CN113475435A - Domestication method for improving ingestion efficiency of daphnia magna - Google Patents

Domestication method for improving ingestion efficiency of daphnia magna Download PDF

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CN113475435A
CN113475435A CN202110678448.9A CN202110678448A CN113475435A CN 113475435 A CN113475435 A CN 113475435A CN 202110678448 A CN202110678448 A CN 202110678448A CN 113475435 A CN113475435 A CN 113475435A
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daphnia magna
improving
daphnia
cysteine
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CN113475435B (en
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金文标
尹诗雨
周旭
迟惠中
韩炜
丁婉晴
涂仁杰
何钟琦
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Shenzhen Graduate School Harbin Institute of Technology
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K61/00—Culture of aquatic animals
    • A01K61/20—Culture of aquatic animals of zooplankton, e.g. water fleas or Rotatoria

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Abstract

The invention provides a domestication method for improving the ingestion efficiency of daphnia magna, which comprises the following steps: adding amino acid into chlorella or scenedesmus obliquus serving as bait, then adjusting the pH to 8-9, adding adult daphnia magna, keeping the environmental temperature at 25-30 ℃, and keeping the illumination at 1500-; wherein the amino acid is at least one of L-proline, D-glutamic acid and L-cysteine. By adopting the technical scheme of the invention, the ingestion efficiency of the daphnia magna on the microalgae can be improved, so that the harvesting efficiency of the microalgae is improved; meanwhile, the nutritive value of the daphnia magna is improved, so that the potential of the daphnia magna serving as a high-value feed for aquaculture is stimulated.

