阅读说明：本技术 一种利用有害蓝藻大规模快速培养草履虫的方法 (Method for rapidly culturing paramecium on large scale by utilizing harmful blue-green algae ) 是由 杨州 张露 徐文杰 孙运菲 黄园 于 2020-11-19 设计创作，主要内容包括：本发明公开了一种利用有害蓝藻大规模快速培养草履虫的方法。该方法将有害群体蓝藻进行富集、干燥和研磨获得单细胞和小群体的蓝藻藻粉,再将其用于草履虫的培养中。本发明不仅缓解了草履虫对群体藻的摄食障碍,在短时间内获得高密度的草履虫种群,同时降低草履虫大规模培养中饵料成本和对水质的污染,避免了复杂的饵料配比环节,藻粉也更适合长期保存。并且,通过草履虫对有害蓝藻的摄食和消化,将有害蓝藻进行有益转化,从而消除富营养化水体中有害蓝藻打捞收集上岸后续处置的环境危害,具有重要的现实意义。本发明中培养草履虫的有机碳源为有害群体蓝藻,该原材料在富营养化水体中常见且易获得。(The invention discloses a method for rapidly culturing paramecium in a large scale by utilizing harmful blue-green algae. The method enriches, dries and grinds the blue algae of a harmful colony to obtain the blue algae powder of a single cell and a small colony, and then the blue algae powder is used for culturing paramecium. The method not only relieves the ingestion obstacle of paramecium by paramecium, obtains high-density paramecium population in a short time, but also reduces the bait cost and the pollution to water quality in large-scale cultivation of paramecium, avoids a complicated bait proportioning link, and is more suitable for long-term storage. In addition, harmful blue-green algae are beneficially transformed by feeding and digesting the harmful blue-green algae by the paramecium, so that the environmental hazard of the subsequent treatment of fishing and collecting the harmful blue-green algae in the eutrophic water body ashore is eliminated, and the method has important practical significance. The organic carbon source for culturing the paramecium is harmful group blue algae, and the raw material is common and easily obtained in eutrophic water.)

1. A method for rapidly culturing paramecium on a large scale by utilizing harmful blue-green algae is characterized by comprising the following steps:

(1) cracking the collected high-density harmful population blue algae, wherein the cracking treatment comprises the following steps: firstly, centrifuging collected algae liquid containing harmful colony blue algae for 10-20min at 4000-; then placing the algae mud in a drying oven at 50-65 ℃ for drying treatment for 24-48 hours; then grinding the dried algae mud, and then sieving the ground algae mud through a sieve with the aperture of 40 mu m to obtain algae powder;

(2) adding algae powder into a low-density paramecium culture solution, wherein the concentration of the algae powder is 0.1-1mg/mL, the initial inoculation amount of the paramecium is not less than 10 ind/mL, culturing is carried out at the temperature of 20-30 ℃, and the concentration of the algae powder in the culture solution is not less than 0.1mg/mL by adding the algae powder every 24 hours in the culturing process;

(3) after culturing the paramecium in the culture medium for 4-7 days or when the paramecium concentration reaches the maximum environment tolerance amount, the paramecium can be collected after enrichment or added into a fresh culture medium for culturing.

2. The method for the large-scale rapid culture of paramecium using harmful cyanobacteria as claimed in claim 1, wherein the paramecium for inoculation is prepared as follows: firstly, collecting paramecium, and separating to obtain pure paramecium; adding a culture solution and algae powder obtained by cracking harmful group blue algae into a culture bottle, wherein the culture solution is a special plankton culture medium diluted by 10-30 times, the addition amount of the algae powder is 5 mg/mL, inoculating the obtained pure paramecium, culturing in 50mL of the culture solution, and directly using as an provenance until the density of the paramecium reaches 300 ind/mL.

3. A process according to claim 1The method for rapidly culturing paramecium on a large scale by utilizing harmful blue-green algae is characterized in that the paramecium is paramecium multiplexParamecium multimicronucleatumParamecium of MeadowrumParamecium caudatumOne or two of them are mixed.

4. The method for rapidly culturing paramecium on a large scale by using harmful cyanobacteria as claimed in claim 1, wherein the initial inoculation amount of paramecium is 10-30 ind/mL.

