Method for promoting microalgae to treat ethanol wastewater and recover lipid by adding acetic acid bacteria

Abstract

The invention discloses a method for promoting microalgae to treat ethanol wastewater to recover lipids by adding acetic acid bacteria, relates to the field of recovering lipids by using microalgae, and particularly relates to a method for simultaneously removing ethanol and recovering lipids by using ethanol wastewater as a substrate and adding acetic acid bacteria (acetobacter pasteurianus CICC 20056) and chlorella (chlorella sorojin chlorella FACHB-24) to construct a bacteria-algae symbiotic system. In the system, the acetobacter pasteurianus metabolizes ethanol in the wastewater into acetic acid by using oxygen generated by photosynthesis of the microalgae, the microalgae store the acetic acid as a substrate into lipid, and the acetic acid and the lipid form a mutualistic symbiotic relationship, so that the ethanol in the ethanol-containing wastewater is efficiently removed, and the lipid accumulation efficiency of the microalgae is remarkably improved.

Description

Technical Field

[0001] The invention relates to the field of microalgae oil production, and in particular to a method for promoting microalgae to process ethanol wastewater and recover lipids by adding acetic acid bacteria. Background Art

[0002] As an important solvent, cleaning agent, extractant and fuel, ethanol plays a key role in many industries such as food and beverage, medicine, chemical industry, fuel, etc. However, with the continuous development of these industries, a large amount of ethanol-containing wastewater has also been generated. In the pharmaceutical industry, ethanol is widely used as a solvent or reaction medium in drug synthesis. The extraction and refining of many drugs and intermediates rely on alcohol solvents, so the ethanol concentration in the wastewater is generally high. Similarly, the production processes of cosmetics, food processing, textiles, automotive fuels and other industries also use a large amount of ethanol, resulting in a large amount of ethanol-containing wastewater in these industries. Developing an efficient and environmentally friendly method for treating ethanol-containing wastewater has important practical significance and broad application prospects.

[0003] At present, the treatment technology of ethanol wastewater mainly includes biodegradation method and physical and chemical method. However, these prior arts still have some shortcomings. The biodegradation method converts the ethanol in the wastewater into carbon dioxide and water by aerobic microorganisms. Although it can remove ethanol, it will release a large amount of carbon dioxide, aggravate greenhouse gas emissions, and is not conducive to achieving the goal of carbon neutrality. In addition, the treatment process adopts an aeration device, which consumes a lot of energy, resulting in high energy consumption in the treatment process, and there is a risk of ethanol stripping. Another technology is nanofiltration technology, which effectively removes ethanol in wastewater through the membrane separation principle, but there are problems of high equipment investment and operating costs. Therefore, it is urgent to develop a kind of efficient, energy-saving and environmentally friendly treatment method to achieve the emission reduction and resource utilization of pollutants in ethanol wastewater.

[0004] In recent years, microalgae have attracted widespread attention due to their carbon emission reduction potential and economic benefits. Microalgae can use organic pollutants in water as the main energy and carbon source through a heterotrophic mode and convert them into microalgae biomass. Among them, lipids, as a biofuel, have important economic value and application prospects. Therefore, combining wastewater treatment with biofuel production is a promising strategy for wastewater resource recovery. However, there are still some limitations of single microalgae systems in wastewater treatment and lipid recovery. For example, the lipid conversion efficiency is not high, especially when treating wastewater containing toxic pollutants, the growth of microalgae is easily inhibited, resulting in unsatisfactory removal effect. Therefore, exploring more efficient synergistic technologies has become an important direction to solve these problems.

[0005] In view of the above problems, the present invention proposes a method for promoting lipid recovery from ethanol wastewater by adding acetic acid bacteria to microalgae. The ethanol removal efficiency in wastewater is enhanced by utilizing the conversion of ethanol to acetic acid by acetic acid bacteria and the photosynthesis of Chlorella vulgaris, while promoting the growth of microalgae and lipid accumulation, improving the resource recovery capacity of the system, and making the wastewater treatment process more cost-effective. Summary of the invention

[0006] The object of the present invention is to provide a method for promoting microalgae to recover lipids from ethanol wastewater by adding acetic acid bacteria, utilizing the synergistic effect of Acetobacter pasteurianus and Chlorella vulgaris to effectively remove ethanol from wastewater and promote lipid accumulation in microalgae, thereby achieving the dual goals of pollutant removal and production of high value-added products.

