CN102978118A - Scenedesmus sp., CHX1 and use thereof - Google Patents

Abstract

The invention discloses a strain of scenedesmus and its application. It was isolated in the pig wastewater treatment pond, named CHX1, and its classification was named Scenedesmus sp.. It has been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, and its preservation number is CGMCC No.6649. For Scenedesmus optimized medium, the following solutes were adjusted to 1 liter: 1ml A ₅ +Co solution, 5g glucose, 3gNaNO ₃ , 0.04gK ₂ HPO ₄ ·3H ₂ O, 0.075gMgSO ₄ ·7H ₂ O, 0.036gCaCl ₂ ·2H ₂ O, 0.006gFe(NH ₄ ) ₃ C ₁₈ H ₁₀ O ₁₄ , 0.001gNa ₂ -EDTA, and water as the balance; Scenedesmus sp., CHX1 was cultured on the optimal medium for Scenedesmus sp., to obtain a seed culture solution. The seed culture fluid can be used to prepare biodiesel and treat swine wastewater. The Scenedesmus CHX1 provided by the present invention grows well in the pig wastewater, and can efficiently remove nitrogen and phosphorus in the wastewater; the Scenedesmus CHX1 itself has relatively high biological and oil yields, and C16-C18 in fatty acid components accounts for more than 70% , suitable for the production of biodiesel.

Description

Scenedesmus and its application

technical field

The invention relates to Scenedesmus and its application, in particular to the application of the Scenedesmus in the fields of bio-energy and purification of pig-raising wastewater.

technical background

Human beings are facing two major crises in the 21st century: energy crisis and water resource crisis. It is estimated that the world's crude oil reserves and natural gas reserves will be exhausted in 40 years and 64 years respectively. Therefore, it is imperative to develop economical and efficient new energy sources, among which bio-new energy is one of the main research hotspots. At the same time, 40% of the countries and regions in the world are facing the problem of water shortage, and the sustainable use of water resources is imminent. Sewage recycling as the second water source of the city provides a new way to solve the shortage of urban water resources, but the high content of nitrogen and phosphorus in the sewage is likely to cause eutrophication of the water body and affect the utilization of sewage. Therefore, finding an efficient and low-cost wastewater ecological restoration technology is one of the important means to solve the current water resource crisis.

Microalgae are a kind of photoautotrophic single-celled organisms, which have the characteristics of rich resources, various types, fast growth, strong adaptability and high oil content. In recent years, with the deepening of human understanding of microalgae, more and more attention has been paid to microalgae in the preparation of biodiesel and deep nitrogen and phosphorus removal of sewage, and a large number of related researches have been carried out. The growth of algae needs nitrogen and phosphorus as substrates, so the use of microalgae can remove pollutants such as nitrogen and phosphorus in sewage; at the same time, microalgae can fix _CO2 into organic carbon (protein, oil, etc.) through photosynthesis, and the oil in algal cells The triacylglyceride is the main raw material for the preparation of biodiesel. Therefore, microalgae can be cultivated in sewage for advanced treatment of nitrogen and phosphorus removal in sewage and biodiesel can be produced at the same time.

At present, there are few domestic research reports on the coupling of advanced sewage treatment and biodiesel production based on microalgae cultivation. Screening microalgae strains suitable for sewage cultivation with high oil yield and easy conversion to biodiesel is the premise and key of this process . However, many high-oil algae species purely for the purpose of producing biodiesel, such as Chlorella and Staphylococcus brancii, do not necessarily grow normally in sewage and accumulate a large amount of oil, and these algal strains generally have high requirements for culture conditions and low biological yield. Problems such as low or low oil yield. Therefore, screening excellent algae species has important practical significance for solving the above problems.

Contents of the invention

The object of the present invention is to provide Scenedesmus and its application.

Scenedesmus, isolated from the pig wastewater treatment pool, named CHX1, and its classification named Scenedesmus sp., has been preserved in the General Microbiology Center of the China Committee for the Collection of Microorganisms, and its preservation number is CGMCC No. 6649.

