Method for treating biogas slurry through phycomycete co-culture and application of method

Abstract

The invention discloses a method for treating biogas slurry through phycomycete co-culture and application of the method. The method comprises the following steps: mixing Sautmophyta and Chinese grease yeast, adding the mixture into pig farm biogas slurry to obtain a treatment system, and culturing the Sautmophyta and the Chinese grease yeast to treat the biogas slurry. Through screening, it is found that the Chinese grease yeast can improve the biogas slurry tolerance of the Sautmophyta, the Chinese grease yeast and the Sautmophyta are combined to treat the biogas slurry, and the biomass and the pollutant removal rate are both higher than those of a contrast treated by single Sautmophyta. Meanwhile, the performance of phycomycetes in a rotating membrane reactor system is superior to that of a bubbling type columnar culture device, the biomass production and pollutant removal capacities of the phycomycetes are higher than that of the bubbling type columnar culture device, the power demand is reduced, and the harvesting cost of algae cells is saved.

Description

Technical Field

[0001] This invention belongs to the field of biological treatment of pollutants, and specifically relates to a method for treating biogas slurry by co-culturing algae and bacteria, and its application. Background Technology

[0002] Pig farming is one of the leading industries in China's animal husbandry sector. In 2017, the output value of the pig industry was nearly 1.3 trillion yuan, accounting for 56.6% of the total output value of livestock and poultry in China (National Bureau of Statistics of China, 2017). Most pig farms use anaerobic digestion technology to treat biogas slurry to control organic matter and pathogens, while generating a large amount of biogas for energy recovery (Garcia et al., Photocatalytic degradation of swine wastewater on aqueous TiO2 supplements: optimization and modeling via Box-Behnken design, Heliyon, 2020, 6, e03293). However, biogas slurry still contains high concentrations of organic and inorganic pollutants, and the content of nutrients such as nitrogen and phosphorus remains high (Hussain et al. Microalgae anecofriendly and sustainable wastewater treatment option: Biomass application in biofuel and bio-fertilizer production. A review. Renewable and Sustainable Energy Reviews, 2021, 137, 110603). The ammonia nitrogen concentration is far higher than the emission limits in the "Emission Standard of Pollutants for Livestock and Poultry Breeding Industry" (GB 18596-2001), requiring further treatment before recycling or discharging into natural water bodies. Therefore, exploring the best way to treat biogas slurry pollution is of great significance to both environmental protection and economic development.

[0003] Numerous studies both domestically and internationally have demonstrated that microalgae can effectively treat nitrogen, phosphorus, and organic matter in wastewater. Compared to traditional processes, microalgae significantly reduce carbon emissions through CO2 fixation via photosynthesis (autotrophic under light) and heterotrophic metabolism (utilizing organic carbon in the absence of light). They directly absorb nitrogen from the wastewater, avoiding the formation of nitrogen oxides, and produce extremely low oxygen levels, further suppressing greenhouse gas emissions. Furthermore, microalgae facilitates resource utilization after wastewater treatment (Hussain et al. Microalgae anecofriendly and sustainable wastewater treatment option: Biomass application in biofuel and bio-fertilizer production. A review. Renewable and Sustainable Energy Reviews, 2021, 137, 110603). However, microalgae technology for treating pig farm wastewater still suffers from drawbacks such as intolerance to high concentrations of wastewater, low COD removal rates, and high costs. Summary of the Invention

[0004] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for treating biogas slurry through co-culture of algae and bacteria.

[0005] Another object of the present invention is to provide the application of the above-mentioned method for treating biogas slurry by co-culturing algae and bacteria.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for treating biogas slurry by co-culturing algae and bacteria includes the following steps: mixing Algae ovalis and Yeast chinensis, adding them to biogas slurry from a pig farm to obtain a treatment system, and culturing Algae ovalis and Yeast chinensis to treat the biogas slurry.

[0008] The algae mentioned is Coelastrum sp. GX03 (hereinafter referred to as GX03), and its accession number is CCTCC NO:M20241314.

[0009] The mixed forms of the algae include solid and liquid forms; preferably, they are mixed in the form of algae liquid.

[0010] The aforementioned *Alternaria solani* is preferably prepared by the following steps: inoculating *Alternaria solani* into an algal culture medium and culturing it to the logarithmic growth phase to obtain *Alternaria solani* algal solution.

[0011] The preferred algal culture medium is BG11 liquid culture medium.

