Scenedesmus Fs and application thereof, scenedesmus solution and culture method and application thereof, and phosphorus-containing wastewater treatment method

Abstract

The invention belongs to the technical field of wastewater treatment and biological energy production, and provides scenedesmus Fs and application thereof, scenedesmus liquid and a culture method and application thereof, and a phosphorus-containing wastewater treatment method. The invention provides a Scenedesmus sp. Fs (Scenedesmus sp.), and the preservation number of the Scenedesmus sp. Fs is CCTCC (China Center for Type Culture Collection) NO: M20242541. The scenedesmus Fs provided by the invention can effectively remove phosphorus in wastewater; meanwhile, the scenedesmus Fs is high in lipid content and has potential application in biodiesel production.

Description

Technical Field

[0001] The present invention relates to the technical field of wastewater treatment and bioenergy production, and in particular to a Scenedesmus Fs and an application thereof, a Scenedesmus algae liquid and a cultivation method and application thereof, and a method for treating phosphorus-containing wastewater. Background Art

[0002] In recent years, with the increasing severity of water eutrophication and energy shortage problems, the development of algae strains that can simultaneously treat wastewater and produce biodiesel has important economic and environmental significance. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a Scenedesmus Fs and its application, Scenedesmus algae liquid and its cultivation method and application, and a method for treating phosphorus-containing wastewater. The Scenedesmus Fs provided by the present invention can efficiently remove phosphorus from wastewater and also has the potential application of generating biodiesel.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The invention provides a Scenedesmus sp. Fs. The preservation number of the Scenedesmus sp. Fs is CCTCCNO: M20242541.

[0006] The present invention also provides a Scenedesmus algae liquid, wherein the Scenedesmus algae liquid comprises the Scenedesmus Fs described in the above technical solution.

[0007] The present invention also provides a method for culturing the Scenedesmus algae liquid described in the above technical solution, comprising the following steps:

[0008] The Scenedesmus Fs described in the above technical solution is placed in a BG11 culture medium and cultured under light conditions.

[0009] Preferably, the intensity of the illumination is 50 to 200 μmolphotons·m - 2 s - 1.

[0010] Preferably, the culture temperature is 20-30°C.

[0011] The present invention also provides the use of the Scenedesmus Fs described in the above technical solution or the Scenedesmus liquid described in the above technical solution in the treatment of phosphorus-containing wastewater.

[0012] The present invention also provides a method for treating phosphorus-containing wastewater, comprising the following steps:

[0013] The Scenedesmus Fs is placed in phosphorus-containing wastewater for treatment;

[0014] The Scenedesmus Fs comes from the Scenedesmus Fs described in the above technical solution or the Scenedesmus algae liquid described in the above technical solution.

[0015] Preferably, the OD of the Scenedesmus Fs in phosphorus-containing wastewater is 680 It is 0.1~0.3.

[0016] Preferably, the total phosphorus content in the phosphorus-containing wastewater is 2-10 mg / L, and the pH is 7-7.5.

[0017] The present invention also provides the use of the Scenedesmus Fs described in the above technical solution or the Scenedesmus liquid described in the above technical solution in the production of biodiesel.

[0018] The present invention provides a Scenedesmus Fs.

[0019] The Scenedesmus Fs provided by the present invention can effectively remove phosphorus in wastewater; at the same time, the Scenedesmus Fs has a high lipid content and has potential application in producing biodiesel.

[0020] Biological Deposit Description

[0021] The Fs strain of Scenedesmus, with the Latin name Scenedesmus sp., was deposited in the China Center for Type Culture Collection (CCTCC) on November 12, 2024. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, Wuhan University, and the deposit number is CCTCC NO: M20242541. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the removal efficiency of total phosphorus (TP) by Scenedesmus Fs in wastewater treatment;

[0023] Figure 2 is the removal efficiency of chemical oxygen demand (COD) by Scenedesmus Fs in wastewater treatment;

[0024] Figure 3 It is the biochemical component of Scenedesmus Fs;

[0025] Figure 4 It is the biodiesel quality indicator of algae strain Fs. DETAILED DESCRIPTION

[0026] The invention provides a Scenedesmus sp. Fs. The preservation number of the Scenedesmus sp. Fs is CCTCCNO: M20242541.

