CN110564645B

CN110564645B - Chlorella endophyte and application thereof in promoting growth of chlorella

Info

Publication number: CN110564645B
Application number: CN201910886058.3A
Authority: CN
Inventors: 孙诗清; 赵永军; 胡长伟; 曹卫星; 刘娟; 孙辰
Original assignee: Jiaxing University
Current assignee: Jiaxing University
Priority date: 2019-09-18
Filing date: 2019-09-18
Publication date: 2020-12-01
Anticipated expiration: 2039-09-18
Also published as: CN110564645A

Abstract

The invention relates to a strain for culturing chlorella, in particular to a chlorella endophyte and application thereof, belonging to the technical field of biology. A chlorella endophyte is a bacterium of the genus Microbacterium (A)Microbacteriumsaccharophilum) The strain S395-2 is preserved in China general microbiological culture Collection center (CGMCC) 30 months and 7 months in 2018, and the number of the strain is CGMCC No. 16182. The method takes Chlorella (Chlorella sp.) as a research object, separates and purifies dominant endophytes from Chlorella, classifies and identifies the dominant endophytes, and researches the growth promoting effect of the endophytes in a Chlorella symbiotic system on the Chlorella by artificially constructing the Chlorella and a Chlorella endophyte symbiotic system. The chlorella endophyte S395-2 can be used as a new microorganism resource for promoting the growth of microalgae and improving the growth quality of microalgae.

Description

Chlorella endophyte and application thereof in promoting growth of chlorella

Technical Field

The invention relates to a strain for culturing chlorella, in particular to a chlorella endophyte and application thereof, belonging to the technical field of biology.

Background

The endophyte is a novel microbial resource which can be continuously developed, and has wide application prospect. The research on plant endophytes at home and abroad has been over a hundred years since kloepper first proposed the concept of 'plant endophytes', but the microorganism group is overlooked by people for a long time because the microorganism group does not show obvious external characteristics and corresponding effects. Until the end of the twentieth century, Stierle and the like separated and screened inside a taxus brevifolia tissue to obtain a plant endophytic fungus capable of producing taxol, and the research is regarded and deeply developed and becomes a research hotspot at home and abroad. Researchers explore the significance of the endophytes from various angles according to different research purposes, greatly promote the research on the diversity of the endophytes, enrich the connotation of the endophytes, and provide valuable reference opinions for the follow-up research.

Chlorella contains abundant nutrients such as protein, dietary fiber, chlorophyll, etc., can promote and maintain human health, and is currently on the market as a health product. At the same time, at present, microalgae is recognized by researchers to be one of high-quality biodiesel raw materials, and the oil production rate of the microalgae is also obviously higher than that of oil crops commonly used in the market at present. Therefore, the development and utilization of microalgae as a third-generation biofuel feedstock are also increasing. Chlorella is utilized for the first time, has the property of high-content protein, the protein content can reach 64.60 percent, and the chlorella can be widely applied to the fields of disease auxiliary treatment, food additives, health care, beauty treatment and the like. Therefore, the key point for developing and applying the chlorella is to improve the growth index of the chlorella.