Description

Domestication method for improving ingestion efficiency of daphnia magna
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a domestication method for improving the ingestion efficiency of daphnia magna.
Background
The large consumption of fossil fuel causes double threats of fuel requirements and climate change, and the effect of sewage resource utilization and energy crisis relief and greenhouse influence can be achieved when the urban sewage is used for culturing microalgae to produce biodiesel. Wherein, the microalgae harvesting link in the preparation process of the microalgae biodiesel becomes the bottleneck for realizing the industrial application due to high cost. Chinese patent CN107794115A discloses a method for extracting microalgae oil by using protozoan ingestion, digestion and wall breaking for assistance, and Chinese patent CN109929672A discloses a method for harvesting oil-producing microalgae and extracting oil by using plankton ingestion. However, at present, ingestion of daphnia will yield microalgae less efficiently.
Disclosure of Invention
Aiming at the technical problems, the invention discloses a domestication method for improving the ingestion efficiency of daphnia magna, which obtains daphnia magna with high-efficiency ingestion capacity through domestication so as to achieve the purpose of improving the ingestion and microalgae harvesting efficiency of daphnia magna.
In contrast, the technical scheme adopted by the invention is as follows:
a domestication method for improving the ingestion efficiency of daphnia magna comprises the following steps:
adding amino acid into algae serving as bait, then adjusting the pH to 8-9 (the pH is influenced by the addition of the amino acid, so that the pH adjustment needs to be carried out after the addition of the amino acid), adding the adult daphnia magna, keeping the environmental temperature at 25-30 ℃, keeping the illumination at 1500-3000Lux, and keeping the light-dark ratio at 6L: 18D-12L: 12D; wherein the amino acid is at least one of L-proline, D-glutamic acid and L-cysteine.
As a further improvement of the invention, the algae are chlorella and scenedesmus obliquus.
As a further improvement of the invention, the amino acids are D-glutamic acid and L-cysteine.
As a further improvement of the invention, the concentration ratio of the D-glutamic acid to the L-cysteine is 1: 3-5.
As a further improvement of the invention, the concentration of the D-glutamic acid is 4X 10-5-6×10-5mol/L, the concentration of the L-cysteine is 1 x 10-4-3×10-4mol/L。
As a further improvement of the invention, the concentration of the D-glutamic acid is 5X 10-5mol/L, the concentration of the L-cysteine is 2 x 10-4mol/L。
As a further improvement of the invention, the initial concentration of the algae liquid is controlled to be 1 multiplied by 106～3×106cells/mL。
As a further improvement of the method, the culture density of the daphnia magna culture is 12-18ind daphnia/30 mL algae liquid. Preferably, the culture density of the daphnia magna culture is 15ind daphnia/30 mL algae liquid.
As a further improvement of the invention, the culture medium of 3/4 is replaced every two days by slightly aerating during the culture period, mainly without causing severe disturbance of the water body.
As a further improvement of the invention, the daphnia magna is harvested once a week, and continuously cultured for more than one month while maintaining the initial culture density.
Compared with the prior art, the invention has the beneficial effects that:
by adopting the technical scheme of the invention, the ingestion efficiency of the daphnia magna on the microalgae can be improved, so that the harvesting efficiency of the microalgae is improved; meanwhile, the nutritive value of the daphnia magna is improved, so that the potential of the daphnia magna serving as a high-value feed for aquaculture is stimulated.
Drawings
FIG. 1 is a graph of an analysis of the effect of different individual amino acids on flea feeding rate in an example of the present invention.
FIG. 2 is a graph showing the effect of different modes of amino acid addition on flea feeding rate in an example of the present invention.
FIG. 3 is a comparison graph of the cultivation of daphnia magna for more than one month in the examples of the present invention and the comparative examples.
Detailed Description
Preferred embodiments of the present invention are described in further detail below.
Example 1
Selecting 100 daphnia of similar size in the same growth period, and placing in 200mL chlorella solution (chlorella solution concentration is (2.0 + -0.2) × 10)6cells/mL), six groups are set, wherein amino acid is not added in one group of algae liquid as a control group, and the concentrations of L-glutamine, L-aspartic acid, L-proline, D-glutamic acid and L-cysteine are respectively controlled to be 10 in five groups-4And (3) adjusting the pH value to 8, placing the six groups at the temperature of 25 ℃, culturing for two days under the conditions of the light intensity of 20% and the light-dark ratio of 6L to 18D, fishing out the daphnia every day of culture, and updating the algae liquid. The step is to lead the water fleas to adapt to the later experimental environment in advance so as to avoid the death of the water fleas from causing great influence on the experimental result due to the unsuitability for the experimental environment. In the third day of experiment, 15 similar-sized Chlorella solution (the concentration of Chlorella solution is (2.0 + -0.2) × 10) is selected from environmental-adapted Daphnia6cells/mL), the treated group and the control group are the same as the above group and are parallel three times, the control group and the five groups of amino acid treated groups are cultured for 24 hours under the conditions of pH 8, temperature 25 ℃, illumination intensity 20%, light-dark ratio 6L:18D, and the flea feeding rate and the water filtration rate are shown in figure 1.
As is evident from FIG. 1, L-aspartic acid is almost non-irritating to the ingestion of daphnia, L-glutamine has a negative effect even on the ingestion of daphnia, L-proline, D-glutamic acid, and L-cysteine can stimulate the ingestion of daphnia, and the stimulation of L-cysteine is superior to that of the other two amino acids.
Example 2
In this embodiment, the daphnia magna is domesticated by the following steps:
a. using Chlorella or Scenedesmus obliquus as bait, controlling initial concentration of algae liquid to be 2 × 106Adding amino acid L-proline with the concentration of 1 multiplied by 10 to about cells/mL-4And (2) mol/L, then adjusting the pH to 8-9 (the pH is influenced by adding amino acid, so the pH adjustment needs to be carried out after adding the amino acid), adding adult daphnia magna into algae liquid with the initial daphnia magna culture density of 15 ind/30 mL, and adjusting environmental parameters: the temperature is 25-30 ℃, the illumination is 2000Lux, and the photoperiod is 6L: 18D.