[0027] In the present invention, the Scenedesmus Fs is preferably isolated from a fecal water sample of a pond in Hubei.

[0028] The present invention also provides a Scenedesmus algae liquid, wherein the Scenedesmus algae liquid comprises the Scenedesmus Fs described in the above technical solution.

[0029] The present invention also provides a method for culturing the Scenedesmus algae liquid described in the above technical solution, comprising the following steps:

[0030] The Scenedesmus Fs described in the above technical solution is placed in a BG11 culture medium and cultured under light conditions.

[0031] In the present invention, the BG11 culture medium is in liquid form.

[0032] In the present invention, the intensity of the light is preferably 50 to 200 μmolphotons·m - 2 s - 1, more preferably 100 to 150 μmol photons·m -2 ·s -1 In the present invention, the culture temperature is preferably 20 to 30°C, more preferably 25°C.

[0033] The present invention also provides the use of the Scenedesmus Fs described in the above technical solution or the Scenedesmus liquid described in the above technical solution in the treatment of phosphorus-containing wastewater.

[0034] The present invention also provides a method for treating phosphorus-containing wastewater, comprising the following steps:

[0035] The Scenedesmus Fs is placed in phosphorus-containing wastewater for treatment;

[0036] The Scenedesmus Fs comes from the Scenedesmus Fs described in the above technical solution or the Scenedesmus algae liquid described in the above technical solution.

[0037] In the present invention, the OD of the Scenedesmus Fs in phosphorus-containing wastewater is 680 It is preferably 0.1 to 0.3, more preferably 0.2, and specifically may be 0.1, 0.15, 0.2, 0.25 or 0.3.

[0038] In the present invention, the total phosphorus content in the phosphorus-containing wastewater is preferably 2 to 10 mg / L; the pH is preferably 7 to 7.5, and more preferably 7.2.

[0039] The present invention also provides the use of the Scenedesmus Fs described in the above technical solution or the Scenedesmus liquid described in the above technical solution in the production of biodiesel.

[0040] In the present invention, the Scenedesmus Fs has a higher lipid content, which gives it a significant advantage in biodiesel production.

[0041] The following detailed description of the Scenedesmus Fs and its application, Scenedesmus algae liquid and its culture method and application, and the treatment method of phosphorus-containing wastewater provided by the present invention is provided in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1 Screening method for algae strain Fs

[0043] The strain Fs was isolated from a fecal water sample collected from a pond in Hubei, China. The isolation process was carried out using the dilution plate method, which involved diluting the sample 5, 10, and 20 times, streaking it onto a BG11 solid medium (1.5% agar) plate, and then culturing it in a light incubator (light intensity of 50 μmol photons·m - 2 s - 1, temperature 25°C), after a single algae colony grows on the solid plate, pick a single algae colony for streaking separation and culture again, and repeat streaking 3 times to obtain a pure algae strain.

[0044] The purified algae were inoculated in BG11 liquid medium and cultured under a light intensity of 100 μmol photons·m - 2 s - 1. Culture in a light incubator at 25°C.

[0045] Example 2 Identification process of algae strains

[0046] The algal strain was identified by 18S rRNA gene sequencing: genomic DNA was extracted, and the 18S rRNA gene was amplified using 18S forward primer 18SF (5′-CCAACCTGGTTGATCCTGCCAGTA-3′, SEQ ID NO.1) and 18S reverse primer 18SR (5′-CCTTGTTAACGACTTCACCTTCCTCT-3′, SEQ ID NO.2). The amplified product was purified and sequenced by Shanghai Sangon Biotechnology Co., Ltd.

[0047] The nucleotide sequence of the 18S rRNA gene of the algae strain Fs is shown in SEQ ID NO.3, and is as follows:

[0048]

[0049] Then, BLASTn was used to compare the sequence homology with the NCBI database. The results showed that the similarity between strain Fs and Scenedemus sp. was 98.08%, the highest homology, so strain Fs was judged to be Scenedemus sp., and Scenedemus was deposited in the China Type Culture Collection of Wuhan University with the deposit number CCTCC NO: M20242541.