[0012] The preferred cultivation conditions are a temperature of 28.0±0.5℃, a light intensity of 10000 lux, and a day-night ratio of 12h:12h.

[0013] The culture time is preferably 5 to 7 days; more preferably 6 days.

[0014] The preferred Chinese oily yeast is Lipomyces chinensis GDMCC2.237.

[0015] The mixed forms of the Chinese oil yeast include solid and liquid forms; preferably, it is mixed in the form of Chinese oil yeast liquid.

[0016] The Chinese oil yeast is preferably prepared by the following steps: inoculating the Chinese oil yeast into a fungal culture medium and culturing it to the logarithmic growth phase to obtain the Chinese oil yeast culture liquid.

[0017] The fungal culture medium is preferably PDB liquid culture medium.

[0018] The preferred culture conditions are 25–30°C and 90–110 rpm in the dark; more preferably, the culture is carried out at 28°C and 100 rpm.

[0019] The culture time is preferably 2 to 3 days; more preferably 2 days.

[0020] The Chinese oil yeast culture and the Algae ovalis culture are mixed at a volume ratio of 2 to 8:1; more preferably at a volume ratio of 4:1.

[0021] The culture treatment includes at least one of conventional culture treatment, culture treatment using a membrane reactor, and culture treatment using a column reactor; preferably, culture treatment using a membrane reactor.

[0022] The aforementioned conventional culture treatment refers to culture treatment using conventional containers, such as bottles, basins, and jars.

[0023] The preferred conditions for the culture treatment are a temperature of 28.0±0.5℃, a light intensity of 10000 lux, and a day-night ratio of 12h:12h.

[0024] The culture treatment time is 6 days to 16 days, preferably 8 to 10 days.

[0025] The above-mentioned method of co-culturing algae and bacteria to treat biogas slurry is applied in environmental protection for the treatment of livestock wastewater, especially biogas slurry, particularly biogas slurry from pig farms.

[0026] The present invention has the following advantages and effects compared with the existing technology:

[0027] This invention provides a method for treating biogas slurry pollutants from pig farms using *Aeromonas chinensis* in combination with *Saccharomyces cerevisiae*. *Saccharomyces cerevisiae* enhances the algae's tolerance to biogas slurry, resulting in higher biomass growth and pollutant removal rates compared to the control group treated with *Aeromonas chinensis* alone. Furthermore, the algae-bacterial combination in a rotating membrane reactor system outperforms a bubble column culture system, exhibiting higher biomass production and pollutant removal capabilities. This method also reduces power consumption and lowers algal cell harvesting costs. Attached Figure Description

[0028] Figure 1 The graph shows the effects of co-treatment of different bacterial species with *Algae ovalis* on biogas slurry; where A represents the algal growth rate OD. 680 B is Chla, C is Chlb, D is Cars, E is Fv / Fm, and F is Pi Abs.

[0029] Figure 2 Figure 1 shows the effect of different inoculation ratios of *Saccharomyces cerevisiae* and *Alternaria solani* on biogas slurry; where A represents the algal growth OD. 680 B stands for Chla, C for Chlb, D for Cars, E for chlorophyll fluorescence parameter Fv / Fm, and F for Pi Abs.

[0030] Figure 3 Figure 1 shows the effect of co-treatment of bacteria and algae on biogas slurry in different reactors; where A is Chla, B is Chlb, C is Cars, D is the chlorophyll fluorescence parameter Fv / Fm, and E is Pi Abs.

[0031] Figure 4 The graph shows the effect of bacteria and algae treating biogas slurry in different reactors; where A represents pH and B represents NH4. + -N, C is TP, D is COD.

[0032] Figure 5 This is a schematic diagram of membrane reactor cultivation.

[0033] Figure 6 Photograph of the culture in the membrane reactor. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0035] The composition of BG11 medium is shown in the table below:

[0036] Table 1. Components of BG11 culture medium

[0037]

[0038] The composition of PDB liquid culture medium is as follows: 5 g / L potato extract powder, 20 g / L glucose, pH 6.0 ± 0.2, and the remainder is water.

[0039] Pig farm biogas slurry stock solution: NH4 + -N 434mg / L, TP 56.2mg / L, COD 1374mg / L, pH=9.5.

