Method for preparing biodiesel by co-culturing algae and bacteria to treat biogas slurry of pig farm

Abstract

The application discloses a method for preparing biodiesel by treating biogas slurry in a pig farm by algae and bacteria co-cultivation, which comprises the following steps: screening a single bacterial strain in the biogas slurry in the pig farm, matching the single bacterial strain with microalgae, configuring an acclimation liquid, acclimating the algae and bacteria in the biogas slurry in the pig farm, adjusting the component proportion of the biogas slurry in the pig farm, treating the biogas slurry in the pig farm by the algae and bacteria, and recovering the microalgae. The single bacterial strain in the biogas slurry in the pig farm is screened first, and then the single bacterial strain is matched with suitable microalgae. The algae and bacteria suitable for survival in the biogas slurry in the pig farm are accurately screened by configuring the acclimation liquid. After the element proportion of the biogas slurry in the pig farm is adjusted, the biogas slurry in the pig farm is fully decomposed and treated by using the principle of the algae and bacteria symbiosis. Finally, the microalgae containing organic matter are recovered, so that the biodiesel is prepared. The method is efficient and rapid, and solves the problem that the existing treatment of the biogas slurry in the pig farm is only harmless treatment and cannot realize element reuse.

Description

Technical Field

[0001] This invention relates to the treatment of biomass wastewater, and more particularly to a method for preparing biodiesel from pig farm biogas slurry through algae-bacteria co-cultivation. Background Technology

[0002] Pig farm biogas slurry is usually rich in nitrogen, phosphorus and organic carbon. Improper treatment of pig farm biogas slurry and discharge into the environment can lead to water pollution, eutrophication, algal blooms, reduced dissolved oxygen (DO), altered aquatic ecosystems, and severe odor problems.

[0003] Existing mature methods for treating pig farm biogas slurry include composting, wastewater stabilization ponds, constructed wetlands for dilution and separation, aquatic plant absorption, and microalgae treatment. Algal biochar is a green biosorbent that can be used for pollutant removal and soil improvement. Because microalgae have high protein, antioxidant, vitamin, and mineral content, they can be used as feed additives in livestock and aquaculture, and biodiesel can be obtained through proper element control. Therefore, using microalgae to treat pig farm biogas slurry to obtain algal products transforms the elements in the wastewater into usable components, rather than simply rendering the wastewater harmless.

[0004] However, because the biogas slurry from pig farms contains a large amount of alkaline substances, especially NH4+, + The main component is NH4+, and the pH of the biogas slurry can approach 10. Different microalgae and different environmental conditions affect the NH4+ content. 4+ Different algae have different tolerances. For most algae, when the pH is 9 to 10, the growth of algae will be severely inhibited. Algae cannot continue to grow in the biogas slurry of pig farms and cannot continuously convert and utilize the elements in the biogas slurry of pig farms. Summary of the Invention

[0005] To address the problem of low survival rates and hindered growth of microalgae in treating pig farm biogas slurry due to their poor tolerance to highly alkaline slurry, thus affecting elemental conversion in the slurry, this invention provides a method for preparing biodiesel through algae-bacteria co-cultivation of pig farm biogas slurry. The method includes several steps: screening single bacterial strains from the pig farm biogas slurry, compatibility and acclimatization of microalgae and single bacterial strains, adjustment of the pig farm biogas slurry, algae-bacteria co-cultivation of the pig farm biogas slurry, and recovery of microalgae. The specific operations are as follows:

[0006] 1) Screening for single bacterial strains in pig farm biogas slurry

[0007] The biogas slurry from the pig farm was gradually diluted with sterile water at a ratio of 1:10 to obtain 10. -4 ~10 -6Diluted biogas slurry was added to LB liquid culture medium with agar, and after heat sterilization, LB solid culture medium was prepared. Diluted biogas slurry was spread onto LB solid culture medium and incubated in an incubator at 37°C for 1-2 days. Single colonies of different morphologies were selected and transferred to new LB solid culture medium. They were isolated and purified by streak plating and single strains were screened.