[0007] The present invention provides a method for promoting microalgae to treat ethanol wastewater and recover lipids by adding acetic acid bacteria, characterized in that acetic acid bacteria and Chlorella vulgaris are added to ethanol-containing wastewater, and the synergistic effect of the two is utilized to achieve efficient removal of ethanol and promote the accumulation of microalgae biomass and lipids. The method comprises the following steps:

[0008] Step 1: Acetobacter pasteurianus and Chlorella sorokinensis are inoculated into seed culture medium respectively until the optical density (OD600) of the Acetobacter pasteurianus bacterial solution reaches 1 and the optical density (OD750) of the Chlorella sorokinensis algae solution reaches 1, to obtain acetic acid bacteria inoculation solution and Chlorella sorokinensis inoculation solution.

[0009] Step 2: Mix the acetic acid bacteria inoculum solution and the Chlorella inoculum solution in a volume ratio of 1:5-1:10 to prepare a composite bacteria-algae system.

[0010] Step 3: Inoculate the composite bacteria-algae system into a transparent reactor containing ethanol wastewater at a volume fraction of 5-20%, wherein the main component of the ethanol wastewater is ethanol, and the final volume concentration is 0.5-2.0% anhydrous ethanol per liter of wastewater.

[0011] Step 4: Use a pH automatic liquid adding machine to control the pH value in the reactor in the range of 6.0-7.0, control the light intensity at 3000-8000 lux through LED lamps, the light cycle is 12 hours of light and 12 hours of darkness, the reaction temperature is controlled at 25-30°C, and the culture time is 7-9 days.

[0012] Step 5: After the culture is completed, the microalgae in the reactor are harvested by centrifugation and lipid extraction is performed. Specifically, lipids are extracted from the freeze-dried microalgae using a mixed solution of chloroform and methanol in a volume ratio of 2:1. The lipid-rich chloroform layer is collected by centrifugation, and the chloroform is evaporated in a 60°C water bath. The mass of the remaining liquid obtained is weighed as the dry weight of lipids.

[0013] The technical principle of the present invention is to use acetic acid bacteria to convert ethanol in wastewater into acetic acid under suitable conditions. Under the metabolic action of acetic acid bacteria, ethanol is converted into acetic acid through redox reaction. The generated acetic acid can provide microalgae with a carbon source that is beneficial to lipid accumulation, thereby promoting the growth of microalgae and lipid accumulation. Chlorella provides oxygen to acetic acid bacteria through photosynthesis, promoting the aerobic metabolism of acetic acid bacteria and improving the degradation efficiency of ethanol.

[0014] The present invention utilizes the mutually beneficial symbiotic relationship between acetic acid bacteria and Chlorella to form a stable bacteria-algae collaborative treatment system. In the system, acetic acid bacteria degrade ethanol and provide nutrients, while Chlorella provides oxygen through photosynthesis and further promotes the degradation of organic matter in wastewater. The synergistic effect of the two not only improves the removal efficiency of ethanol, but also enhances the biomass and lipid yield of microalgae, providing valuable guidance for the resource management of ethanol-containing wastewater.

[0015] The beneficial effects of the present invention are:

[0016] (1) Through the bacterial-algal synergistic treatment technology, the volatile organic compound ethanol in ethanol-containing wastewater can be significantly removed, achieving the treatment of harmful pollutants.

[0017] (2) The conversion of ethanol to acetic acid promotes the growth and lipid accumulation of microalgae. These microalgae products can be further used for the production of biofuels or high-value bioproducts, achieving the dual benefits of wastewater treatment and resource recovery.

[0018] (3) The bacteria-algae co-culture system provides oxygen through the photosynthesis of microalgae, supports the aerobic metabolism of bacteria, reduces the energy consumption of traditional aeration systems, reduces treatment costs, and embodies the significant advantages of low carbon and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0020] Figure 1 Schematic diagram of the reactor

[0021] LED light (1) Temperature probe (2) Temperature controller (3) pH control panel (4) pH probe (5) Reactor (6) Magnetic rotor (7) Magnetic stirrer (8)

[0022] Figure 2 This is a schematic diagram of the present invention. DETAILED DESCRIPTION

[0023] The following examples are provided to further illustrate the present invention, but not to limit the present invention. Unless otherwise specified, the materials or instruments used in the examples can be easily obtained from commercial companies.

[0024] The microorganism sources used in the present invention are as follows:

[0025] Acetic acid bacteria were purchased from Acetobacter pasteurianus CICC 20056 from the China Industrial Microbiological Collection.

[0026] The microalgae was Chlorella sorokinensis FACHB-24, which was purchased from the freshwater algae seed bank of the Chinese Academy of Sciences.

[0027] Example 1

[0028] A method for promoting microalgae to treat ethanol wastewater and recover lipids by adding acetic acid bacteria. In this embodiment, the co-cultivation effect of Chlorella and acetic acid bacteria was studied under the conditions of a final ethanol volume concentration of 1%, a biomass ratio of acetic acid bacteria to Chlorella of 1:10, a pH control of 6.5, and a light intensity of 5000 lux, to explore the removal effect of the composite bacterial solution on ethanol and the accumulation of Chlorella biomass and lipids.