5. The method as claimed in claim 1, wherein the low-density paramecium culture solution is lake water or clear water which is filtered by a filter membrane or filter with a thickness not less than 1 μm and does not contain other active organisms, or culture solution obtained by diluting 10-30 times with plankton-dedicated culture medium sterilized at high temperature and high pressure, and the nitrogen content of the culture solution is not higher than 25 mg/L.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for rapidly culturing paramecium in a large scale by utilizing harmful blue-green algae.

Background

Paramecium is a common planktonic protozoa in water, has simple cell structure and rapid propagation, takes phytoplankton, bacteria, organic debris and the like as food, is a main food resource of aquatic animals, is often used as natural bait for cultivating aquatic economic species seedlings and ornamental fishes, and can play a role in purifying water quality because the paramecium can take the organic debris as food. In scientific research, paramecium has the characteristics of easy culture and easy observation, and is also often used as an important species for scientific experiments, such as genetic experiments, water quality monitoring and the like. Therefore, the rapid culture of paramecium on a large scale is a key technology in the use of paramecium as natural bait and as scientific research material.

At present, organic carbon sources generally adopted in the conventional culture of paramecium include straw leachate, bacteria, beef extract, wheat grain culture solution, rice culture solution, fresh milk, peptone culture solution and the like, and although the materials of the culture solution can culture the high-density paramecium in a short time, the cost is high, the quality is easy to deteriorate to influence the water quality, and the addition ratio is not easy to control, so that the method is not suitable for the long-term large-scale culture of the paramecium. In addition, because organic matters in lake water are limited, the abundance of paramecium obtained by culturing the boiled lake water is low, and the requirement of obtaining high-density paramecium in a short time cannot be met. Microalgae is a natural bait for paramecium, but no technical disclosure of large-scale cultivation of paramecium by adopting harmful blue-green algae is found at present. The harmful blue algae are proliferated in a large quantity to form algal blooms, generally exist in a colony form, the micro phytophagous aquatic animals are limited by factors such as feeding modes and small bodies, the feeding efficiency of the colony form microcystis is relatively low, and the harmful blue algae cannot be efficiently transformed and utilized. The common harmful blue algae treating method includes mainly spraying chemical reagent, mechanical operation and other steps to make algae cell cracking and deactivating.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides a method for rapidly culturing paramecium in a large scale by utilizing harmful blue-green algae.

The invention aims to provide a method for rapidly culturing paramecium on a large scale by utilizing harmful blue-green algae, which is characterized by comprising the following steps:

(1) cracking the collected high-density harmful population blue algae, wherein the cracking treatment comprises the following steps: firstly, centrifuging collected algae liquid containing harmful colony blue algae for 10-20min at 4000-; then placing the algae mud in a drying oven at 50-65 ℃ for drying treatment for 24-48 hours; then grinding the dried algae mud, and then sieving the ground algae mud through a sieve with the aperture of 40 mu m to obtain algae powder;

(2) adding algae powder into a low-density paramecium culture solution, wherein the concentration of the algae powder is 0.1-1mg/mL, the initial inoculation amount of the paramecium is not less than 10 ind/mL, culturing is carried out at the temperature of 20-30 ℃, and the concentration of the algae powder in the culture solution is not less than 0.1mg/mL by adding the algae powder every 24 hours in the culturing process;

(3) after culturing the paramecium in the culture medium for 4-7 days or when the paramecium concentration reaches the environmental tolerance, the paramecium can be collected after enrichment or added into a fresh culture medium for culturing.

The paramecium for inoculation is prepared as follows: firstly, collecting paramecium, and separating to obtain pure paramecium; adding a culture solution and algae powder obtained by cracking harmful group blue algae into a culture bottle, wherein the culture solution is a special plankton culture medium diluted by 10-30 times, the addition amount of the algae powder is 5 mg/mL, inoculating the obtained pure paramecium, culturing in 50mL of the culture solution, and directly using as an provenance until the density of the paramecium reaches 300 ind/mL.

The paramecium is paramecium multiplexParamecium multimicronucleatumParamecium of MeadowrumParamecium caudatumOne or two of them are mixed.

The initial inoculation amount of the paramecium is 10-30 ind/mL.

The low-density paramecium culture solution is lake water or clear water which is filtered by a filter membrane or a filter with the thickness not less than 1 mu m and does not contain other active organisms, or is obtained by diluting 10-30 times by using a special plankton culture medium sterilized at high temperature and high pressure, and the nitrogen content of the culture solution is not higher than 25 mg/L.