[0008] 2) Synergistic combination of microalgae and pig farm biogas slurry bacteria

[0009] Dilute the microalgae culture medium with sterile water, spread the diluted solution on LB solid medium, and incubate for 4-6 days. Then, pick single microalgae colonies for streak plating and separate them. After 3-4 generations, pure algal colonies are obtained. Incubate the pure algal colonies to the logarithmic growth phase, collect the culture medium, centrifuge at 6000-8000 rpm for 10-20 min, collect the supernatant, and filter the supernatant through a 0.15-0.25 μm sterile filter membrane to obtain metabolite solutions secreted by different microalgae. Add each metabolite solution to the LB solid medium of each single strain screened in step 1), and incubate for 36-48 h. Select the combination of bacteria that successfully grows.

[0010] 3) Prepare acclimatization solution

[0011] The components and concentrations in the acclimatization solution are as follows: glucose 0.5–2 g / L, Na₂CO₃ 0.05–0.2 g / L, NaHCO₃ 0.05–0.15 g / L, Na₃PO₄ 0.15–0.25 g / L, urea 0.1–0.3 g / L, (NH₄)₂SO₄ 0.5–2 g / L, KNO₃ 0.1–0.4 g / L, and NaNO₂ 0.1–0.35 g / L.

[0012] 4) Co-cultivation and domestication of algae and bacteria in biogas slurry from pig farms

[0013] Microalgae and bacteria in the logarithmic growth phase were inoculated into an acclimatization solution to adapt them to the biogas slurry environment. After 4 days of cultivation, COD, TP, and NH4 in the acclimatization solution were measured. + The concentration of -N determines whether the compatible combination of microalgae and bacteria can be co-cultured in the acclimatization solution.

[0014] 5) Adjust the composition ratio of biogas slurry in pig farms

[0015] Sampling and testing of C, N, and P elements in pig farm biogas slurry; adding soluble nitrates and / or soluble phosphates to adjust C / N and N / P ratios.

[0016] 6) Algae and Bacteria Co-culture Treatment of Pig Farm Biogas Slurry

[0017] Add the logarithmic growth phase microalgae and bacteria to the pig farm biogas slurry after the component ratio is adjusted in step 4) according to the quantity ratio, and culture at room temperature for 4 to 6 days. During this period, take samples regularly to test the biogas slurry indicators. Stop the culture after the indicators are stable.

[0018] 7) Recycling microalgae

[0019] Filter the treatment solution from step 6) to recover the microalgae.

[0020] In step 1) of this invention, single strains of five bacteria, namely Staphylococcus sciuri, Kurthiagibsonii, Rhizobium sp. MN13, Staphylococcus saprophyticus, and Bacillus megaterium, were screened from the biogas slurry of pig farms. In step 2) four microalgae, namely C. vulgaris, C. pyrenoidos, Desmodesmus sp., and Desmodesmus sp. G41-M, were selected and combined with the above single strains respectively. The compatibility results showed that Staphylococcus sciuri and Kurthia gibsonii could grow in the supernatant of C. vulgaris culture medium; Kurthia gibsonii and Rhizobium sp. MN13 could grow in the supernatant of C. pyrenoidosa culture medium; Staphylococcus sciuri, Staphylococcus saprophyticus and Bacillus megaterium could grow in the supernatant of Desmodesmus sp. culture medium; Staphylococcus sciuri, Kurthia gibsonii and Rhizobium sp. MN13 could grow in the supernatant of Desmodesmus sp. G41-M culture medium.

[0021] Furthermore, the successfully combined algae and bacteria were subjected to acclimatization to pig farm biogas slurry in an acclimatization solution simulating pig farm biogas slurry. The acclimatization results showed that Desmodesmus sp. and Bacillus megaterium could be stably co-cultured in the acclimatization solution, as could C. vulgaris and Staphylococcus sciuri.

[0022] Finally, the elements of the biogas slurry in the pig farm were fine-tuned according to the composition of the domestication solution, so that the C / N ratio in the biogas slurry in the pig farm was 100:(15-80) and the N / P ratio was (15-40):1, which is suitable for the stable growth of the domesticated algae and bacteria combination.

[0023] This invention utilizes the synergistic effect of microalgae and bacteria to comprehensively treat pig farm biogas slurry. Through a rational combination of microalgae and bacteria, the bacteria can lower the pH value of the biogas slurry, creating a sustainable environment for the microalgae to survive. The metabolic products of the microalgae, in turn, provide nutrients for the bacteria. Therefore, the microalgae-bacteria co-culture method of this invention can rapidly and effectively treat pig farm biogas slurry. Controlling the ratio of microalgae to bacteria to (3-15):1 maximizes the transfer of elements from the biogas slurry to the microalgae.