[0050] Example 3 Application of Scenedesmus Fs in Treatment of Phosphorus-Containing Wastewater

[0051] Artificially synthesized municipal wastewater was used, and its composition was as follows: glucose 200.0 mg / L, sucrose 200.0 mg / L, protein 68.3 mg / L, ammonium sulfate 66.7 mg / L, ammonium chloride 10.9 mg / L, potassium dihydrogen phosphate 4.44 mg / L, dipotassium hydrogen phosphate 9.0 mg / L, magnesium sulfate heptahydrate 21.0 mg / L, manganese sulfate (II) heptahydrate 2.7 mg / L, sodium bicarbonate 30.00 mg / L, calcium chloride hexahydrate 19.74 mg / L, iron (III) chloride hexahydrate 0.14 mg / L. Initial pH 7.2.

[0052] The algae strain Fs was placed in the above-mentioned synthetic municipal wastewater. The initial OD of the algae strain Fs in the synthetic municipal wastewater was 680 is 0.2.

[0053] After the artificial synthetic municipal wastewater was treated with Scenedesmus Fs for different time periods (12, 24, 48 and 72 h), the treated artificial synthetic municipal wastewater was centrifuged at 8000 rpm for 10 min and filtered through a 0.45 μm nylon filter membrane. The concentrations of total phosphorus (TP) and chemical oxygen demand (COD) in the treated artificial synthetic municipal wastewater were measured. The determination method was based on the national standard scheme: "Water and Wastewater Monitoring and Analysis Methods", Fourth Edition, 2002. The removal efficiency of Scenedesmus Fs on total phosphorus and chemical oxygen demand was calculated. The results are shown in Figure 1 and Figure 2 As shown, Figure 1 is the removal efficiency of total phosphorus (TP) by Scenedesmus FS in wastewater treatment, Figure 2 is the removal efficiency of chemical oxygen demand (COD) by Scenedesmus Fs in wastewater treatment. Figure 1 It can be seen that Scenedesmus Fs has a very high efficiency in removing total phosphorus (TP) from wastewater, achieving 90% phosphorus removal in 12 hours and almost all phosphorus removal within 72 hours. Figure 2 It can be seen that the removal efficiency of chemical oxygen demand (COD) in wastewater by Scenedesmus Fs gradually increased over time, and more than 90% of chemical oxygen demand (COD) removal was achieved within 72 hours.

[0054] Example 4 Application of Scenedesmus Fs in Biodiesel Production

[0055] (1) Protein, carbohydrate and fatty acid content in Scenedesmus Fs

[0056] 1.1 Determination of total lipid content

[0057] Take freeze-dried Scenedesmus Fs powder (hereinafter referred to as algae powder) and grind it thoroughly in a mortar. Then weigh 100 mg of the ground algae powder into a 50 mL centrifuge tube, add 9.5 mL of a mixed solvent of chloroform: methanol: distilled water with a volume ratio of 1:2:0.8, and vortex for 5 minutes. Centrifuge at 3000 rpm for 3 minutes, collect the supernatant, and repeat the above operation twice for the remaining precipitate. Add chloroform and distilled water to the collected supernatant so that the volume ratio of chloroform: methanol: distilled water in the solution is 1:1:0.9. Mix well and let stand. Centrifuge at 5000 rpm for 10 minutes. The solution is stratified, and the lower layer is chloroform, which contains the target lipids. Collect the chloroform layer in a glass centrifuge tube with a pre-weighed weight (M1), blow dry the solvent with a nitrogen blower, and measure the total content (M2). The total lipid weight is M2-M1. The results are shown in Figure 3 .

[0058] 1.2 Protein content determination

[0059] Determination was performed using the BCA kit (PC0020) from Solebao: First, prepare the BCA reagent: Cu reagent in a ratio of 50:1 (V / V) according to the kit instructions, and mix thoroughly to prepare the BCA working solution. Take 10 μL of BSA standard and dilute it to 100 μL with PBS to make the final concentration of BSA standard 0.5 mg / mL. Add 0, 2, 4, 6, 8, 12, 16, and 20 μL of the standard to a 96-well plate, and add PBS to make up to 20 μL. Add 200 μL of BCA working solution to each well and place at 37°C for 30 minutes. Use an enzyme reader to measure the absorbance at a wavelength of 562 nm to obtain a standard curve of protein concentration.