Many microorganisms have been shown to survive in plant tissues and establish a good symbiotic relationship with plants, and most promote plant growth. The plant endophyte can play a role in promoting the growth of the plant by producing plant growth hormone substances, and the plant growth regulating hormones mainly comprise auxin, gibberellin, zeatin, cytokinin and the like and can directly promote the growth of the plant. The researches of Chua scholan and the like find that endophyte BS-2 has obvious growth promoting effect on pepper seedlings, and after the pepper seedlings are treated by the strain, the content of plant growth regulating hormones such as IAA, GA3 and the like of a host plant body is increased, so that the growth of the pepper seedlings is promoted, and the yield of pepper is increased. The tobacco seedlings are subjected to greenhouse pot experiment by utilizing endophytic bacillus subtilis B-001 with gold and the like, and the increase of the content of gibberellin, zeatin nucleoside and indoleacetic acid in the tobacco is found in the endogenous hormone detection process, so that the growth of the tobacco seedlings is promoted. Researches show that the co-culture of bacteria and microalgae not only can improve the growth amount of algae cells by 30-300%, but also can obviously increase the growth indexes of chlorella, such as the content of algae powder, the content of pigments such as chlorophyll a, chlorophyll b and carotenoid of the algae cells, increase the oil yield and greatly reduce the cost of the traditional culture. Under the background, the search of new microbial resources for improving the bacterial-algae co-culture technology has great prospect. The plant body is a complex micro-ecological system, the healthy plant body contains a large number of endophytes, the endophytes are microorganisms which can colonize in the plant body and establish a harmonious symbiotic relationship with the host plant, and the endophytes can promote the growth of the host plant, keep the plant in a good growth state and improve the disease resistance of the plant. The method explores the growth promotion effect of endophyte on chlorella as an original host, and has great significance for the large-scale production of chlorella.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a chlorella endophyte.

The invention also aims to provide the application of the chlorella endophyte in promoting the growth of chlorella.

The technical problem of the invention is implemented by the following technical scheme:

a chlorella endophyte is a strain S395-2 of Microbacterium saccharophilum which is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 7 and 30 months. Address: the institute of microbiology, national academy of sciences No.3, Xilu No.1, Beijing, Chaoyang, Beijing.

An application of the chlorella endophyte in promoting the growth of chlorella. Preferably, in the application, chlorella endophyte and chlorella are co-cultured, and the cell concentration ratio of chlorella to chlorella is 1-10: 1.

preferably, the chlorella endophyte is cultured in a LB liquid culture medium in a constant temperature shaking table at 37 + -1 deg.C and 200 + -50 rpm in the dark, and the obtained somatic cells are used for culturing chlorella.

Preferably, the strain culture solution is cultured in a light incubator with the light intensity of 1500-.

The method takes Chlorella (Chlorella sp) as a research object, obtains dominant endophyte by separating and purifying the Chlorella, and researches the growth promoting effect of the endophyte to the Chlorella in a Chlorella symbiotic system by artificially constructing the Chlorella and Chlorella endophyte symbiotic system after classifying and identifying the dominant endophyte, and simultaneously explores and develops the feasibility of the plant endophyte as a new microorganism resource for promoting the growth of the microalgae and improving the biochemical quality of the microalgae.

The inventor separates and purifies the chlorella liquid cultured in a laboratory to obtain the endophyte S395-2, the appearance is yellow, the colony is round, the texture is sticky and moist, and the colony distribution is relatively independent. By staining and molecular characterization, it belongs to Pseudomonas sp⁺And (5) bacteria. The cell concentration ratio of the bacteria and the algae is 10: 1 hour, endophyte S395-2 can effectively promote the growth efficiency of chlorella, and the dry weight of chlorella under different gradients is A>Blank group>B>C (A is the ratio of the cell concentration of the bacteria to the algae of 10: 1, B is the ratio of the cell concentration of the bacteria to the algae of 1:1, and C is the ratio of the cell concentration of the bacteria to the algae of 1: 10). When the chlorella is cultured to the 6 th day, the chlorella endophyte starts to obviously promote the growth of the chlorella, when the chlorella endophyte is cultured to the 10 th day, the growth rate starts to slow down, the total content of chlorophyll a and carotenoid reaches the maximum difference value compared with a blank control group when the chlorella endophyte is cultured to the 12 th day, the total pigment content of a chlorella co-culture system in the group A is increased by 33.3 percent compared with the control group, and the group B and the group C have no obvious growth promoting effect on the chlorella, which shows that the growth promoting effect has certain correlation with.

In a co-culture system, the chlorella endophyte S395-2 and the chlorella have obvious interaction effect, and within a certain limit, the higher the chlorella endophyte concentration is, the more obvious the growth promoting effect is; when the concentration is lower, the growth rate is reduced individually, but the growth rate is within the error range, and no obvious inhibition effect exists. After the co-culture volume is increased, the biomass of the chlorella, the chlorophyll a content and the carotenoid content can be more obviously increased, and the growth rate of the chlorella is increased. The chlorella endophyte S395-2 can be used as a new microorganism resource for promoting the growth of microalgae and improving the growth quality of microalgae.