For the Scenedesmus optimized medium, the following solutes were adjusted to 1 liter: 1ml A ₅ +Co solution, 5g glucose, 3g NaNO ₃ , 0.04g K ₂ HPO ₄ ·3H ₂ O, 0.075g MgSO ₄ ·7H ₂ O, 0.036g CaCl ₂ ·2H ₂ O, 0.006g Fe(NH ₄ ) ₃ C ₁₈ H ₁₀ O ₁₄ , 0.001g Na ₂ -EDTA, the balance is water; the A ₅ +Co solution is the following solute Dilute to 1 liter to obtain: 2.86 g H ₃ BO ₃ , 1.81 MnCl ₂ ·H ₂ O, 0.222 ZnSO ₄ ·7H ₂ O, 0.079 CuSO ₄ ·5H ₂ O, 0.39 Na ₂ MoO ₄ ·2H ₂ O, 0.049 Co(NO ₃ ) ₂ ·6H ₂ O, the balance is water; the initial pH of the optimized medium is 8, and the initial concentration of the optimized medium after inoculation with Scenedesmus culture solution is OD ₆₉₀ =0.16.

The application of the Scenedesmus optimized medium is used to cultivate Scenedesmus sp., CHX1, to obtain a seed culture solution.

The dry weight biomass and total oil yield of the seed culture solution are respectively: 814.1 mg/liter.day (range is 809.6~818.6 mg/liter.day) and 128.3 mg/liter.day (range is 127.5~129.0 mg/liter.day) mg/L.day); the content of C16-C18 components in the fatty acids that make up the total oils and fats accounts for 70%, which can be used to prepare biodiesel.

The application of the Scenedesmus in the treatment of swine wastewater.

The pig raising wastewater is treated to remove nitrogen and/or phosphorus.

Beneficial effects of the present invention: Scenedesmus has low requirements for culture conditions, grows well in sewage, and can efficiently remove nitrogen and phosphorus in sewage. Scenedesmus has fast growth rate, high biological yield and high oil yield, and can be used to prepare biodiesel. In summary, Scenedesmus CHX1 has broad application prospects in the application of pig wastewater treatment coupled with high-value biomass production.

Description of drawings

Figure 1 is a phylogenetic tree constructed based on the partial sequence of 18S rDNA.

Figure 2 shows the optimal absorption peak selection of Scenedesmus CHX1.

Figure 3 shows the growth of Scenedesmus CHX1 under different culture methods.

Figure 4 shows the growth of Scenedesmus CHX1 under different carbon sources.

Figure 5 shows the growth of Scenedesmus CHX1 under different glucose dosages.

Fig. 6 is the biomass yield of Scenedesmus CHX1 orthogonal optimization experiment.

Detailed ways

The following examples facilitate a better understanding of the present invention, but the present invention is not limited to the examples described. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

Example 1 Isolation and identification of Scenedesmus CHX1

1. Sample collection

In July 2011, it was collected from a wastewater pond of a farm in Xiaoshan District, Hangzhou City, Zhejiang Province containing

Water sample of green algae.

2. Isolation and purification of Scenedesmus CHX1

Separation and purification steps: Centrifuge and concentrate the collected water samples (5000 r/min, 4°C), dilute with sterile water, pass through a 60-micron mesh protozoan sieve, and then pass through a 0.45-μm filter membrane. Rinse the filter membrane several times with sterile water, transfer the remaining cells on the filter membrane to a 12-well cell culture plate (each well contains 1ml of sterilized BG-11 culture solution, and the sterilization method is high-pressure steam sterilization at 121°C 20 minutes), placed in a light incubator for enrichment culture (cultivation conditions are full-day light, light intensity is 40μmol photon/m ² . s, temperature is 25°C). After 7 days of enrichment culture, properly dilute the algae solution, draw the diluted algae solution and smear it on the BG-11 solid medium containing 1.5% agar, and place it in a light incubator for several days (cultivation conditions: full-day light, light intensity of 40μmol photon/m ² . s, the temperature is 25℃), and then pick a single colony in the liquid BG-11 culture medium and then expand the culture (cultivation conditions: full-day light, light intensity 40μmol photon/ m ² . s, temperature 25°C, shaker speed 150 r min ^-1 ). A pure algae strain was obtained and named CHX1.