b. During the culture period, the culture medium of 3/4 is replaced every two days, mainly because the culture medium is slightly aerated and does not cause the severe disturbance of the water body.
c. Harvesting daphnia magna once a week, always keeping the culture density of 15ind daphnia magna/30 mL algae liquid, and continuously culturing for more than one month.
Example 3
Based on example 2, in this example, L-cysteine was added as amino acid at a concentration of 1X 10-4mol/L。
Example 4
Based on example 2, in this example, D-glutamic acid was added as amino acid at a concentration of 5X 10-5mol/L。
Example 5
Based on example 2, in this example, the amino acids added were D-glutamic acid and L-cysteine at concentrations of 5X 10-5mol/L，2×10-4mol/L。
Example 6
Based on example 2, in this example, the amino acids added are L-cysteine and L-proline, the concentrations of which are 1X 10 respectively-5mol/L，1×10-4mol/L。
Example 7
Based on example 2, in this example, the amino acids D-glutamic acid and L-proline were added at concentrations of 5X 10-5mol/L，1×10-4mol/L。
The ingestion of the daphnia magna is influenced by weather and seasons, so when the concentration of a single amino acid and the concentration of two amino acids are compared to influence the ingestion of the daphnia magna, the same batch of daphnia magna needs to be selected for domestication in the same environment. Examples 2 to 7 select the same batch of daphnia magna to acclimate in the same environment, the amino acid adding conditions of the examples 2 to 7 are shown in table 1, fig. 2 shows the influence of different amino acid adding modes on the intake of the daphnia magna, wherein the control group is the condition without adding amino acid.
TABLE 1 amino acid addition condition tables of examples 2 to 7
Examples Kind of amino acid Amino acid concentration (mol/L)
Example 2 L-proline 1×10-4
Example 3 L-cysteine 1×10-4
Example 4 D-glutamic acid 5×10-5
Example 5 D-glutamic acid: l-cysteine 5×10-5:2×10-4
Example 6 L-cysteine: l-proline 1×10-5:1×10-4
Example 7 D-glutamic acid: l-proline 5×10-5:1×10-4
As shown in the results of FIG. 2, L-proline has a less stimulatory effect on flea feeding than D-glutamic acid and L-cysteine, and L-cysteine has the best stimulatory effect; similarly, when two amino acids act simultaneously, the combined effect of D-glutamic acid and L-cysteine is superior to that of the other two combinations and that of D-glutamic acid and L-cysteine alone. Therefore, in the case of laboratory and outdoor scale-up acclimatization, 5 × 10 g/L-cysteine, D-glutamic acid, can be used-5mol/L:2×10-4The adding mode of mol/L is used for obtaining the daphnia with higher feeding efficiency.
Example 8
The following was chosen to use D-glutamic acid, L-cysteine ═ 5X 10-5mol/L:2×10-4And carrying out a verification test on the adding mode of mol/L.
Transferring daphnia magna collected from Daohuacun in the plateau mountain region of Shenzhen, Guangdong province into a laboratory, and performing acclimation culture according to the following steps:
a. using Chlorella or Scenedesmus obliquus as bait, controlling initial concentration of algae liquid to be 2 × 106About cells/mL, amino acid, D-glutamic acid are added according to the following concentrations: l-cysteine-5 × 10-5mol/L：2×10-4mol/L (the concentration of amino acid added is lower, the concentration of the prepared amino acid is 1 multiplied by 10-2Diluting and adding mother liquor of D-glutamic acid and L-cysteine at mol/L, wherein the L-cysteine has low solubility, so that the mother liquor needs to be fully mixed on a shaking table during preparation), then adjusting the pH to 8-9 (the pH is influenced by adding amino acid, so that the pH adjustment needs to be carried out after adding the amino acid), adding daphnia magna adult into algae liquor with the initial daphnia magna culture density of 15 ind/30 mL, and adjusting environmental parameters: the temperature is 25-30 ℃, the illumination is 2000Lux, and the photoperiod is 6L: 18D.
b. Slightly aerating during cultivation, mainly not causing severe disturbance of water, replacing 3/4 culture medium every two days, harvesting daphnia magna once every week, always keeping the culture density of 15ind daphnia/30 mL algae liquid, and continuously culturing for more than one month. The filter screen can be used for filtering when the culture medium is changed and the daphnia magna is harvested, the filter screen with the length of 1.40mm can be used for selecting the daphnia magna with the larger body length, and the filter screen with the length of 0.85mm can intercept all individual daphnia magna, so that the culture medium can be conveniently replaced.
Meanwhile, the control group was prepared without adding amino acid.
The comparative picture of the daphnia magna cultured for more than one month in proportion to the control group is shown in fig. 3, compared with the daphnia magna cultured for more than one month without adding amino acid shown in the left side of fig. 3, the daphnia magna cultured by adding amino acid in the right side of fig. 3 is darker, the endurance of cladocera to hypoxia is due to the existence of heme in plasma, and the daphnia magna red is more resistant to hypoxia and more active than the daphnia magna grey.
In addition, the amino acid composition of daphnia magna cultured for one month in this example and comparative example was analyzed, as shown in table 2. Compared with the daphnia magna cultured for one month without adding amino acid, the ingestion rate of the daphnia magna combining D-glutamic acid and L-cysteine is improved by 24.93 percent compared with the ingestion rate of the daphnia magna without adding amino acid, and simultaneously the total content of 18 amino acids in the daphnia magna is increased by 2.43 percent.
TABLE 2 comparative analysis table of amino acid composition
Figure RE-GDA0003221095760000051
The comparison between the examples and the comparative examples shows that the daphnia magna is popular in the field of toxicology due to the sensitivity to environmental changes, and a proper amount of amino acid concentration can generate stress response to the daphnia magna or generate phagostimulant through metabolic reaction to stimulate the ingestion of daphnia magna, but not all amino acids can promote the ingestion of daphnia magna. And proper amino acid is adopted for feeding, so that the ingestion rate of daphnia magna and the content of total amino acid in the body can be improved.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims (10)