Drawings

FIG. 1 is a gram-stained microscopic image of endophyte S395-2;

FIG. 2 is a gel electrophoresis chart of endophyte S395-2;

FIG. 3 is a phylogenetic tree of the 16SrDNA sequence of Chlorella endophyte S395-2;

FIG. 4 is a standard curve of cell number and OD value of chlorella endophyte;

FIG. 5 is a standard curve of the number of chlorella cells versus OD value;

FIG. 6 is a dry weight chart of Chlorella under different gradients;

FIG. 7 is a diagram showing the measurement of biomass in co-cultivation of bacteria and algae;

FIG. 8 is a graph of the change of biomass of Chlorella under different gradients;

FIG. 9 is a graph showing the change in the chlorophyll-a content of the culture broth;

FIG. 10 is a graph showing the change in the carotenoid content in the culture broth.

Detailed Description

The technical solution of the present invention will be further specifically described below by way of specific examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.

In the present invention, all parts and percentages are by weight, unless otherwise specified, and the equipment and materials used are commercially available or commonly used in the art. The methods in the following examples are conventional in the art unless otherwise specified.

Example isolation, purification and characterization of Chlorella endophytes

1 materials of the experiment

The experimental sample is taken from a laboratory chlorella liquid, and the chlorella liquid is cultured in an intelligent illumination incubator with the illumination intensity of 2000lux and the temperature of 37 +/-1 ℃.

The main components of LB culture medium adopted in the bacterial culture medium experiment are shown in the table 1:

TABLE 11 LB Medium Main reagent Table

After the solvent is dissolved (agar is added into the solid culture medium by 15.0g), the volume of distilled water is fixed to 1L, the pH value is adjusted to about 7.6 by using 1mol/LNaOH, a 250mL conical bottle is packaged, and the conical bottle is sterilized for 20min under high pressure for later use.

2 method of experiment

2.1 screening, isolation and purification of Chlorella endophytes

1) Inverted plate

The solid LB medium, which was sterilized in advance, was heated to melt, and about 25ml of LB medium was poured into each dish, and then cooled to solidify for use.

2) Extracting endophyte from Chlorella (aseptic operation)

a, taking a bottle of chlorella with good growth vigor, shaking up, sucking the chlorella liquid by using a liquid transfer gun to a 45mL centrifuge tube (total 8 tubes), and centrifuging (centrifuging at 4000rpm for 10 min).

And b, pouring out the supernatant, combining every 4 tubes into one tube, washing the precipitate with sterile water, centrifuging again (repeating for three times), and removing bacteria in the algae liquid.

c, taking out the precipitate (adding a small amount of sterile water) after three times, and grinding the precipitate to light, medium and heavy degrees respectively (sterilizing a grinding pan and a grinding rod in advance).

d pouring 20ul of ground material (lightly ground) into the solidified LB plate, spreading evenly with a triangular coating bar, and marking (medium and heavy ground treatment as above). Three parallel sets were set for each degree of abrasion.

e, putting the mixture into a dark incubator at the temperature of 28 +/-1 ℃ for 24-48 h.

Supplementing: the original chlorella solution is streaked, and the effects of comparison and foreign bacteria elimination are achieved.

3) Flat plate scribing

a, after 24-48h of culture (according to the growth condition of strains), taking colonies with better growth in a culture medium by using an inoculation sterilization ring, and streaking chlorella endophytes with different grinding degrees for 3 times respectively.

b, placing the mixture into a dark incubator at the temperature of 28 +/-1 ℃ for inverted culture for 24-48 h.

4) Picking single colony

Good results generally appear in the C area, a small amount of thallus is selected from typical single colonies with obvious morphological characteristics and is put into an LB solid culture medium, and the primarily separated pure chlorella endophyte is obtained after culture.