[0060] Take the freeze-dried algae powder and grind it thoroughly in a mortar, then weigh 10 mg of the ground algae powder into a 15 mL centrifuge tube, add 10 mL of deionized water to mix and dissolve it completely. Take the above 1 mL sample and dilute it appropriately, add 20 μL to the sample wells of the 96-well plate, add 200 μL of BCA working solution to each well, and place it at 37°C for 30 minutes. Use an enzyme reader to measure the absorbance value at a wavelength of 562 nm, and calculate the sample protein concentration based on the standard curve. The results are as follows: Figure 3 shown.

[0061] 1.3 Determination of carbohydrate content

[0062] Determination of carbohydrate content by 620nm sulfuric acid anthrone method: prepare 100mL of sulfuric acid (70% concentrated sulfuric acid) anthrone reagent in a brown reagent bottle, take 2mg of vacuum-frozen algae powder in a 5mL centrifuge tube, add 1mL of deionized water, and place the centrifuge tube on a vortex oscillator for 5min, then place the centrifuge tube in a water bath at 80℃ for 10min, then centrifuge at 12000rpm for 3min, take the supernatant into another 5mL clean centrifuge tube, and add 1mL of deionized water to the original centrifuge tube again, repeat the above process, and collect the supernatant obtained from the two operations. Take 0.2mL of supernatant in a clean 1.5mL black EP tube, take 1mL of anthrone sulfate reagent in a black EP tube under light-proof conditions, and then put the black EP tube in a 100℃ water bath. After 10 minutes of water bath, quickly take it out and put it in the refrigerator. Take it out after cooling to room temperature, mix the sample and use a pipette to draw 200μL into a 96-well plate, adjust the wavelength of the microplate reader to 620nm, and use the blank water sample as a reference to determine the absorbance value. The standard curve is determined by using the blank sample as a reference to determine the absorbance values ​​of glucose standard solutions with different concentration gradients at 620nm, and based on the relationship between concentration and absorbance, a standard curve is obtained. The absorbance value at a wavelength of 620nm is determined by an microplate reader, and the carbohydrate concentration of the sample is calculated based on the standard curve. The results are shown in Figure 3 .

[0063] Figure 3 The biochemical components of Scenedesmus Fs are Figure 3 It can be seen that the fatty acid content in Scenedesmus Fs is as high as 60% of the cell dry weight, and it is a potential algae strain for high production of fatty acids or biodiesel.

[0064] (2) After the Scenedesmus Fs in Example 3 was treated in the artificial synthetic municipal wastewater for 72 h, the Scenedesmus Fs cells were collected, freeze-dried, and sent to Suzhou Baiqu Biotechnology Co., Ltd. for fatty acid GC-MS detection.

[0065] The steps of GC-MS detection are as follows:

[0066] 1. Take the sample into a 2mL EP tube, add 500μL of extraction solution (isopropanol: n-hexane, volume ratio 2:3, containing internal standard 0.2mg / L), add steel beads, and vortex mix for 30s.

[0067] 2.40 Hz grinder treatment for 4 min, ultrasonic treatment for 5 min (ice water bath), repeat 3 times.

[0068] 3. Centrifuge the sample at 4°C, 12000 rpm for 15 min, and transfer about 450 μL of the supernatant into a new 1.5 mL EP tube.

[0069] 4. Add 500 μL of extraction solution (isopropanol: n-hexane, volume ratio 2:3, containing internal standard 0.2 mg / L) to the remaining sample, vortex mix for 30 seconds, and repeat steps 2 and 3.

[0070] 5. Take about 450 μL of the supernatant, combine it with the supernatant in step 3, and vortex for 10 seconds.

[0071] 6. Take 800 μL of supernatant and combine them in a 2 mL EP tube and blow dry with nitrogen.

[0072] 7. Add 500 μL methanol:trimethylsilyldiazomethane solution (the volume ratio of methanol to trimethylsilyldiazomethane is 1:2) and let stand at room temperature for 30 min;

[0073] 8. Dry with nitrogen.

