algae seeds once by hand at regular time every day, taking the algae seeds as test algae when the algae basically reach a synchronous growth stage and are in a logarithmic phase, and observing the growth condition of the algae seeds under a microscope at each inoculation.

1.3 determination of initial inoculation algal cell concentration: counting the number of algae cells of pure culture stock solution of the crescent moon algae and the chlorella pyrenoidosa in logarithmic growth phase under a microscope by using a blood cell counting method, wherein each algae is counted for not less than three times, and taking an average valueAnd diluting the original algae solution to the same algae cell concentration of 5 × 10 by using BG11 culture medium⁵one/mL.

1.4 algal species mixed culture design: the combined experiments of the crescent moon algae (Sc) and the Chlorella pyrenoidosa (Cp) are provided with 3 treatment groups and 2 control groups in total, each treatment group in the experiments is provided with 3 parallel groups, and in a Sc-Cp mixed system, the initial total density of mixed culture and pure culture is set to be 5 multiplied by 10⁵one/mL, and the total culture volume is 120mL, which is specifically designed as shown in Table 2.

TABLE 2 treatment of two kinds of green algae mixed culture experiment

1.5 algal species mixed culture: under aseptic operation, the initial inoculation algae cell concentration determined under pure culture condition is 5 × 10⁵The Sc and Cp algae are uniformly mixed according to a designed proportion, 3 treatment groups and 2 control groups are arranged, 3 parallel treatments are arranged in each experiment, the mixture is placed in a 250mL conical flask for culture, the mouth of the flask is sealed, the flask is also placed in an illumination incubator for culture, the culture conditions of the incubator are the same as the pure culture conditions, the flask is shaken once by hand every day in three stages, the position of the flask is changed in a crossed manner, the illumination is uniform, and the culture is carried out for 15 d.

1.6 determination of related indexes of mixed culture algae

1.6.1 measurement of algal cell Density

The algal cell density of green algae was measured with a hemocytometer. Taking 1mL of the uniformly mixed algae solution, then dropwise adding 1% of Luge reagent for fixing, measuring the algae cell density of each green algae in unit volume by using a blood counting plate after uniformly mixing, measuring each sample for three times, and calculating the average value to obtain the algae cell density of each sample. The first day from the day of inoculation, counts were measured every other day, each at the same time point. The results are shown in FIG. 1.

1.6.2 determination of chlorophyll-a

Ethanol-sonication was used. Day after day of self-inoculationThe measurement was performed 3 times on the first day, namely, the 5 th day, the 10 th day and the 15 th day. Collecting 20mL of the algae solution, and mixing the algae solution at 10000r.min^-1Centrifuging for 10min, and pouring out supernatant after centrifuging. Adding 95% ethanol solution, ultrasonic crushing for 20min, shielding from light for 24 hr, and ultrasonic crushing at 10000r.min^-1Centrifuging for 10min, pouring the centrifuged supernatant into a cuvette, measuring absorbance at 665nm and 649 nm, and measuring chlorophyll a content in mg.L by using the following formula^-1. The results are shown in FIG. 2.

Chl a=13.95×OD₆₆₅-6.88×OD₆₄₉ (1)

1.6.3 determination of Absorbance value

Full-wave scanning is carried out on the crescent moon algae and the chlorella pyrenoidosa by using an ultraviolet visible spectrophotometer, the absorption peaks are all between 680 and 684nm, 681nm is taken in the embodiment, 3mL of uniformly mixed algae liquid is taken, and the OD value of the algae liquid is measured by using the ultraviolet visible spectrophotometer. The first day from the day of inoculation, counts were measured every other day, each at the same time point. The results are shown in FIG. 3.

1.6.4 determination of Biomass

Drying the 0.22 μm filter membrane in an incubator, and determining the mass W of the filter membrane after drying₀. Filtering the cultured algae solution 5 m L of 15d with 0.22 μm oven-dried filter membrane, oven-drying the filter membrane in 80 deg.C constant temperature incubator, and determining the mass of the oven-dried filter membrane

W₁. Biomass of each algae per unit volume W =200 × (W)₁- W₀) The biomass is measured in g.L^-1. The results are shown in FIG. 4.