And carrying out the next identification.

2.2 preservation of the strains

The experiment adopts glycerol preservation:

a, culturing the separated pure strains in an LB liquid culture medium in a shake flask to a logarithmic phase (the pure strains are turbid in a culture medium system by naked eyes).

b diluting glycerol to 50% (equal volume of distilled water plus equal volume of glycerol, glycerol needs to be slowly absorbed).

And c, sterilizing the diluted glycerol, a 2mL centrifuge tube, a gun head and other experimental articles at 121 ℃ for 20 min.

d, mixing chlorella endophyte bacterial liquid with 50% glycerol 1:1 equal volume was mixed in the centrifuge tube with a final glycerol concentration of 25%.

e, labeling and storing in a refrigerator at the temperature of-76 ℃.

2.3 identification of endophytic bacteria

2.3.1 Observation of the morphology of endophytic bacteria

The cultured pure endophytic strain is observed by naked eyes in shape, size, color and the like, and photographed and recorded.

2.3.2 gram stain visualization

Dropping a drop of sterile water in the center of a clean glass slide, selecting a small amount of pure chlorella endophyte, mixing the water drop with the pure chlorella endophyte, coating the mixture into a thin mycoderm, drying the smear in the air, putting an alcohol lamp on the alcohol lamp for three times, and dropping a dye after cooling. After primary dyeing, mordant dyeing, decoloring, secondary dyeing and microscopic observation, photographing and recording.

2.3.3 identification of Chlorella endophyte 16S rDNA molecule

Activating and passaging the preserved strain for 1-2 generations, enhancing the strain activity, purifying the product, sequencing, performing PCR amplification by using primers 7F 1540R 5'-CAGAGTTTGATCCTGGCTAGGAGGTGATCCAGCCGCA-3' (the nucleotide sequence is SEQ ID No.2) and 27F 1492R 5 '-AGTTTGATCMTGGCTCAGGGTTACCTTGTTACGACTT-3' (the nucleotide sequence is SEQ ID No.3) (the PCR reaction system is shown in table 2), performing PCR reaction and circulation, performing gel electrophoresis, performing electrophoresis in 1% agarose, observing electrophoresis in 150V and 100mA 20min at temperature of 100 and performing sequencing on the product, wherein [96 ℃ 1min → (96 ℃ 10sec → 50 ℃ 5sec → 60 ℃ 4min) x 25 circulation → 4 ℃ heat preservation ].

TABLE 1 PCR Standard reaction System

2.4 results and analysis

2.4.3 separation and purification of Chlorella endophyte and morphological identification

The morphological identification of chlorella endophytes is analyzed from colony morphology, white and yellow strains exist on an LB culture medium, most of the white strains are at the beginning, and yellow strains are gradually increased at the later stage to become dominant growth bacteria. Compared with the coating condition of chlorella prototheca liquid, the two bacteria are chlorella endophytes. The white colony is numbered S395-1 (not discussed in the present invention) and the yellow colony is numbered S395-2. The results observed after multiple streaking purifications are shown in table 3.

TABLE 3 morphological observation and record table of endophyte

Gram-positive bacteria are gram-positive bacteria, and endophyte S395-2 is purple thick arrowhead after gram staining and microscopic observation (see figure 1).

2.4.4 sequencing of 16S rDNA and phylogenetic Tree analysis of endophytic bacteria S395-1 and S395-2

The gel electrophoresis pattern of the endophyte S395-2 is shown in FIG. 2.

S395-2 sequencing result shows that the length of the bacterial sequence is about 1471bp, and the nucleotide sequence is shown as SEQ ID No. 1.

The sequences of S395-2 bacteria were aligned in the ribosome database (http:// rdp. cme. msu. edu/index. jsp) and a phylogenetic tree was constructed (see FIG. 3). The results show that: the S395-2 strain has 99% homology with the 16SrDNA sequence of Microbacterium sp, and can be identified as a genus Microbacterium.

