3.1 Collection of microalgae
100 L of water was collected from Yamdroktso lake (25 times by water collector), the phytoplankton were filtered through a phytoplankton net of 500 mesh, and the water sample was poured into a teflon bucket of 50 L for preservation. 200g of sediment from the bottom of the lake was randomly collected at various points, and stored in a TEflon bottle with a breathable film tied at the mouth. Transport to the laboratory within 6 hours.
3.2 preparation method of culture medium
Preparation method of liquid culture medium: weigh the BG-11culture medium1.7g add it into a 1000 mL conical flask, add water to a constant volume,shake until they are completely dissolved, use 1M HCl or NaOH to adjust the pH to 7.2, cover the bottle mouth of the conical flask with a rubber stopper, and then use a 0.22 µm sterilization membrane for filtration in a clean workbench for later use.
Preparation method of solid culture medium: add 25 g of nutrient agar medium to the liquid culture medium prepared above, heat it to 60℃ in water bath, and shake it until it is completely dissolved. Then use 0.22 µm sterilization filter membrane to filter in a clean workbench, and use it for later use. After the culture medium is cooled to 35℃, it is poured into a sterile Petri dish and cooled continuously to obtain a solid culture medium.
3.3 Pre-culture after filtration
Under the condition that the purification workbench is opened, the collected water samples are poured into the Brinell funnel in turn, and filtered under the suction filtration of the water ring vacuum pump, and the filter paper is a 0.45 µm water-based filter membrane.
A water sample with a total volume of 100 L is filtered for three times, and a new filter membrane is replaced each time, and the replaced filter membrane is put into a liquid culture medium prepared in advance for culture. Three groups of parallel cultures can be obtained by three times of filtration. There is no need to filter the bottom mud collected from the water bucket, and about 30 g of bottom mud is taken from each group and put into the liquid culture medium separately, which requires three groups in parallel. The top of the conical flask is sealed with a breathable sealing film to prevent external bacteria from entering.
Shake it three times a day in a light incubator with a temperature of 25 ± 1℃ (light-dark ratio of 12 h:12 h, light intensity of 2000 Lx), and place each sample at random so that the sample can receive light evenly. After culturing for 7–15 days until the whole solution system is yellow-green or green, microscopic examination and dilution are carried out to separate algae strains.
3.4 Isolation and purification of microalgae
The preparation method of culture medium adopts the solid medium separation method. When the purification workbench is turned on, dip the above-mentioned fresh algal liquid at the end of logarithmic growth for about 10 days with the inoculation ring, and draw a dense line on the solid medium. Invert it into a 25 ± 1 ℃ light incubator for culture (light dark ratio 12h: 12h, light intensity 2000 Lx). The formation of algae strains with large biomass can be seen in 7–10 days. Pick out large algae colonies for microscopic examination. If a single species is detected by microscope, it indicates that the separation is successful. At this time, the purpose of separation and purification has been achieved, and expanded culture can be carried out.
3.5 The scale-up cultivation of Desmodesmus sp.
① in a 1,000 mL beaker, prepare 500 mL BG-11 liquid medium according to the formula of BG-11 medium, and adjust the pH to 7.1. 200 mL of BG-11 liquid medium is filled in each 500 mL conical flask, or 50 mL of BG-11 liquid medium is filled in 150 mL conical flask;
② in the operation of ① above, the BG-11 culture medium should be filter–ed by 0.22 micron sterilization filter membrane under the clean workbench, and then the bottle mouth should be bandaged for later use; gas permeable membrane, sterile filter membrane, filter device, conical flask and inoculation ring, etc. should be sterilized by steam pressure sterilization pot at 101.33 kPa and 121℃ for 30 minutes before use;
③ under the clean workbench, select the microalgae with good growth by sterile inoculation ring and put them into 10 mL BG-11 liquid culture medium after sterilization and filtration, at 25 ± 1℃, the light intensity is 2,000 Lx (light ratio is 12 h:12 h), shake or stir for 2–3 times every 12 h, and the culture time is 7–15 days; when the biomass is obviously increased (> 106 /L), amplification culture can be carried out in time;
④ under the clean workbench, 10 mL of cultured algae liquid is added into the liquid medium containing 20 mL BG-11 for culture (1:2 amplification),the temperature is 25 ± 1℃, the light intensity is 2,000 Lx (the light ratio is 12 h:12 h), and the culture time is 7–15 days by shaking or stirring for 2–3 times every 12 h, when the bottle mouth is sealed with a gas permeable membrane and the biomass is obviously increased;
⑤ normal culture conditions: temperature 25 ± 1℃, light intensity 3,000 Lx (light ratio 12h: 12 h), shaking or stirring for 2–3 times every 12 h; when the culture volume is greater than 1 L, consider adding stirring to assist the culture, and the culture time is 7–15 days; when microalgae species with long culture period are used, the culture period can be appropriately prolonged;
⑥ then, add the culture solution in ⑤ into 150 mL BG-11 medium for amplif-ication culture (1:5 amplification), and the culture period is 15–20 days; when microalgae species with long culture period are used, the culture period can be appropriately extended to about 30 days;
⑦ when the volume of amplification culture medium is greater than 1 L, the amplification culture conditions are 25 ± 1℃, the light intensity is 3,000 Lx (light ratio is 12 h: 12 h) and 100–200 r/min; when microalgae species with long culture period are used, the culture period can be appropriately extended to about 30 days;
⑧ after the logarithmic phase of the growth of plateau green algae, when the final optical density OD689 value is ≥ 0.700 (the maximum absorption wavelength of each algae species can be obtained by full-wavelength scanning with UV-spectro-photometer), take the algae solution after amplification culture, centrifuge or filter, and wash it with BG-11 culture solution for 3 times for later use.