3. Identification of Scenedesmus CHX1

1. Morphological identification of Scenedesmus CHX1

The cells are green and often aggregated into clusters; a single cell is round or ellipsoid in shape, with a diameter of 3-6 μm; the pigment body is perichrome and contains lipid droplets; the cell contains a clearly visible protein nucleus.

2. Molecular identification of Scenedesmus CHX1

Cells in the exponential growth phase were collected, and genomic DNA was extracted with a plant genomic DNA extraction kit (Nanjing Jiancheng Biotechnology Co., Ltd.), and the 18S rDNA gene fragment was amplified using it as a template. The PCR amplification system is 20 μl, which contains 1 μl 50ng DNA template, 0.2 μl 1.6-Unit Taq polymerase, 2 μl 10xPCR reaction system, 2 μl 2.5mM dNTP, 0.5 μl each of forward and reverse primers (0.2 μM), double-distilled sterilized water 13.8 μl. The PCR amplification program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 40 s, annealing at 58°C for 35 s, extension at 72°C for 35 s, a total of 36 cycles; and extension at 72°C for 10 min.

The primers used to amplify 18S rDNA are specific primers for eukaryotic 18S ribosomal DNA, and the sequence is as follows:

  Forward primer (18SF): 5'-CAGGTCTGTGATGCCC-3';

Reverse primer (18SR): 5'-ACGGGCGGTG TGTAC -3';

 The PCR amplification products were separated by 1% agarose gel electrophoresis, recovered with a gel recovery kit, and delivered to Shanghai Huada Gene Sequencing Co., Ltd. for sequencing. The PCR amplification product is a partial sequence of 18S rDNA, and the sequencing results are shown in the sequence list.

The sequencing results were compared and analyzed by BLAST on the NCBI website. The Query coverage value of CHX1 and Scenedesmaceaesp . (AY197639) reached 100%, and the Max-Ident was 96%. Multi-sequence homology analysis was performed with MEGA 4.0 software, and a phylogenetic tree was constructed according to the NJ method (see Figure 1, the value on the branch is bootstrap 100). Microalgae CHX1 and Scenedesmaceaesp . well clustered in one branch.

Based on the results of morphological identification and molecular identification, CHX1 was identified as Scenedesmus sp., and it was deposited in the General Microorganism Center of China Committee for the Collection of Microorganisms on October 10, 2012, and its preservation number is CGMCC No. 6649 .

Example 2 Optimization of Growth Conditions of Scenedesmus CHX1

1. Determination of Optimal Absorbance Density Value of Scenedesmus CHX1

In order to better describe the growth of Scenedesmus CHX1 and determine the optimal absorbance value of Scenedesmus CHX1, the BG11 culture solution containing microalgae was scanned automatically in the range of 600-700nm. The scan results are shown in Figure 2.

2. Optimization and determination of the growth conditions of Scenedesmus CHX1

In order to make Scenedesmus CHX1 grow faster and better, the combination of culture mode, carbon source, glucose dosage and culture conditions was optimized on the basis of BG-11 medium.

1. Optimal determination of culture methods

Based on the BG-11 medium, three culture methods were set up: photoautotrophic, heterotrophic and polytrophic. The photoautotrophic culture condition was light + BG-11 culture medium; the heterotrophic culture condition was BG-11 culture medium + 1g/L glucose; the mixed culture condition is light + BG-11 culture solution + 1g/L glucose. Scenedesmus CHX1 was inoculated into each culture solution respectively (Scenedes CHX1 with an OD ₆₉₀ value of 0.637 was inoculated in each culture solution at a volume ratio of 10%, and the OD ₆₉₀ value of Scenedesmus in the culture solution was all about 0.067) , cultured at 25°C for 3 days, and the light intensity was 40μmol photon/m ² .s. The OD ₆₉₀ value in each culture solution was detected, and the biomass yield was calculated according to the conversion relationship between OD ₆₉₀ and the dry weight of algae cells. The results are shown in Figure 3.