Example Chlorella endophyte application of chlorella endophyte to chlorella growth promotion

1 test reagent

(1) Strain: chlorella endophyte algae liquid: chlorella liquid

(2) Culture medium: LB liquid culture Medium

(3) BG11 formulation is shown in Table 4(1000ml for example)

Subpackaging 250ml conical bottles, sealing, sterilizing in a table pressure sterilizing pan at 121 ℃, and cooling for later use.

TABLE 4 BG11 liquid Medium formulation (1L)

The system pH was adjusted to 7.1 with 1M NaOH or HCl solution.

2 method of experiment

2.1 Standard Curve for determining cell number and OD value of Chlorella endophyte

(1) Culturing strains: and (3) transferring a single colony of the 1-ring chlorella endophyte through aseptic operation, inoculating the single colony into a liquid LB culture medium, and standing overnight. (2) Subpackaging the culture solution: and (5) performing sterile operation. (3) Correcting zero point: the culture solution without inoculated bacterial liquid is selected to have a wavelength of 600nm to correct the zero point. (4) Inoculation: the bacterial liquid inoculation amount is about 10%. (5) OD value at zero measurement: the OD value immediately after the inoculation of the seed solution was measured. (6) And (5) shaking culture. (7) Measuring the growth amount: OD values and cell numbers were determined every 1.5 h. (8) And (5) cleaning. (9) And drawing a standard curve of the cell number and the OD value of chlorella endophyte. And measuring the standard curve of the cell number and the OD value of the chlorella in the same way.

2.2 Co-culture of Chlorella with endophytes

Inoculating endophytic bacteria to LB liquid culture medium, culturing at 37 deg.C and 230rpm in dark in a constant temperature shaking table, and determining OD_600nmAbout 0.6, the strain grows in log phase according to the growth curve, and the concentration of the bacterial liquid is about 1.26 multiplied by 10⁸cells/mL. Taking 45mL of bacterial liquid in a 2-tube, centrifuging at the room temperature of 4000rpm for 15min, washing precipitates with sterile water for 3 times, centrifuging for 10min again, collecting thalli cells, and suspending the thalli cells by using 45mLBG11 liquid culture medium for later use. Inoculating bacteria and algae co-culture systems with different concentration ratios, respectivelyThe ratio of the cell concentration of algae (S395-2 to that of chlorella) is 10: 1,1: 1,1: 10 three concentration gradients. The test group is a fungus-algae co-culture test group, pure algae culture is set as a control group, and 3 parallel tests are set in each group. Culturing the strain culture solution in an intelligent illumination incubator at the illumination intensity of 2000lux and the temperature of 37 +/-1 ℃, 8:00 in the morning and 12 in the noon: 00 at night 6: 00 are shaken once each.

2.3 Chlorella biomass determination

The biomass of chlorella is determined by dry weight method. The experiment was performed with 4 sets of culture medium, 3ml of each flask was aseptically pipetted using a pipette, labeled, and centrifuged at 8000rpm for 10 min. The supernatant was decanted, dried under vacuum at 60 ℃ and weighed.

2.4 Chlorella chlorophyll-a and carotenoid extraction and content determination

An ultrasonic extraction method is utilized:

(1) taking 4ml of culture solution from each conical flask, adding 4ml of ethanol extractant, placing in an ultrasonic cleaner, performing ultrasonic treatment for 30min, centrifuging at 8000rpm for 10min in a centrifuge, collecting supernatant, measuring absorbance at 470nm, 645nm and 663nm, and recording.

(2) C content of chlorophyll a is calculated according to a chlorophyll a calculation formula_a＝0.0127A₆₆₃-0.00269A₆₄₅，

Calculating the Carotenoid concentration C_b＝0.0229A₆₄₅-0.00468A₆₆₃Carotenoid concentration ═

(1000A₄₇₀-3.27C_a-104C_b)/229。

3 results and analysis

The experiment mainly researches the growth promoting effect of S395-2 chlorella endophyte on chlorella.