[0024] This invention first screens single bacterial strains from pig farm biogas slurry, then selects suitable microalgae to combine with them. By preparing an acclimatization solution, it accurately screens algae-microbe combinations suitable for survival in pig farm biogas slurry. After adjusting the elemental ratio of the biogas slurry, it utilizes the principle of algae-microbe symbiosis to fully decompose the biogas slurry. Finally, it harvests microalgae containing organic matter to produce bio-petroleum. This invention is highly efficient and rapid, solving the problem that existing pig farm biogas slurry treatment methods only achieve harmlessness and cannot achieve elemental reuse. Attached Figure Description

[0025] Figure 1 The curves showing the COD changes of the Ds-Bm algae-bacteria combination and the Cv-Ss algae-bacteria combination of the present invention after co-cultivation in the acclimatization solution for 4 days are shown.

[0026] Figure 2 The curves showing the change in TP (tumor activity) after 4 days of co-cultivation in the acclimatization solution for the Ds-Bm and Cv-Ss algae combinations of this invention.

[0027] Figure 3 The Ds-Bm algae and bacteria combination and the Cv-Ss algae and bacteria combination of the present invention were co-cultured in the acclimatization solution for 4 days with NH4. + The curve of -N variation.

[0028] Figure 4 The curves showing the changes in COD of pig farm biogas slurry treated with different ratios of Ds-Bm algae and bacteria combinations according to the present invention are shown.

[0029] Figure 5 The curves showing the changes in total phosphorus (TP) in pig farm biogas slurry treated with different ratios of Ds-Bm algae and bacteria combinations according to the present invention are shown.

[0030] Figure 6 This is a bar chart showing the organic matter content in microalgae after treating pig farm biogas slurry with the pure culture system of Desmodesmus sp. and the 9:1 Ds-Bm co-culture system of this invention. Detailed Implementation

[0031] The present invention will be described below with reference to examples. These examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0032] The algal species and sources used in this invention are as follows: *Chlorella vulgaris* (FACHB-1072), *Chlorella pyrenoidosa* (FACHB-10), and *Desmodesmus* sp. (FACHB-2919) were purchased from the freshwater algae strain bank of the Wuhan Institute of Hydrobiology, Chinese Academy of Sciences, and subsequently isolated and purified by the Marine Microalgae Biology Laboratory for later use; *Desmodesmus* sp. G41-M is a mutant algal strain with high biomass and high lipid production, isolated from inland river samples in Xinjiang by the Marine Microalgae Biotechnology Laboratory. The biogas slurry was obtained from a pig farm in Yantai City, Shandong Province.

[0033] Microalgae were screened and purified. 1.6% agar (by weight) and 25 U / mL penicillin were added to BG11 liquid medium. The mixture was heated to 45°C, stirred to solidify, and then allowed to stand to solidify into BG11 solid medium. *Chlorella vulgaris*, *Chlorella pyrenoidosa*, and *Desmodesmus sp., Desmodesmus sp. G41-M* were diluted with sterile water. The diluted solutions were spread onto the solid medium and cultured for 6 days. Single algal colonies were then streaked onto plates for separation. After four generations, pure single algal colonies were obtained. The solid medium containing single algal colonies was stored at 4°C for later use. The formulation of BG11 liquid medium is shown in Table 1.

[0034] Table 1. Formulation of BG11 liquid culture medium

[0035]

[0036] The biogas slurry from the pig farm was gradually diluted with sterile water at a ratio of 1:10 to obtain 10. -4 ~10 -6 Diluted biogas slurry was added to LB liquid culture medium with 1.5% agar by weight. After heat sterilization, LB solid culture medium was prepared. The diluted biogas slurry was spread onto the LB solid culture medium and incubated at 37°C for 2 days. Single colonies of different morphologies were selected and transferred to new LB solid culture medium for isolation and purification using the streak plating method until single bacteria were screened. The formulation of LB liquid culture medium is shown in Table 2.