2. Carbon source optimization and determination

Different carbon sources were added on the basis of removing the sodium carbonate component in the BG-11 medium, and the addition amount of each carbon source was set according to the carbon content of equimolar concentration. Inoculate Scenedesmus CHX1 into each culture solution respectively (inoculate Scenedesmus CHX1 with an OD ₆₉₀ value of 1.70 in each culture solution at a volume ratio of 20%, and the OD ₆₉₀ value of Scenedesmus in the culture solution is about 0.38) , cultured at 25°C for 3 days, and the light intensity was 40μmol photon/m ² .s. The OD ₆₉₀ value in each culture solution was detected, and the biomass yield was calculated according to the conversion relationship between OD ₆₉₀ and the dry matter weight of algae cells. The _results are shown in Figure 4.

3. Optimal determination of glucose dosage

On the basis of determining the optimal carbon source, glucose was used instead of sodium carbonate as carbon source in BG-11 culture, and different glucose dosages were set. Inoculate Scenedesmus CHX1 into each culture solution (inoculate Scenedesmus CHX1 with an OD ₆₉₀ value of 0.8 in each culture solution at a volume ratio of 10%, and the OD ₆₉₀ values of Scenedesmus in the culture solution are all about 0.08) , cultured at 25°C for 3 days, and the light intensity was 40μmol photon/m ² .s. The OD ₆₉₀ value in each culture solution was detected, and the biomass yield was calculated according to the conversion relationship between OD ₆₉₀ and the dry matter weight of algae cells. The results are shown in Figure 5.

4. Determination of the optimal combination of culture conditions

L ₉ (3 ⁴ ) orthogonal experiment table was used to further optimize the combination of culture conditions, and the dosage of glucose, nitrate, initial pH and inoculum concentration were used as indicators to screen the optimal combination of conditions. The specific design scheme is shown in Table 1 . Scenedesmus CHX1 was inoculated into each culture solution, cultured at 25°C for 3 days, and the light intensity was 40μmol photon/m ² .s. The OD ₆₉₀ value in each culture solution was detected, and the biomass yield was calculated according to the conversion relationship between OD ₆₉₀ and the dry matter weight of algae cells. The specific results are shown in Figure 6.

Table 1 L ₉ (3 ⁴ ) Orthogonal Experiment Table Design Scheme

According to the above optimization results, the optimized culture conditions and optimized medium of Scenedesmus CHX1 were determined. The optimized medium is obtained by distilling the following solutes to 1 liter with water: adding 1ml A ₅ +Co solution, 5g glucose, 3g NaNO ₃ , 0.04g K ₂ HPO ₄ ·3H ₂ O, 0.075g MgSO ₄ ·7H ₂ O , 0.036g CaCl ₂ ·2H ₂ O, 0.006g Fe(NH ₄ ) ₃ C ₁₈ H ₁₀ O ₁₄ , 0.001g Na ₂ -EDTA.

A ₅ +Co solution is obtained by distilling the following solutes to 1 liter with water: 2.86 g H ₃ BO ₃ , 1.81 MnCl ₂ ·H ₂ O, 0.222 ZnSO ₄ ·7H ₂ O, 0.079 CuSO ₄ ·5H ₂ O, 0.39 Na ₂ MoO ₄ ·2H ₂ O, 0.049 Co(NO ₃ ) ₂ ·6H ₂ O. The initial pH of the medium was 8, and the inoculation volume was 10% (v/v, OD ₆₉₀ =0.16).

Example 3 Growth and Oil Production of Scenedesmus CHX1

1. Cultivation of Scenedesmus CHX1

Scenedesmus CHX1 was cultured according to the optimized conditions and medium in Example 2, at a culture temperature of 25° C. and under the conditions of 40 μmol photon/m ² .s of light throughout the day for 3 days.

2. Determination of Growth and Oil Production of Scenedesmus CHX1

1. Biomass determination of Scenedesmus CHX1

Take the culture medium and place it in a centrifuge tube, centrifuge for 10 minutes (10000 r/min, 4°C), discard the supernatant, transfer the concentrated biomass to tin foil, and freeze-dry. 2.44g of dry algae were obtained per liter of culture medium (the average biomass yield was 814.1 mg/L.d, and the range was 809.6~818.6 mg/L.day), and the biomass yield was relatively high.