3.1 Chlorella endophyte growth curve

A standard curve of the number of chlorella endophyte cells and the OD value is shown in figure 4, and the number of chlorella endophyte cells and the OD value are basically in direct proportion.

3.2 Chlorella growth Curve

A standard curve of the number of chlorella cells and the OD value is shown in FIG. 5, and the number of chlorella cells and the OD value are substantially proportional.

3.3 Chlorella biomass determination

Taking 3ml of culture solution, and measuring dry weight change of chlorella under different concentrations, wherein A is a cell concentration ratio of 10: 1 and B is the ratio of the cell concentration of bacteria and algae to 1:1 and C is the ratio of the cell concentration of the bacteria and the algae to the cell concentration of 1: 10. from Table 3-2, the dry weight of Chlorella vulgaris can be analyzed in comparison to A > blank > B > C. The blank group has little difference with the B and C groups, and has no obvious growth promoting effect or obvious inhibiting effect within an error range. The dry weight of Chlorella under different gradients is shown in FIG. 6, the dry weight change of Chlorella under different gradients is shown in Table 5, the biomass measurement of bacteria co-culture is shown in FIG. 7, and the biomass change curve of Chlorella under different gradients is shown in FIG. 8.

TABLE 5 Chlorella dry weight change under different gradients

According to the experimental result, when the concentration ratio of bacteria to algae is 10: 1, the biomass of the chlorella can be increased, and the concentration ratio of the chlorella is 1: the growth promoting effect is not different from that of the blank group at 1 or 1:10, within the error range, the dry weight content of the blank group is basically consistent with that of the A and B groups at the 14 th day, and no inhibiting effect exists.

3.4 Effect of Chlorella endophytes on Chlorella chlorophyll a and Carotenoid content

Comparing the change of chlorophyll a and carotenoid content in the culture solution of the fungus algae co-culture group and the pure algae culture control group, the results are shown in fig. 9 and fig. 10. From day 2 to 14, the culture broth gradually increased in the contents of chlorophyll a and 2 kinds of pigments of carotenoids, which was consistent with the continuous growth of algal cells. The ratio of bacteria to algae in the bacteria-algae co-culture group culture solution is 10: in the step 1, the contents of chlorophyll a and carotenoid are slightly higher than those of a control group, the difference between the contents of chlorophyll a and carotenoid is not obvious overall, the chlorella endophyte starts to obviously promote the growth of the chlorella when cultured to the 6 th day, the growth rate starts to slow down when cultured to the 10 th day, the maximum content phase difference value exists when cultured to the 12 th day, the total pigment content of a group A chlorella co-culture system is increased by 33.3 percent compared with that of the control group, and the difference is obvious. The experimental result shows that when the concentration of chlorella endophyte and chlorella liquid is 10: 1, the chlorophyll content in the algae can be obviously increased, and the concentration ratio of bacteria to algae is 1: the growth promoting effect is weaker at 1 or 1:10, and is basically the same as that of the blank group.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.

Sequence listing

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<120> chlorella endophyte and application thereof in promoting growth of chlorella

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Claims

1. A chlorella endophyte is a bacterium of the genus MicrobacteriumMicrobacterium saccharophilumThe strain S395-2 is preserved in China general microbiological culture Collection center (CGMCC) at 7 months and 30 days in 2018, and the number of the strain is CGMCC No. 16182.

2. Use of the chlorella endophyte of claim 1 to promote growth of chlorella.

3. Use according to claim 2, characterized in that: in the application, chlorella endophyte and chlorella are co-cultured, and the cell concentration ratio of chlorella is 1-10: 1.

4. use according to claim 2, characterized in that: the chlorella endophyte is cultured in a LB liquid culture medium in a constant temperature shaking table at 37 +/-2 ℃ and 237 +/-50 r/min in the dark, and the obtained thallus cells are used for culturing the chlorella.

5. Use according to claim 2, characterized in that: culturing the algae culture solution in a light culture box with the light intensity of 1500-.