Has the advantages that: the drying method of the harmful group blue algae and the step of cracking the group blue algae in the invention are simple and easy to operate, and are beneficial to popularization and implementation. Meanwhile, on the basis that paramecium is a main predator of algae, harmful blue-green algae can be efficiently ingested and digested, so that the rapid growth of the population per se can be realized in a short time, and repeated steps such as transitional culture and culture solution replacement are not needed in the process of adding organic substances, so that the large-scale paramecium culture method disclosed by the invention avoids the problems of high cost and low benefit of an organic carbon source in the original method, and can be used for long-term large-scale paramecium culture. In addition, the method utilizes the resources of the harmful blue-green algae, realizes the large-scale and quick culture of the paramecium with high benefit, solves the problems of the removal and the effective utilization of the harmful blue-green algae, lightens the adverse effect of the harmful blue-green algae on the aquatic environment, and has important practical significance.

Drawings

FIG. 1 is a schematic view showing the operation of the present invention for rapidly culturing paramecium in large scale by using harmful cyanobacteria, wherein the harmful cyanobacteria is colony cyanobacteria.

FIG. 2 shows paramecium polycarya in example 1 of the present inventionParamecium mutimicronucleatumYZ growth in microcystis treatment of different populations is shown schematically.

FIG. 3 shows paramecium roridum in example 2 of the present inventionParamecium caudatum YZ growth in microcystis treatment of different populations is shown schematically.

FIG. 4 shows paramecium polycarya in example 3 of the present inventionParamecium mutimicronucleatumThe growth of YZ in different anabaena treatments is shown schematically.

Detailed description of the invention

In order to facilitate an understanding of the invention, the invention will be described more fully hereinafter with reference to the accompanying drawings and specific examples, but the scope of the invention is not limited to the specific examples below.

The following examples used the formulation of modified BG-11 medium: contains 25mg NaNO per liter₃，1mgK₂HPO₄，75mgMgSO₄.7H₂O，36mgCaCl₂.2H₂O, 6mg citric acid, 6mg ferric ammonium citrate, 1mg EDTA, 20mg Na₂CO₃1mL of a trace element solution (2.86 mgH3BO3, 1.81 mgMnCl)₂.4H₂O，0.222mgZnSO₄.7H₂O，0.39mgNaMoO₄.5H₂O，0.079mgCuSO₄.5H₂O，0.0494mgCo(NO₃)₂.6H₂O), and 1mL vitamin B₁₂。

Paramecium for inoculation in the following examples was obtained by the following treatment: firstly, collecting paramecium, and separating to obtain pure paramecium; adding a culture solution and algae powder obtained by cracking harmful group blue algae into a culture bottle, wherein the culture solution is a special plankton culture medium diluted by 10-30 times, the addition amount of the algae powder is 5 mg/mL, inoculating the obtained pure paramecium, culturing in 50mL of the culture solution, and directly using as an provenance until the density of the paramecium reaches 300 ind/mL.

Example 1

The paramecium in the embodiment is a multi-small-core paramecium separated and purified from leachate of lakes, Taihu lakes and straws in the Xianlin school of south Beijing universityParamecium multimicronucleatumYZ, the collected paramecium is treated and then respectively inoculated into four groups of culture solutions marked as a treatment group 1, a control group 2 and a control group 3 at the addition of 10 cells/mL, the four groups of culture solutions are cultured at room temperature (20-30 ℃), and the maximum value of the paramecium population and the paramecium growth rate are counted, as shown in figure 2.

The treatment group 1 is 1/20 modified BG-11 medium added with 15mg harmful microcystis algae powder, the volume of the culture solution is 150mL, the algae powder is added every 24 hours in the culture process to make the concentration of the algae powder in the culture solution reach 0.1mg/L, and the algae powder is continuously added for 5 days. Firstly, centrifuging collected algae liquid of harmful microcystis colony for 10min at 4000rpm to obtain algae mud; then placing the algae mud in a 50 ℃ oven for drying treatment for 24 hours; then grinding the dried algae mud, and then sieving the ground algae mud through a sieve with the aperture of 40 mu m to obtain algae powder. The processed algae powder is a mixture of single-cell microcystis and microcystis colony with particle size less than 40 μm.