[0029] Chlorella sorokinensis was inoculated into a triangular flask containing 200 mL of growth medium 1 and cultured under the conditions of 12 hours of light time and 12 hours of dark time. The light intensity was 5000 lux, and it was placed in a water bath shaker at 25°C and a speed of 120 rpm for 7 days to obtain Chlorella algae liquid.

[0030] The formula of growth medium 1 is 1 mL of acetic acid, 0.375 g of ammonium chloride, 0.288 g of dipotassium hydrogen phosphate, 0.144 g of potassium dihydrogen phosphate, 0.1 g of magnesium sulfate heptahydrate, 0.05 g of calcium chloride dihydrate, 1 mL of trace element mother solution, add water to 1000 mL, adjust the pH to 7.0 with NaOH or HCl. Dispense in 250 mL Erlenmeyer flasks and autoclave at 121°C for 20 minutes. The formula of trace element mother solution includes 3.58 g of Al2(SO4)3·18H2O, 12.98 g of MnCl2·4H2O, 1.83 g of CuSO4·5H2O, 3.20 g of ZnSO4·7H2O, add water to 1000 mL, and autoclave at 121°C for 20 minutes.

[0031] Under sterile conditions, the Acetobacter pasteurianus cells were picked from the solid culture medium 1 with an inoculation loop and inoculated into the growth medium 2. The cells were placed in a shaking incubator at 30° C. and 120 rpm for 72 hours to obtain an acetic acid bacteria solution.

[0032] The formula of solid culture medium 1 is 10.0 g of yeast extract, 2.0 g of calcium carbonate, 100.0 g of glucose, and 15.0 g of agar. Water is added to make the volume 1000 mL, and the pH is adjusted to 7.0. After the above ingredients are mixed evenly, they are sterilized at 121° C. for 20 minutes for use.

[0033] The formula of growth medium 2 is 10 g glucose, 10 g yeast extract, 20 mL anhydrous ethanol, and water is added to make up to 1000 mL, and then sterilized by high pressure at 121°C for 20 minutes before use.

[0034] The method for preparing the composite bacterial liquid comprises the following steps:

[0035] 1) Centrifuge a certain amount of acetic acid bacteria liquid, discard the supernatant, resuspend with sterile water, repeat 3 times, and adjust the OD600 to 1.0 with sterile experimental culture medium to obtain acetic acid bacteria inoculum.

[0036] 2) Centrifuge a certain amount of Chlorella algae liquid, discard the supernatant, resuspend with sterile water, repeat 3 times, and adjust the OD750 to 1.0 with sterile experimental culture medium to obtain Chlorella inoculum liquid.

[0037] 3) Mixing the inoculum solutions prepared in step 1) and step 2) at a volume ratio of 1:10, and mixing them evenly to obtain a composite bacterial solution.

[0038] The formula of the experimental culture medium includes: 10 mL of anhydrous ethanol, 1 g of ammonium sulfate, 0.288 g of dipotassium hydrogen phosphate, 0.127 g of sodium dihydrogen phosphate, 0.1 g of magnesium sulfate heptahydrate, 0.05 g of calcium chloride dihydrate, 1 mL of trace element stock solution, add water to make up to 1000 mL, and sterilize by high pressure at 121°C for 20 minutes.

[0039] The pretreated composite bacterial liquid in the above steps was inoculated into 1000 mL of experimental culture medium and cultured at 30°C. The pH value was controlled at 6.5 by an automatic liquid addition pH adjustment device. The light cycle was 12 hours of light and 12 hours of darkness, and the light intensity was 5000 Lux. The experiment was repeated three times, and after 7 days of culture, the ethanol removal rate, Chlorella biomass and lipid accumulation were determined.

[0040] The experimental results showed that on the 7th day of cultivation, the ethanol removal rate of the composite bacterial liquid reached 96.8%, the maximum biomass yield reached 572.5 mg / L, and the lipid yield reached 26.7% dry weight.

[0041] Example 2

[0042] A method for promoting microalgae to treat ethanol wastewater and recover lipids by adding acetic acid bacteria. In this embodiment, the co-cultivation effect of Chlorella and acetic acid bacteria was studied under the conditions of a final volume concentration of ethanol of 1%, a biomass ratio of acetic acid bacteria to Chlorella of 1:10, a pH control of 6.5, and a light intensity of 8000 lux, to explore the removal effect of the composite bacterial solution on ethanol and the accumulation of Chlorella biomass and lipids.

[0043] Chlorella sorokinensis was inoculated into a triangular flask containing 200 mL of growth medium 1 and cultured under the conditions of 12 hours of light time and 12 hours of dark time. The light intensity was 5000 lux, and it was placed in a water bath shaker at 25°C and a speed of 120 rpm for 7 days to obtain Chlorella algae liquid.