2. Determine the oil yield of Scenedesmus CHX1

Weigh a certain amount of freeze-dried algae sample (weight is W ₀ ), add solvent (chloroform:methanol=2:1, volume ratio), 30°C ultrasonic water bath for 30min, let it stand for a while, collect the supernatant and pass it through a glass fiber filter , transfer the filtrate to a centrifuge tube containing 0.9% NaCl solution, shake well and then centrifuge at 1000 r/min for 10 minutes, read the volume of the lower layer liquid (marked as V ₀ ), draw a certain volume (marked as V ₁ ) of the lower layer liquid and transfer Put it into a new centrifuge tube that has been dried and weighed (marked as W ₁ ), blow nitrogen until the organic solvent evaporates to dryness, weigh the weight of the centrifuge tube (marked as W ₂ ), and calculate the Scenedesmus oil content (%) = ( W ₂ - W ₁ )* V ₀ /( W ₀ * V ₁ )*100, oil yield (mg/L.d) = oil content (%)*biomass yield (mg/L.d). The results showed that the average oil yield was 128.3 mg/L.d, and the range was 127.5~129.0 mg/L.day (oil content was 15.8%).

3. Determination of Fatty Acid Composition in Scenedesmus CHX1

Weigh 0.1g of freeze-dried algae powder into a hydrolysis tube with a screw cap, add 8ml of 0.5M KOH-CH ₃ OH solution, fill with N ₂ for 1min, seal it, and use SCQ-250 ultrasonic cleaner (33kHz, 250W) to sonicate Extract for 5min. Place the fully mixed sample in a water bath at 100°C for 15 minutes, cool to room temperature, adjust the pH to <2 with 0.7M HCl-CH ₃ OH solution, and bathe again in a water bath at 100°C for 15 minutes. After cooling to room temperature, 2 ml of saturated NaCl solution was added, and then extracted twice with 2 ml of n-hexane. The combined extracts were placed in a centrifuge tube with a stopper and blown with nitrogen to 20-50 μl for GC-MS analysis.

The GC-MS instrument contains a DB-5-MS capillary chromatographic column and an ion flame detector. The analysis program is that the column temperature is 80°C, the temperature is kept constant for 5 minutes, and then the temperature is raised to 290°C at a rate of 4°C/min, and the temperature is kept constant for 5 minutes. The injector temperature was 250°C and the detector temperature was 230°C. The carrier gas is helium, and the flow rate of the carrier gas is 1ml/min. The results showed that the fatty acid composition of Scenedesmus CHX1 was dominated by short-chain fatty acids, of which C16-C18 accounted for 70% of the total fatty acid content, which was suitable for biodiesel production. See Table 2 for details.

Table 2 Fatty acid composition analysis of Scenedesmus CHX1

fatty acid type The content of total fatty acids (%) C12:0 0.13 C14:0 0.44 C16:0 28.3 C16:2 1.61 C18:0 0.86 C18:1 28.1 C18:2 8.91 C18:3 2.18 C20:1 0.62 C22:0 0.13 Unknown 28.72

Embodiment 4, Scenedesmus CHX1 grows in pig raising wastewater and removes nitrogen and phosphorus

Pig breeding wastewater was taken from the stable pond effluent of a pig breeding factory in Xiaoshan District, Hangzhou City. The air blowing method was used in the laboratory (aeration volume 1500 ml/min, aeration time 12 h/d, continuous aeration 7 days) to treat the breeding wastewater. Pretreatment reduces the content of ammonia nitrogen and reduces the growth inhibitory effect on Scenedesmus. The pretreated aquaculture wastewater was passed through a 0.45 μm filter membrane without sterilization. The water quality indicators are shown in Table 3.

Table 3 Water Quality Index of Pig Raising Wastewater

1. Cultivation of Scenedesmus CHX1

Pig wastewater needs to be pretreated before being used for microalgae cultivation to remove the growth inhibitory effect of high ammonia nitrogen concentration on microalgae. Inoculate the BG-11 culture solution containing Scenedesmus CHX1 (OD ₆₉₀ =1.24) at 20% (v/v) into 500ml Erlenmeyer flasks (each Erlenmeyer flask contains 250ml), light all day (light intensity 100μmol photo/ m ² .s) while blowing air containing 5% CO ₂ (the aeration rate is 100ml/min, continuous aeration), and the incubation time is 8 days at 27°C.