[0040] Example 1: Construction of an algae-bacteria system

[0041] (1) Algal strain activation culture

[0042] The algae *Coelastrum* sp. GX03 was isolated and purified by our project group and deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M20241314 and deposit date of June 20, 2024. It has been published in the national invention patent application No. 202410935512.0 entitled "A microalga capable of tolerating high concentrations of carbon dioxide and its application." The algal strain was deposited on BG11 solid agar plates. A small amount was taken from the plate and added to a 50 mL Erlenmeyer flask containing 20 mL of sterile BG11 medium. The culture was activated by incubation at 28°C, 10000 lx light intensity, and a 12 h:12 h light-dark cycle.

[0043] (2) Activation and culture of microbial strains

[0044] The bacterial strains used were *Aspergillus oryzae* GDMCC 3.471 (AO), *Bacillus velezensis* GDMCC 1.135 (BV), *Rhizobium rhizogenes* GDMCC 1.141 (RR), and *Lipomyces chinensis* GDMCC 2.237 (LC), all purchased from the Guangdong Microbial Culture Collection Center. Four strains were inoculated into 100 mL of NB (BV), YMB (RR), and PDB (AO, LC) liquid media, respectively, and cultured on a shaker in the dark for approximately two days (25℃, 110 rpm). Culture medium components: NB (NaCl 5g / L, peptone 10g / L, beef extract 3g / L), YMB (KH2PO4 0.5g / L, MgSO4·7H2O 0.2g / L, NaCl 0.1g / L, yeast extract 1g / L, mannitol 10g / L) and PDB (potato 200g / L, glucose 20g / L); laboratory purified water was used as the solvent.

[0045] (3) Construction and screening of bacterial and algal cells

[0046] Wastewater from pig farms was mixed with pure water at a 1:1 ratio and then sterilized to obtain treated diluted wastewater. GX03 algae were added to the treated diluted wastewater as a control, and the initial biomass was controlled as follows: the algal cell density in the culture medium after inoculation was approximately 2.6 × 10⁻⁶. 5 The control group consisted of cells / mL. The treatment group consisted of GX03 algal culture and four different bacterial cultures mixed at a 1:1 volume ratio, centrifuged at 25°C and 8000 rpm, and the resulting algal and bacterial cells were inoculated into diluted treated pig farm wastewater. The initial algal cell density in the culture medium after inoculation was approximately 2.6 × 10⁻⁶ cells / mL. 5 The cell density of the four bacterial strains was approximately 1.1 × 10⁻⁶ cells / mL. 5 The algae were cultured at a concentration of [number] cells / mL under the same conditions as the control group. The culture and activation conditions were the same, with the algae shaken three times daily. On day 6 of culture, samples were taken to measure the OD (exponential growth rate) of the algal solution. 680 Values, photosynthetic pigments, and chlorophyll fluorescence.

[0047] OD 680 Measurement: 4 mL of algal solution was taken at 0, 2, 4, 6 and 8 days after inoculation and the absorbance at 680 nm was measured.

[0048] Determination of photosynthetic pigment content: Take 6 mL of algal sample, centrifuge at 5000 rpm for 10 minutes, remove the supernatant, add 6 mL of 80% acetone solution, and then refrigerate the mixture at 4°C overnight. Finally, centrifuge again at 5000 rpm for 10 minutes, and measure the absorbance of the supernatant at 663 nm, 646 nm, and 470 nm using a spectrophotometer. Calculate the concentrations (mg / L) of chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids (Cars) in the algal solution according to the formulas shown below:

[0049] C Chla =12.21×OD 663 -2.81×OD 646

[0050] C Chlb =20.13×OD 646 -5.03×OD 663

[0051]

[0052] Note: OD 663 OD 646 OD 470 These are the absorbances of the filtrate at wavelengths of 663 nm, 646 nm, and 470 nm, respectively.

[0053] Chlorophyll fluorescence value determination: Microalgal chlorophyll fluorescence was measured using a handheld chlorophyll fluorometer (AquaPen AP-P100). The sample algal solution was diluted with deionized water, and a small amount of algal membrane was scraped from the membrane reactor and diluted to determine its OD value. 680 The value was 0.24. After dark treatment for 20 minutes, its OJIP value was measured directly in the dark. The data was exported and analyzed using FluorPen software. The maximum photosynthetic efficiency Fv / Fm and photochemical performance index Pi_abs of PSⅡ were recorded.

[0054] Experimental results are as follows Figure 1 As shown, the concentrations of Chla and Cars in the co-treatment with *Saccharomyces cerevisiae* and GX03 were significantly higher than those in the control group and other treatment groups. Combined with Fv / Fm and Pi-Abs data, it was determined that the co-growth promotion effect of *Saccharomyces cerevisiae* and *GX03* was optimal. Therefore, subsequent experiments selected *Saccharomyces cerevisiae* for co-culture experiments to explore the optimal growth conditions and algae-saccharomyces mixing ratio.