[0074] 9. Add 160 μL of n-hexane to dissolve, centrifuge at 12000 rpm for 1 min, and collect the supernatant into a vial.

[0075] 10. GC-MS detection was used to obtain a fatty acid content profile of Scenedesmus Fs. The fatty acid content of Scenedesmus Fs is shown in Table 1.

[0076] Table 1 Content of fatty acids in Scenedesmus Fs (%)

[0077] Fatty acid type Content / wt% Decanoic acid 0.05 Myristic acid 0.18 Pentadecanoic acid 0.08 Hexadecanoic acid (palmitic acid) 10.90 Heptadecanoic acid 0.10 Octadecanoic acid (stearic acid) 2.85 Trans-9-octadecenoic acid (elaidic acid) 0.17 Cis-9-octadecenoic acid (oleic acid) 10.49 cis-11-octadecenoic acid 1.41 cis-9,12-linoleic acid 32.50 Gamma-linolenic acid 0.21 α-linolenic acid 40.31 Eicosanoic acid (arachidic acid) 0.05 cis-11-eicosenoic acid 0.04 Behenic acid 0.19 Lignoceric acid 0.19 cis-4,7,10,13,16,19-Docosahexaenoic acid 0.27

[0078] It can be seen from Table 1 that the content of polysaturated fatty acids such as α-linolenic acid and cis-9,12-linoleic acid in Scenedesmus Fs is high, accounting for more than 70% of the total fatty acid content.

[0079] The saponification value (SN), iodine value (IN) and cetane number (CN) were calculated based on the content of each fatty acid in Scenedesmus Fs in Table 1 to evaluate the quality of the algae strain as biodiesel. These values ​​were calculated based on the fatty acid profile using the following equations:

[0080] SN=∑(560×Pi) / MWi

[0081] IN=∑(254×D×Pi) / MWi

[0082] CN=46.3+5458 / SN-0.225×IN

[0083] In the equation, Pi represents the weight percentage of each fatty acid, MWi represents the molecular weight of each fatty acid, and D represents the number of double bonds in each oil.

[0084] The results are as follows Figure 4 As shown, Figure 4 is the biodiesel quality index of algae strain Fs, from Figure 4It can be seen that although Fs showed a slightly lower CN, it also exhibited a higher saponification value (SN), which contributed to efficient biodiesel conversion; the higher iodine value (IN) of Fs indicated that its fatty acid unsaturation was higher, which may affect the oxidative stability, but blending Fs biodiesel with more stable oils or introducing antioxidants could address this limitation.

[0085] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Scenedesmus sp. Fs, characterized in that: The deposit number of the Scenedesmus Fs is CCTCC NO: M20242541.

2. A Scenedesmus algae liquid, characterized in that: The Scenedesmus algae liquid includes the Scenedesmus Fs described in claim 1.

3. The method for culturing Scenedesmus algae liquid according to claim 2, characterized in that: The following steps are involved: The Scenedesmus Fs described in claim 1 is placed in a BG11 culture medium and cultured under light conditions.

4. The culture method according to claim 3, characterized in that The intensity of the illumination is 50 to 200 μmolphotons·m - 2 s - 1.

5. The culture method according to claim 3, characterized in that: The culture temperature is 20-30°C.

6. Use of the Scenedesmus Fs described in claim 1 or the Scenedesmus liquid described in claim 2 in the treatment of phosphorus-containing wastewater.

7. A method for treating phosphorus-containing wastewater, characterized in that: The following steps are involved: The Scenedesmus Fs is placed in phosphorus-containing wastewater for treatment; The Scenedesmus Fs is derived from the Scenedesmus Fs described in claim 1 or the Scenedesmus algae liquid described in claim 2.

8. The processing method according to claim 7, characterized in that: OD of the Scenedesmus Fs in phosphorus-containing wastewater 680 It is 0.1~0.

3.

9. The processing method according to claim 7, characterized in that: The total phosphorus content in the phosphorus-containing wastewater is 2-10 mg / L, and the pH is 7-7.

5.

10. Use of the Scenedesmus Fs according to claim 1 or the Scenedesmus liquid according to claim 2 in biodiesel production.

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