[0037] Table 2. Formulation of LB liquid culture medium

[0038] serial number reagents The amount of reagent added in 1L of culture medium 1 peptone 10g 2 Yeast extract 5g 3 NaCl 10g

[0039] Single algal colonies were cultured to the logarithmic growth phase, the culture medium was collected, centrifuged at 8000 rpm for 10 min, and the supernatant was collected. The supernatant was filtered through a 0.20 μm sterile filter membrane to obtain metabolite solutions secreted by different microalgae. Each metabolite solution was added to LB solid medium of each selected single strain. After culturing for 48 h, the combination of bacteria that successfully grew was selected. This microalgae-bacteria combination can be used to treat biogas slurry from pig farms. The synergistic compatibility results of microalgae and bacteria in this invention are as follows: Staphylococcus sciuri and Kurthia gibsonii can grow in the supernatant of C. vulgaris culture medium; Kurthia gibsonii and Rhizobium sp. MN13 can grow in the supernatant of C. pyrenoidosa culture medium; Staphylococcus sciuri, Staphylococcus saprophyticus and Bacillus megaterium can grow in the supernatant of Desmodesmus sp. culture medium; Staphylococcus sciuri, Kurthia gibsonii and Rhizobium sp. MN13 can grow in the supernatant of Desmodesmus sp. G41-M culture medium.

[0040] The C, N, and P element ratios in the biogas slurry from the pig farm to be treated were analyzed. An acclimatization solution was prepared with the following components and concentrations: glucose 1 g / L, Na₂CO₃ 0.1 g / L, NaHCO₃ 0.1 g / L, Na₃PO₄ 0.2 g / L, urea 0.2 g / L, (NH₄)₂SO₄ 1.5 g / L, KNO₃ 0.3 g / L, and NaNO₂ 0.25 g / L. Logarithmic growth phase microalgae (3 × 10⁻⁶) were then added. 5 cells / mL) and bacteria (1×10⁻⁶) 5 Microalgae (3 × 10⁻⁶ cells / mL) were co-inoculated into a conical flask containing acclimatized liquid, while microalgae (3 × 10⁻⁶ cells / mL) were inoculated separately. 5 As a control group, cells / mL were used, and the cells were cultured for about 4 days under the following conditions: temperature 25±2℃, light intensity 45μmol / mL. 2 The light / dark cycle was 14h / 10h, and the algae solution was shaken three times a day to ensure thorough mixing. Three replicates were performed, and samples were collected daily. The following parameters were monitored: COD (chemical oxygen demand), TP (total phosphorus), and NH4+. + -N concentration.

[0041] Starting from the day of inoculation, collect 5 mL of sample from each conical flask daily, centrifuge at 10000g for 10 min, collect the supernatant and dilute it appropriately for COD and NH4 concentration analysis. + The concentrations of -N and TP were determined. The detection methods for each pollutant were based on the "Methods for Monitoring and Analysis of Water and Wastewater". The pollutant removal rate (Ri, %) was calculated using the following formula: Ri = (Si0 - Si,t) / Si0, where Ri (%) represents the removal rate of substrate i (COD, TP, NH4+). + -N), Si,t, and Si0 (mg / L) are the concentrations of i at time t and the initial time, respectively.

[0042] COD concentration detection

[0043] The concentration of COD in samples was determined using a rapid digestion spectrophotometric method. The principle is that the COD value in a sample is directly proportional to the increase in absorbance (OD600) of trivalent chromium produced by the reduction of K2CrO4. This method has a detection range of 15-1000 mg / L for COD. The main detection methods are as follows:

[0044] (1) Reagent preparation: Prepare H2SO4 solution (10%), Ag2SO4-H2SO4 solution (10 g / L), HgSO4 solution (0.24 g / mL), K2CrO4 standard solution (0.5 mol / L), pre-filled mixed reagent (0.5 mL K2CrO4 standard solution + 0.25 mL HgSO4 solution + 3 mL Ag2SO4-H2SO4 solution), C8H5KO4 standard stock solution (COD value of 5000 mg / L), and C8H5KO4 standard series working solutions (COD values ​​of 100, 200, 400, 600, 800 and 1000 mg / L respectively).

[0045] (2) Standard curve: Preheat the COD digester to 165±2℃; take several pre-filled mixed reagents, shake well, and open the cap; slowly add 1.5mL of C8H5KO4 standard series stock solution along the tube wall, tighten the cap and invert several times to mix well; place the digestion tube into the COD digester, wait for the temperature to rise back to 165±2℃, start timing, and heat for 15min; after digestion, wait for cooling to 60℃, invert several times to mix well; wait for cooling to room temperature, use water as the reference solution, and detect OD600. The COD value of the COD standard series solution corresponds to the difference between its OD600 and the OD600 of the blank test, and plot the standard curve.