1. A domestication method for improving the ingestion efficiency of daphnia magna is characterized by comprising the following steps:
adding amino acid into algae serving as bait, then adjusting the pH value to 8-9, adding adult daphnia magna, keeping the environmental temperature at 25-30 ℃, keeping the illumination at 1500-; wherein the amino acid is at least one of L-proline, D-glutamic acid and L-cysteine.
2. The domestication method for improving the feeding efficiency of daphnia magna according to claim 1, characterized in that: the amino acids are D-glutamic acid and L-cysteine.
3. The domestication method for improving the feeding efficiency of daphnia magna according to claim 2, characterized in that: the concentration ratio of the D-glutamic acid to the L-cysteine is 1: 3-5.
4. The domestication method for improving the feeding efficiency of daphnia magna according to claim 3, characterized in that: the concentration of the D-glutamic acid is 4 x 10-5-6×10-5mol/L, the concentration of the L-cysteine is 1 x 10-4 -3×10-4 mol/L。
5. The domestication method for improving the feeding efficiency of daphnia magna according to claim 4, characterized in that: the concentration of the D-glutamic acid is 5 x 10-5mol/L, the concentration of the L-cysteine is 2 x 10-4 mol/L。
6. The domestication method for improving the feeding efficiency of daphnia magna according to claim 1, characterized in that: controlling the initial concentration of algae solution to 1 × 106 ~3×106 cells/mL。
7. The domestication method for improving the feeding efficiency of daphnia magna according to claim 1, characterized in that: the culture density of the daphnia magna culture is 12-18ind daphnia/30 mL algae liquid.
8. The domestication method for improving the feeding efficiency of daphnia magna according to any one of claims 1 to 7, characterized by comprising the following steps: aeration is carried out during the culture period, and 3/4 culture medium is replaced every two days.
9. The acclimatization method for improving the feeding efficiency of daphnia magna according to claim 8, characterized in that: harvesting daphnia magna once a week, maintaining the initial culture density, and continuously culturing for more than one month.
10. The acclimatization method for improving the feeding efficiency of daphnia magna according to claim 8, characterized in that: the algae is Chlorella and Scenedesmus obliquus.
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CN108040937A (en) * 2017-11-21 2018-05-18 佛山科学技术学院 A kind of cultural method that may advantageously facilitate Daphnia magna growth and breeding

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CN108040937A (en) * 2017-11-21 2018-05-18 佛山科学技术学院 A kind of cultural method that may advantageously facilitate Daphnia magna growth and breeding

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尹诗雨: "水蚤驯化及收获微藻工艺优化研究", 《万方数据》 *

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