Control group 1 contained 5X 10⁶ 1/20 modified BG-11 culture medium containing cells/mL untreated harmful microcystis, the volume of culture solution is 150mL, and the untreated harmful microcystis is added every 24 hours in the culture process to make the concentration of microcystis in the culture solution reach 5 × 10⁶ cells/mL, 5 days of continuous addition. Untreated, harmful populations of microcystis are large populations of microcystis, usually in macroscopic particulate form, collected directly from the field and without any treatment.

The control group 2 is 1/20 modified BG-11 culture medium added with 15mg harmful microcystis algae powder, the volume of the culture solution is 150mL, the harmful microcystis algae powder is added every 24 hours in the culture process to make the concentration of the algae powder in the culture solution reach 0.1mg/L, and the culture solution is continuously added for 5 days. The preparation method of the algae powder comprises the following steps: firstly, centrifuging collected algae liquid of harmful microcystis colony for 10min at 4000rpm to obtain algae mud; then directly grinding the algae mud, and passing through a screen with the aperture of 40 mu m to obtain the algae mud.

The control group 3 is 1/20 modified BG-11 culture medium added with 15mg harmful microcystis algae powder, the volume of the culture solution is 150mL, the harmful microcystis algae powder is added every 24 hours in the culture process to make the concentration of the algae powder in the culture solution reach 0.1mg/L, and the culture solution is continuously added for 5 days. The preparation method of the algae powder comprises the following steps: firstly, centrifuging collected algae liquid of harmful microcystis colony for 10min at 4000rpm to obtain algae mud; and then placing the algae mud in a 50 ℃ oven for drying treatment for 24 hours to obtain the algae mud.

The results show that: paramecium caudatum of control group 1, control group 2, control group 3 and treatment group 1Paramecium multimicronucleatumYZ respectively achieves the maximum abundance of the population of 191 + -8 ind/mL, 1011 + -29 ind/mL, 166 + -4 ind/mL and 2524 + -129 ind/mL, and the population number of the paramecium cultured by the harmful cyanobacteria algae powder in the treatment group 1 in the example is increased by at least one relative to the initial inoculation amount within 6 days250 times, and the number of paramecium populations is respectively 13 times, 2 times and 15 times higher than that of the control group 1, the control group 2 and the control group 3, and only a small amount of the population algae in the control group 1 is eliminated by the paramecium, which indicates that the untreated population blue algae cannot be effectively utilized by the paramecium, but the microcystis algae powder obtained by treating the population microcystis by adopting the method of the invention can obviously promote the growth of the paramecium population and can rapidly obtain the high-density paramecium population in a short time. Particularly, in the treatment group, the paramecium can reach the maximum peak value within 6 days by continuously supplementing the algae powder every day, and can obtain high-density paramecium of about 2500ind/mL, the paramecium has strong phagocytic activity and can be directly used in further expanded culture, or after the paramecium reaches 1-2 days of the maximum peak value, the paramecium can completely digest harmful algae cells in the body and can be used as bait after the paramecium is enriched.

The data for control 2 and treatment 1 show that: if the centrifuged algae mud is not dried immediately, the algae mud is easy to rot and decompose, and then the algae mud is ground again to cause algae nutrition loss;

the data for control 3 and treatment 1 show that: if the dried algae mud is not ground, the algae mud is in a large block shape which can be seen by naked eyes, and the paramecium still can not be ingested;

based on the ingestion characteristics of paramecium, high-density harmful group blue algae is dried, single-cell or small-group-shaped algae powder is obtained by grinding and cracking the harmful blue algae in group form, and the natural microalgae algae powder is used for large-scale rapid culture of paramecium, so that the ingestion obstacle of paramecium to group algae is relieved, high-density paramecium population is obtained in a short time, meanwhile, the bait cost and the pollution to water quality in large-scale culture of paramecium are reduced, a complex bait proportioning link is avoided, and the algae powder is more suitable for long-term storage. In addition, the paramecium ingests the harmful blue-green algae, so that the harmful blue-green algae is beneficially transformed and utilized, the adverse effects caused by the harmful blue-green algae in eutrophication of culture water and some natural lakes can be reduced, and the method has important practical significance.

Example 2

In this example, paramecium roridum will be collectedParamecium caudatum The resulting YZ-treated pellets were inoculated at an addition rate of 30/mL to a medium containing about 4X 10⁶ cells/mL of 1/20 modified BG-11 medium containing untreated harmful microcystis cells (control group) and harmful microcystis powder (treatment group) of 100mg, with a culture medium volume of 1L, cultured at room temperature. In this example, the maximum value of paramecium population and the paramecium growth rate were counted, as shown in fig. 3.