[0046] (3) Sample detection: Take 1.5 mL of sample diluted with an appropriate concentration and test OD600 according to the above steps.

[0047] (4) Blank test: Take 1.5 mL of water instead of the sample and test OD600 according to the above steps. The blank test should be performed at the same time as the sample test.

[0048] TP concentration detection

[0049] The concentration of phosphorus (TP) in the sample was determined using ammonium molybdate spectrophotometry. The specific principle is that phosphorus (P) in the sample is oxidized to PO4 by K2S2O8. 2- It reacts with ammonium molybdate to form phosphomolybdic acid, which is then reduced by ascorbic acid to a blue complex. The absorbance (OD700) of this complex is directly proportional to the TP content. The detection range for TP using this method is 0.01-0.6 mg / L. The main detection methods are as follows:

[0050] (1) Reagent preparation: Prepare sulfuric acid solution (50%), K2S2O8 solution (50g / L), ascorbic acid solution (100g / L), molybdate solution, phosphorus standard stock solution (50μg / mL), and phosphorus standard working solution (2μg / mL) respectively.

[0051] (2) Standard Curve: Measure 0, 0.25, 0.5, 1.5, 2.5, 5, and 7.5 mL of phosphorus standard working solution into 25 mL stoppered ground glass tubes, respectively, and dilute with water to 12.5 mL; then add 2 mL of K₂S₂O₈ solution, and tighten the cap; place in an autoclave at 121℃ for 30 min; after cooling to room temperature, dilute with water to 25 mL; then add 0.5 mL of ascorbic acid solution and mix thoroughly; after standing for 30 s, add 1 mL of molybdate solution and mix thoroughly; after standing for 15 min, use water as a reference solution and measure OD₇₀. Plot the standard curve by subtracting the absorbance of the blank test and the corresponding TP content.

[0052] (3) Sample detection: Take 12.5 mL of sample diluted with an appropriate concentration and test OD700 according to the above steps.

[0053] (4) Blank test: Take 12.5 mL of water instead of the sample and test OD700 according to the above steps. The blank test should be performed at the same time as the sample test.

[0054] NH4 + -N concentration detection

[0055] NH4 in the sample was determined using Nessler's reagent spectrophotometry. + The concentration of -N. The specific principle is that the NH4+ in the sample... + -N reacts with Nessler's reagent to form a pale reddish-brown complex, the absorbance (OD420) of which is similar to that of NH4+. + The -N content is directly proportional to the method for NH4 + The detection range for -N is 0.1-2 mg / L.

[0056] The main detection methods are as follows:

[0057] (1) Reagent preparation: Prepare potassium sodium tartrate solution (500g / L) and NH4+ solution respectively. + -N standard stock solution (1000 μg / mL), NH4 + -N standard working solution (10 μg / mL).

[0058] (2) Standard curve: 0, 0.25, 0.5, 1, 2, 3, 4 and 5 mL of NH4+ were measured respectively. + Dilute the -N standard working solution to 25 mL in a stoppered ground glass test tube with water to a final volume of 25 mL; then add 0.5 mL of potassium sodium tartrate solution and mix thoroughly; next, add 0.5 mL of Nessler's reagent and mix thoroughly; let stand for 10 min, and using water as a reference solution, measure OD420. Plot the absorbance after blank correction as the ordinate, and then plot the corresponding NH4+. + Plot a standard curve with -N content on the x-axis.

[0059] (3) Sample detection: Take 25 mL of sample diluted with an appropriate concentration and test OD420 according to the above steps.

[0060] (4) Blank test: Take 25 mL of water instead of the sample and test OD420 according to the above steps. The blank test should be performed at the same time as the sample test.

[0061] The tests revealed that the combination of *Desmodesmus sp.* and *Bacillus megaterium* (Ds-Bm) and the combination of *C. vulgaris* and *Staphylococcus sciuri* (Cv-Ss) could coexist in the acclimatization solution. Both combinations had a positive effect on COD, TP, and NH4 in the acclimatization solution. + The removal of -N is as follows: Figures 1-3 As shown in the figure, the co-culture of algae and bacteria resulted in higher COD and TP consumption in the acclimation solution compared to microalgae or bacteria alone. Table 3 shows the removal rates of various pollutants in the acclimation solution by the two algae-bacteria combination. The data in Table 3 indicate that the Ds-Bm algae-bacteria combination exhibited the highest COD and TP consumption.