3.6 Species identification of 18S rRNA
Using the extracted microalgae DNA as the template, primers (positive 1143-510-2-F(AATTGACGGAAKGGCA) and reverse 1637-510-2-R (CGACGGGCGGTGTGTA ) were designed by primer premier 5.0 software.The 18S rRNA gene was amplified by PCR. 25µL PCR reaction system: DNA template 1µL. Primer each 1µL, dNTP 1µL,Taq Buffer (with MgCl2)2.5µL. Taq enzyme 0.25µL, add ddH2O to 25µL. The PCR reaction conditions were: pre denaturation at 95℃ for 5 min, denaturation at 94℃ for 30s, annealing at 63℃ for 30s (0.5℃ per cycle), extension at 72℃ for 30s, 10 cycles; Denaturation at 95℃ for 30s, annealing at 58℃ for 30s, extension at 72℃ for 30s, 30 cycles; Recover and extend at 72℃ for 10min and keep warm at 4℃. After PCR products were detected, the sequences were blast compared in GenBank database. Construct a neighbor-joining phylogenetic tree based on 18S rRNA sequences.
3.7 Determination of Amplification Culture Cycle
Microalgae need to consume nutrients such as N, P and heavy metals in the growth process(Xenopoulos et al. 2002; Shekarabi et al, 2021). Considering the easy sedimentation characteristics of Desmodesmus sp., the logarithmic logarithmic phase of the growth, transfer and harvest time of this species are determined by detecting the change trend of total nitrogen, total phosphorus, pH and conductivity in the culture solution of Desmodesmus sp. in the amplification culture stage, so as to avoid the error caused by the optical density method(Tan et al.2020).
The water quality multi parameter tester(KN-MUL20, kenuokeyi instrument, Beijing) and intelligent digestion instrument(KN-HEA12, kenuokeyi instrument,Beijing) are used to determine the total nitrogen and total phosphorus. The water samples for the determination of total nitrogen and total phosphorus need to be filtered with 0.22 µm filter in advance. The conductivity (cond) and pH were measured with a multi parameter test pen (PCSTestr-35).
3.8 Experiment on correlation between algae concentration and absorbance and turbidity
For the determination method of algal concentration, the blood cell counting plate is used to count under the optical microscope, the average value is taken for three counts, and the average deviation is controlled within 10%. Before counting, the algal solution needs to be vibrated with an oscillator to ensure the uniform dispersion of microalgae, and the initial algal concentration (PCS / L) is determined by taking the stock solution of Desmodesmus sp. culture solution as the test water sample.
Method for preparing water sample containing algae. Dilute 10mL of the same volume of algal solution with ultrapure water successively, and the volume is 20mL, 40mL, 80mL, 100mL and 200mL. Dilute it into a series of algal solutions with different concentrations at constant volume for cell counting and turbidity detection, there are three parallel samples. Dilute 10mL of the same volume of algal solution with ultrapure water to 20mL, 30mL, 40mL, 50mL and 100mL respectively, and dilute it into a series of algal solutions with different concentrations at constant volume for cell counting and absorbance (689nm), there are three parallel samples.
According to the above values, fit the absorbance algae concentration curve, establish the linear regression equation, fit the turbidity algae concentration curve, and establish the linear regression equation(Tsenkova 2020; O'Donoghue et al. 2022).