[0055] Example 2: Optimizing the inoculation ratio of bacteria and algae for treating biogas slurry

[0056] The algae and bacteria activation and inoculation methods were the same as in Example 1, with a control CK (algae density 2.6 × 10⁻⁶) treated with a single algae. 5 The inoculation was conducted using four different bacterial-algae inoculation ratios (1:1, 2:1, 4:1, and 8:1, volume ratios). The initial algal cell density in the biogas slurry after inoculation was approximately 2.6 × 10⁻⁶ cells / mL. 5 Cells / mL, the cell density of *Saccharomyces cerevisiae* in the four different bacterial-algae ratio treatments was approximately 1.12 × 10⁻⁶ cells / mL. 5 cells / mL, 2.24 × 10 5 4.48 × 10⁻⁶ cells / mL 5 8.96 × 10⁻⁶ cells / mL 5 Cells / mL. Culture conditions were the same as in Example 1. The effects of different inoculum ratios on biogas slurry OD, pigment, and chlorophyll fluorescence parameters were investigated.

[0057] When the bacteria-to-algae ratio was 4:1, after 8 days of culture, the concentrations of Chla, Chlb, and Cars were significantly higher than those of the control group and other treatment groups, increasing by 103%, 106%, and 45% respectively compared to the control group. The Fv / Fm value increased by approximately 76% compared to the control group, and the Pi-Abs value increased by approximately 976% compared to the control group. In summary, the growth and physiological indicators of the experimental group with a bacteria-to-algae ratio of 4:1 were significantly higher than those of other treatment groups. Therefore, a bacteria-to-algae ratio of 4:1 was selected for subsequent experiments.

[0058] Example 3: Comparison of the effects of different reactors for co-treating biogas slurry with bacteria and algae

[0059] The scale-up experiment of biogas slurry treatment based on bacterial-algae co-culture was conducted in a 10L rotating membrane reactor and a bubble column reactor (model: PZ10L, Shanghai Binzhi Acrylic Glass Co., Ltd.). The rotating membrane reactor was a custom-made incubator from Dongguan Jieguan Conveying Equipment Co., Ltd., consisting of a membrane (1.4mm thick cotton canvas) and a motor to drive the membrane movement. The membrane moves in a ladder-like motion driven by the motor. A schematic diagram of the reactor is shown below. Figure 5 See the experimental diagram. Figure 6 The culture conditions were: temperature 28.0±0.5℃, light intensity 10000 lux, and day-night ratio of 12h:12h.

[0060] The main parameters of the membrane reactor include: motor power 25w, brand: Taisong; membrane size: 800mm×200mm, rotation speed: 4cm / s.

[0061] The membrane reactor is operated as follows: the membrane is driven by a motor on the reactor and rotates clockwise at a speed of 4 cm / s. The bottom 1 / 3 of the membrane is in contact with the algal solution (control group) or the bacterial-algae mixture (experimental group).

[0062] The operating parameters of the bubbling column reactor are as follows: The column reactor is a cylindrical hard acrylic container with an outer diameter of 19cm and a height of 40cm; there is an air outlet at the bottom connected to an air pump, the air pump brand is Haili, the power is 25W, and the bubbling rate is 45L / min.

[0063] When the bacteria-algae inoculation was performed at the optimal ratio of 4:1, the algae density in the biogas slurry was 2.6 × 10⁻⁶. 5 The bacterial density is 4.48 × 10⁶ cells / mL. 5 Cells / mL. The bacterial and algal growth, the effect on the removal of pollutants from the biogas slurry, and the biomass accumulation capacity are respectively shown in [the table below]. Figure 3 , Figure 4 Tables 1 and 2.

[0064] (1) Growth of bacteria and algae in different reactors

[0065] Figure 3 This indicates that microalgae grew well in all culture media, with the rate of increase in Chla content consistently higher in the membrane reactor than in the column reactor. Under algal-bacterial treatment, the rate of increase in Chla content was higher in the microalgae than in the single-algae treatment. In both reactors, the trends of Fv / Fm and Pi-Abs values ​​were essentially the same, showing a steady upward trend. The values ​​in the membrane reactor were stable and consistently higher than those in the column reactor, while the column reactor exhibited an initial decrease followed by an increase.