[0062] Table 3. Removal rates of various pollutants in the acclimation solution by the combination of two algae and bacteria.

[0063] training system COD removal rate TP removal rate <![CDATA[NH4 + -N removal rate]]> Ds 31.88％±7.67％ 24.08％±0.06％ 8.16％±1.75％ Bm 27.98％±6.27％ 14.74％±4.42％ —— Ds-Bm 77.12％±2.14％ 34.40％±1.90％ —— Cv 40.07％±3.91％ 24.57％±0.75％ 14.34％±0.00％ Ss —— —— —— Cv-Ss 38.94％±0.53％ 29.04％±1.77％ 22.01％±1.02％

[0064] Taking the Ds-Bm algae-bacterial combination as an example, different ratios of algae and bacteria were used to treat pig farm biogas slurry. Samples of the pig farm biogas slurry were taken to determine its C, N, and P element content. The C / N ratio was adjusted to 5:2 and the N / P ratio to 15:1 by adding ammonium nitrate and ammonium dihydrogen phosphate. Logarithmic growth phase microalgae *Desmodesmus* sp. (Ds) and bacteria *Bacillus megaterium* (Bm) were used to form algae-bacterial co-culture systems at ratios of 3:1, 6:1, 9:1, 12:1, and 15:1, respectively. These systems were added to the pig farm biogas slurry after element adjustment and cultured at room temperature. From the sixth day onwards, samples were taken to measure the concentrations of COD and TP. Figure 4 and Figure 5 As shown.

[0065] Figure 4 In the study, the COD removal rates of the various co-culture systems Ds:Bm (3:1, 6:1, 9:1, 12:1, and 15:1) reached 58%, 75%, 81%, 72%, and 76% respectively on day 6, with the Ds:Bm (9:1) co-culture system showing the best COD removal effect. As the microalgae to bacteria inoculation ratio increased from 3:1 to 6:1, the COD removal rate of the co-culture system significantly improved. When the inoculation ratio was further increased to 15:1, the COD removal rate did not increase further, but it still remained at a high level, 95-110% higher than the Desmodesmus sp. pure culture system. After treatment with the Ds:Bm (3:1, 6:1, 9:1, 12:1, and 15:1) co-culture systems, the COD concentration in the biogas slurry met the requirements of the "Emission Standard of Pollutants for Livestock and Poultry Breeding Industry GB 18596-2001".

[0066] Figure 5 In the study, the removal rate of total phosphorus (TP) and the chlorophyll content of microalgae showed similar trends in different culture systems. The TP concentration first decreased and then increased with the extension of culture time, reaching its lowest value on day 6. The TP removal rate significantly increased with the increase of the proportion of microalgae in the co-culture system. When the microalgae to bacteria inoculation ratio was 12:1, the TP removal rate reached its highest level (43%), which was 2.68 times that of the pure culture system of *Desmodesmus* sp. The amount of TP removed also increased significantly with the increase of the proportion of microalgae in the co-culture system. The increase in TP concentration in the later stage of the Ds-Bm co-culture system may be related to microbial disintegration. Under normal circumstances, the intracellular substances of microorganisms will be consumed by themselves under starvation, and cell decomposition will release phosphorus back into the culture medium, resulting in a decrease in the system's ability to treat TP.

[0067] Therefore, considering the overall factors of time, input, and return, a Ds-Bm ratio of 9:1 and a processing time of 6 days are most reasonable.

[0068] A Ds-Bm ratio of 9:1 was used to treat pig farm biogas slurry after element adjustment at the production level. The microalgae *Desmodesmus* sp. was recovered, and the organic matter composition was analyzed. Figure 6 As shown. From Figure 6 It can be seen that, compared with pure *Desmodesmus* sp., the algae obtained by co-culturing *Ds-Bm* algae and bacteria showed significantly increased C16:0 and C18:0 contents. The main fatty acid components in biodiesel are: hexadecanoic acid (C16:0), octadecanoic acid (C18:0), oleic acid (C18:1n9c), linoleic acid (C18:2n6c), and linolenic acid (C18:3n3). The organic matter in the microalgae obtained by co-culturing *Ds-Bm* algae and bacteria meets the requirements for carbon chain length (C15-C22) for biodiesel.