The treatment group is 1/20 modified BG-11 culture medium containing 60mg harmful microcapsule algae powder, the volume of the culture solution is 1L, the algae powder is added every 24 hours during the culture process to make the concentration of the algae powder in the culture solution reach 0.1mg/L, and the culture solution is continuously added for 5 days. Firstly, centrifuging collected algae liquid of harmful microcystis colony for 10min at 4000rpm to obtain algae mud; then placing the algae mud in a 50 ℃ oven for drying treatment for 24 hours; then grinding the dried algae mud, and then sieving the ground algae mud through a sieve with the aperture of 40 mu m to obtain algae powder. The processed algae powder is a mixture of single-cell microcystis and microcystis colony with particle size less than 40 μm.

Control group 1 contained 4X 10⁶ 1/20 modified BG-11 culture medium containing cells/mL untreated harmful microcystis, the volume of the culture solution is 1L, and the untreated harmful microcystis is added every 24 hours in the culture process to make the concentration of microcystis in the culture solution reach 4 × 10⁶ cells/mL, 5 days of continuous addition. Untreated, harmful populations of microcystis are large populations of microcystis, usually in macroscopic particulate form, collected directly from the field and without any treatment.

The results show that: paramecium caudatum of control group and treatment groupParamecium caudatum YZ respectively achieves the maximum abundance of the populations of 142 +/-4 ind/mL and 2608 +/-119 ind/mL, and the population number of the paramecium cultured by the harmful cyanobacteria algae powder in the example is increased by at least 86 times relative to the initial inoculation amount within 7 days. The paramecium of the control group is increased by about 4 times compared with the initial inoculation amount, and only a small amount of colony algae in the control group is eliminated by the paramecium, which indicates that the untreated colony blue algae can not be effectively utilized by the paramecium, but the method of the invention is adopted to treat the colony blue algaeThe microcystis powder obtained from microcystis can remarkably promote the growth of paramecium population, and high-density paramecium population can be rapidly obtained in a short time. In this embodiment, when the paramecium population reaches the maximum peak value, the paramecium has a strong phagocytic activity and can be directly used in further expanded culture, or after the paramecium population reaches the maximum peak value for 1-2 days, the paramecium can completely digest the harmful algal cells in the body and enrich the paramecium for use as a bait.

Example 3

The harmful blue algae in the embodiment is anabaena enriched from Taihu lake, and the culture solution is lake water of Taihu lake. Culturing paramecium caudatumParamecium multimicronucleatumThe resulting YZ-treated pellets were inoculated at an amount of 10 cells/mL to a medium containing about 5X 10 cells⁶ cells/mL untreated anabaena and 15mg anabaena powder in filtered lake water, and culturing at room temperature. The example makes statistics of the maximum value of paramecium and the paramecium growth rate.

The treatment group is filtered lake water added with 15mg of harmful anabaena algae powder, the volume of the culture solution is 500mL, the algae powder is added every 24 hours in the culture process to ensure that the concentration of the algae powder in the culture solution reaches 0.1mg/L, and the algae powder is continuously added for 5 days. Firstly, centrifuging collected algae liquid of the harmful anabaena colony for 10min at 4000rpm to obtain algae mud; then placing the algae mud in a 50 ℃ oven for drying treatment for 24 hours; then grinding the dried algae mud, and then sieving the ground algae mud through a sieve with the aperture of 40 mu m to obtain algae powder. The processed algae powder is a mixture of single-cell anabaena and anabaena small population with the particle size less than 40 mu m.

The control group contained 5X 10⁶ cells/mL untreated harmful anabaena colony filtering lake water, the volume of the culture solution is 500mL, and the untreated harmful anabaena colony is added every 24 hours in the culture process to ensure that the concentration of anabaena in the culture solution reaches 4 x 10⁶ cells/mL, 5 days of continuous addition. The untreated harmful population of anabaena is a large population of anabaena collected directly from the field and without any treatment, usually in macroscopic particulate form.

The results show that: the maximum population density of the paramecium cultured by the anabaena algae powder treated by the method can reach 940 +/-44 ind/mL; the anabaena was directly fed to paramecium as shown in fig. 4, and the population density of the control group reached 30ind/mL at the maximum.