2. Determination of nitrogen and phosphorus removal rate

Take the wastewater culture solution for total nitrogen determination (GB11894-89, the determination of total nitrogen concentration in water quality is determined by alkaline potassium persulfate digestion ultraviolet spectrophotometry), and the determination of ammonia nitrogen (HJ535-2009, the determination of ammonia nitrogen concentration in water quality is determined by Nessler's reagent spectrophotometry) and total phosphorus determination (GB/T 11893-1989, determination of water quality total phosphorus concentration using ammonium molybdate scenery photometry), the results are shown in Table 4 (average of three repetitions).

Table 4 Nitrogen and phosphorus content in water before and after Scenedesmus CHX1 culture

Total nitrogen content before cultivation (mg/L) Total nitrogen content after cultivation (mg/L) Total nitrogen removal rate (%) 110.3 13.4 87.9 Ammonia nitrogen content before cultivation (mg/L) Ammonia nitrogen content after cultivation (mg/L) Ammonia nitrogen removal rate (%) 109.3 3.0 97.3 Total phosphorus content before cultivation (mg/L) Total phosphorus content after cultivation (mg/L) Total phosphorus removal rate (%) 8.53 0.58 93.2

3. Determination of biomass and oil content

Determination method is with the step of embodiment 2. The average biomass yield is 869 mg/L.d (range is 867.4~870.6 mg/L.d), the oil content is 13.6%, and the average oil yield is 118.2 mg/L.d (range is 118.0~118.4 mg/ L.d).

The results show that Scenedesmus CHX1 lives in pretreated aquaculture wastewater, has high biomass and oil yield, and has a strong removal effect on nitrogen and phosphorus. Microalgae with great potential in applications.

Claims (6)

1. Scenedesmus sp. is characterized in that: it is separated in the swine wastewater treatment pond, is named after CHX1, and its classification is named after Scenedesmus sp., and has been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, and its preservation number is CGMCC No. 6649. 2. An optimized medium for Scenedesmus as claimed in claim 1, characterized in that: the following solutes are adjusted to 1 liter: 1ml A ₅ +Co solution, 5g glucose, 3g NaNO ₃ , 0.04g K ₂ HPO ₄ 3H ₂ O, 0.075g MgSO ₄ , 7H ₂ O, 0.036g CaCl ₂ , 2H ₂ O, 0.006g Fe(NH ₄ ) ₃ C ₁₈ H ₁₀ O ₁₄ , 0.001g Na ₂ -EDTA, the balance is water; The A ₅ +Co solution is obtained by distilling the following solutes to 1 liter: 2.86 g H ₃ BO ₃ , 1.81 MnCl ₂ ·H ₂ O, 0.222 ZnSO ₄ ·7H ₂ O, 0.079 CuSO ₄ ·5H ₂ O , 0.39 Na ₂ MoO ₄ ·2H ₂ O, 0.049 Co(NO ₃ ) ₂ ·6H ₂ O, and the balance is water; the initial pH of the optimized medium is 8, and the initial concentration of the optimized medium after inoculation with Scenedesmus culture solution is OD ₆₉₀ =0.16. 3. An application of the Scenedesmus optimized medium as claimed in claim 2, characterized in that: the Scenedesmus optimized medium is used to cultivate Scenedesmus sp., CHX1, to obtain a seed culture solution. 4. The application according to claim 3, characterized in that: the dry weight biomass and the total oil yield of the seed culture solution are respectively: 809.6-818.6 mg/liter.day and 127.5-129.0 mg/liter. days; C16-C18 components account for 70% of the fatty acids that make up the total oils and fats, which are used to prepare biodiesel. 5. an application of Scenedesmus as claimed in claim 1 in the treatment of swine wastewater. 6. The application according to claim 5, characterized in that: the pig raising wastewater is treated to remove nitrogen and/or phosphorus.

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