[0069] In the pure culture system of *Desmodesmus* sp., the contents of the aforementioned fatty acids in the microalgae were 42%, 9%, 7%, and 29%, respectively. Co-culturing *Desmodesmus* sp. with *B. megaterium* had a certain impact on the fatty acid composition of the microalgae, with the contents of C16:0 and C18:0 increasing by 12% and 12%, respectively, while the contents of C18:2n6c and C18:3n6 decreased by 1% and 15%, respectively. In the pure culture system of *Desmodesmus* sp., the total content of fatty acid components suitable for biodiesel production (C16:0, C18:0, and C18:2n6c) in the microalgal cells was 58%. After co-culturing with *B. megaterium*, the content of these fatty acids in the microalgal cells increased to 81%, a 40% increase.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing biodiesel from pig farm biogas slurry through algae-bacteria co-cultivation, characterized in that, Includes the following steps: 1) Screening for single bacterial strains in pig farm biogas slurry The biogas slurry from the pig farm was gradually diluted with sterile water at a ratio of 1:10 to obtain 10. -4 ~10 -6 Diluted biogas slurry was added to LB liquid culture medium with agar, and after heat sterilization, LB solid culture medium was prepared. Diluted biogas slurry was spread onto LB solid culture medium and incubated in an incubator at 37°C for 1-2 days. Single colonies of different morphologies were selected and transferred to new LB solid culture medium. They were isolated and purified by streak plating and single strains were screened. The LB liquid culture medium is prepared as follows: peptone: yeast extract: NaCl = 10:5:10; 2) Synergistic combination of microalgae and pig farm biogas slurry bacteria Dilute the microalgae culture medium with sterile water, spread the diluted solution on LB solid medium, and incubate for 4-6 days. Then, pick single microalgae colonies for streak plating and separate them. After 3-4 generations, pure algal colonies are obtained. Incubate the pure algal colonies to the logarithmic growth phase, collect the culture medium, centrifuge at 6000-8000 rpm for 10-20 min, collect the supernatant, and filter the supernatant through a 0.15-0.25 μm sterile filter membrane to obtain metabolite solutions secreted by different microalgae. Add each metabolite solution to the LB solid medium of each single strain screened in step 1), and incubate for 36-48 h. Select the combination of bacteria that successfully grows. 3) Prepare acclimatization solution The components and concentrations in the acclimatization solution are as follows: glucose 0.5~2g / L, Na2CO3 0.05~0.2g / L, NaHCO3 0.05~0.15g / L, Na3PO4 0.15~0.25g / L, urea 0.1~0.3g / L, (NH4)2SO4 0.5~2g / L, KNO3 0.1~0.4g / L, and NaNO2 0.1~0.35g / L. 4) Co-cultivation and domestication of algae and bacteria in biogas slurry from pig farms Microalgae and bacteria in the logarithmic growth phase were inoculated into an acclimatization solution to adapt them to the biogas slurry environment. After 4 days of cultivation, COD, TP, and NH4 in the acclimatization solution were measured. + The concentration of -N was used to determine whether the compatible microalgae and bacteria combination could be co-cultured in the acclimation solution; Desmodesmus sp. and Bacillus megaterium were stably co-cultured in the acclimation solution; 5) Adjust the composition ratio of biogas slurry in the pig farm Sampling and testing of C, N, and P elements in pig farm biogas slurry; adding soluble nitrates and / or soluble phosphates to adjust C / N and N / P ratios. 6) Algae and Bacteria Co-culture Treatment of Pig Farm Biogas Slurry Add the logarithmic growth phase microalgae and bacteria to the pig farm biogas slurry after the component ratio is adjusted in step 4) according to the quantity ratio, and culture at room temperature for 4-6 days. During this period, take samples regularly to test the biogas slurry indicators. Stop the culture after the indicators are stable. 7) Recycling microalgae Filter the treatment solution from step 6) to recover the microalgae.

2. The method according to claim 1, characterized in that, In step 5), adjust C / N to 100:(15~80) and N / P to (15~40):

1.

3. The method according to claim 1, characterized in that, In step 6), the ratio of microalgae to bacteria is (3~15):1.

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