1.7 screening for optimal combinations

Based on the results of the determination of the index, the optimal combination is selected by comparing the results with the control group.

From the results of fig. 1 ~ 4, it is known that, when the crescent moon algae and the chlorella pyrenoidosa are mixed and cultured in a proper inoculation ratio under a proper condition, the cell density, chlorophyll a, light absorption value and biomass of the crescent moon algae and the chlorella pyrenoidosa in the mixed culture system are improved compared with those in pure culture, the growth of the crescent moon algae and the chlorella pyrenoidosa is mutually promoted to different degrees, and the chlorella pyrenoidosa becomes a dominant species, wherein, when the mixing ratio of the crescent moon algae and the chlorella pyrenoidosa is 40:80, the cell density, the chlorophyll a, the light absorption value and the biomass of the crescent moon algae in the mixed culture process reach the maximum value, and the best combination for screening is the combination of the crescent moon algae and the chlorella pyrenoidosa with the mixing ratio of 40: 80.

The above description is only for the purpose of illustrating the present invention, and is not intended to limit the present invention in any way. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. It is intended that any person skilled in the art can use the above disclosed methods and techniques to make many possible variations and modifications to the disclosed embodiments, or to modify an equivalent embodiment to an equivalent variation without departing from the spirit and scope of the present invention. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.

Claims (1)

1. A method for screening the best green algae combination mixed culture is characterized by comprising the following specific steps:

(1) the algae seed pure culture, purchasing algae seed and preparing corresponding culture medium, after receiving the algae seed, directly transferring 5 ~ 10mL of algae seed into a 250mL conical flask under aseptic operation, adding prepared fresh culture medium to a constant volume of 100mL, sealing the bottle mouth, placing the bottle mouth in a lighting incubator for culture, diluting and transferring the algae seed according to a ratio of 1:1 every 2 ~ 3d, transferring the algae seed to 3 generations to serve as test algae, and observing the growth condition of the algae seed under a microscope during each inoculation;

(2) and (3) algal strain mixed culture design: the two algae species combination experiments are provided with 3 treatment groups and 2 control groups in total, each algae species in the experiments is arranged to be treated in 3 parallel, and the initial algae cell density of pure culture and mixed culture is set in a two algae species mixed system;

(3) and (3) algae seed mixed culture: selecting two types of test algae in logarithmic growth phase under the pure culture condition obtained in the step (1), calculating the cell concentration of the algae by a blood counting method, diluting with a culture medium to ensure the same initial inoculation cell concentration of the two types of algae, and uniformly mixing and culturing according to a proportion;

(4) and (3) measuring the related indexes of the mixed culture algae: measuring 4 indexes of algae cell density, chlorophyll a, light absorption value and biomass at the same time point in each measurement from the next day of inoculation;

(5) screening an optimal combination: screening out the optimal combination of high algae cell density, high chlorophyll a content, large light absorption value and large biomass according to the relevant index result measured in the step (4) and compared with a control group;

the algae seeds in the step (1) specifically refer to crescent moon algae and chlorella pyrenoidosa, the culture temperature of the illumination incubator is 25 ℃, the illumination condition is 1000 ~ 2000lux, and the time is 12Hr day and 12Hr night;

the 3 treatment groups in the step (2) are set according to 3 different proportions, and the 2 comparison groups are respectively pure cultured crescent moon algae and pure cultured chlorella pyrenoidosa under the same conditions;

the initial inoculation cell concentration of the two kinds of algae in the step (3) is about 5 multiplied by 10⁵Per mL;

measuring the cell density of the green algae in the step (4) by using a blood counting chamber; the chlorophyll a is determined by an ethanol-ultrasonic method; the light absorption value is measured by an ultraviolet visible spectrophotometer UV 9000S; biomass was determined by dry weight method.