[0066] (2) Pollutant removal from pig farm biogas slurry in different reactors

[0067] Changes in pollutants in biogas slurry treated by different reactors are as follows: Figure 4 As shown. The pH under algae treatment fluctuated upwards, with no significant difference. On the last day, the pH in the membrane reactor was 9.43, and the pH in the column reactor was 9.93. The pH in the membrane reactor under algae treatment fluctuated downwards, but the pH in the column reactor fluctuated upwards with significant differences. NH4 under different treatments + The decreasing trends of -N concentration were basically the same in both reactors, and the effects of NH4+ in the biogas slurry were similar in both types of reactors. + -N removal was significantly effective, with noticeable differences between the two methods, achieving removal rates of 99.3% and 97.1%, respectively. Both reactors showed significant TP removal effects from the biogas slurry, with noticeable differences between them, achieving removal rates of 95.6% and 83.6%, respectively. After 10 days of treatment, the COD concentrations in the membrane reactor and column reactor were 134 mg / L and 329 mg / L, respectively, with removal rates of 80.4% and 52.2%, showing a significant difference. Under algal cell treatment, after 10 days, the COD concentrations in the membrane reactor and column reactor were 119 mg / L and 239 mg / L, respectively, with removal rates of 82.7% and 65.2%, showing a significant difference.

[0068] In summary, the removal efficiency of GX03 and its algal cells from biogas slurry in both reactors demonstrates that the membrane reactor exhibits significantly superior performance compared to the column reactor.

[0069] (3) Assessment of biomass accumulation capacity

[0070] The biomass accumulation capacity in different reactors is shown in Tables 1 and 2. As can be seen from the tables, the total biomass accumulation in the membrane reactor with co-treatment of bacteria and algae was significantly higher than that in other treatments, reaching 69.9 g / m³. 2 The biomass in the membrane reactor was nearly twice that of algae-only treatment, and the biomass accumulation in the column reactor co-treatment of algae and bacteria was also significantly higher than that of algae-only treatment.

[0071] Table 1 Biomass accumulation in biogas slurry under membrane reactor treatment.

[0072]

[0073] Note: * in the same column indicates significant differences between treatments (P<0.05), the same applies below.

[0074] Table 2 Biomass accumulation in biogas slurry under column reactor treatment.

[0075]

[0076] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for treating biogas slurry by co-culturing algae and bacteria, characterized in that The method comprises the following steps: mixing Coelastrum sp. and Chinese oil yeast, adding the mixture into pig farm biogas slurry to obtain a treatment system, and culturing Coelastrum sp. and Chinese oil yeast to treat the biogas slurry.

2. The method according to claim 1, wherein the method is characterized in that: the Coelastrum sp. is Coelastrum sp. GX03, and the preservation number is CCTCC NO: M20241314； the Chinese oil yeast is Chinese oil yeast GDMCC 2.

237.

3. The method according to claim 1, wherein the method is characterized in that: the Coelastrum sp. is prepared by the following steps: inoculating Coelastrum sp. into an algal culture medium, and culturing the Coelastrum sp. to logarithmic growth phase to obtain Coelastrum sp. algal liquid; the Chinese oil yeast is prepared by the following steps: inoculating Chinese oil yeast into a fungal culture medium, and culturing the Chinese oil yeast to logarithmic growth phase to obtain Chinese oil yeast bacterial liquid.

4. The method according to claim 3, wherein the method is characterized in that: the algal culture medium is BG11 liquid culture medium; the fungal culture medium is PDB liquid culture medium.

5. The method according to claim 3, wherein the method is characterized in that: the Coelastrum sp. algal liquid is cultured at a temperature of 28.0±0.5℃, illumination of 10000 lux, and day-night ratio of 12h:12h; the Chinese oil yeast bacterial liquid is cultured at a temperature of 25-30℃, 90-110 rpm, and in dark.

6. The method according to claim 5, wherein the method is characterized in that: the Chinese oil yeast bacterial liquid and the Coelastrum sp. algal liquid are mixed at a volume ratio of 2-8:

1.

7. The method according to claim 1, wherein the method is characterized in that: the culturing treatment comprises at least one of general culturing treatment, culturing treatment using a membrane reactor, and culturing treatment using a columnar reactor.

8. The method according to claim 7, wherein the method is characterized in that: the general culturing treatment is culturing treatment using a conventional container; the culturing treatment is carried out at a temperature of 28.0±0.5℃, illumination of 10000 lux, and day-night ratio of 12h:12h.

9. Use of the method according to any one of claims 1-8 in treating aquaculture wastewater.

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