[0044] The formula of growth medium 1 is 1 mL of acetic acid, 0.375 g of ammonium chloride, 0.288 g of dipotassium hydrogen phosphate, 0.144 g of potassium dihydrogen phosphate, 0.1 g of magnesium sulfate heptahydrate, 0.05 g of calcium chloride dihydrate, 1 mL of trace element mother solution, add water to 1000 mL, adjust the pH to 7.0 with NaOH or HCl. Dispense in 250 mL Erlenmeyer flasks and autoclave at 121°C for 20 minutes. The formula of trace element mother solution includes 3.58 g of Al2(SO4)3·18H2O, 12.98 g of MnCl2·4H2O, 1.83 g of CuSO4·5H2O, 3.20 g of ZnSO4·7H2O, add water to 1000 mL, and autoclave at 121°C for 20 minutes.

[0045] Under sterile conditions, the Acetobacter pasteurianus cells were picked from the solid culture medium 1 with an inoculation loop and inoculated into the growth medium 2. The cells were placed in a shaking incubator at 30° C. and 120 rpm for 72 hours to obtain an acetic acid bacteria solution.

[0046] The formula of solid culture medium 1 is 10.0 g of yeast extract, 2.0 g of calcium carbonate, 100.0 g of glucose, and 15.0 g of agar. Water is added to make the volume 1000 mL, and the pH is adjusted to 7.0. After the above ingredients are mixed evenly, they are sterilized at 121° C. for 20 minutes for use.

[0047] The formula of growth medium 2 is 10 g glucose, 10 g yeast extract, 20 mL anhydrous ethanol, and water is added to make up to 1000 mL, and then sterilized by high pressure at 121°C for 20 minutes before use.

[0048] The method for preparing the composite bacterial liquid comprises the following steps:

[0049] 1) Centrifuge a certain amount of acetic acid bacteria liquid, discard the supernatant, resuspend with sterile water, repeat 3 times, and adjust the OD600 to 1.0 with sterile experimental culture medium to obtain acetic acid bacteria inoculum.

[0050] 2) Centrifuge a certain amount of Chlorella algae liquid, discard the supernatant, resuspend with sterile water, repeat 3 times, and adjust the OD750 to 1.0 with sterile experimental culture medium to obtain Chlorella inoculum liquid.

[0051] 3) Mixing the inoculum solutions prepared in step 1) and step 2) at a volume ratio of 1:10, and mixing them evenly to obtain a composite bacterial solution.

[0052] The formula of the experimental culture medium includes: 10 mL of anhydrous ethanol, 1 g of ammonium sulfate, 0.288 g of dipotassium hydrogen phosphate, 0.127 g of sodium dihydrogen phosphate, 0.1 g of magnesium sulfate heptahydrate, 0.05 g of calcium chloride dihydrate, 1 mL of trace element stock solution, add water to make up to 1000 mL, and sterilize by high pressure at 121°C for 20 minutes.

[0053] The pretreated composite bacterial liquid in the above steps was inoculated into 1000 mL of experimental culture medium and cultured at 30°C. The pH value was controlled at 6.5 by an automatic liquid addition pH regulator. The light cycle was 12 hours of light and 12 hours of darkness, and the light intensity was 8000 Lux. The experiment was repeated three times, and after 9 days of culture, the ethanol removal rate, Chlorella biomass and lipid accumulation were determined.

[0054] The experimental results showed that on the 9th day of cultivation, the ethanol removal rate of the composite bacterial liquid reached 92.4%, the maximum biomass yield reached 546.6 mg / L, and the lipid yield reached 21.5% dry weight.

[0055] Example 3

[0056] A method for promoting microalgae to treat ethanol wastewater and recover lipids by adding acetic acid bacteria. In this embodiment, the co-cultivation effect of Chlorella and acetic acid bacteria was studied under the conditions of a final ethanol volume concentration of 0.5%, a biomass ratio of acetic acid bacteria to Chlorella of 1:10, a pH control of 6.5, and a light intensity of 5000 lux, to explore the removal effect of the composite bacterial solution on ethanol and the accumulation of Chlorella biomass and lipids.

[0057] Chlorella sorokinensis was inoculated into a triangular flask containing 200 mL of growth medium 1 and cultured under the conditions of 12 hours of light time and 12 hours of dark time. The light intensity was 5000 lux, and it was placed in a water bath shaker at 25°C and a speed of 120 rpm for 7 days to obtain Chlorella algae liquid.

[0058] The formula of growth medium 1 is 1 mL of acetic acid, 0.375 g of ammonium chloride, 0.288 g of dipotassium hydrogen phosphate, 0.144 g of potassium dihydrogen phosphate, 0.1 g of magnesium sulfate heptahydrate, 0.05 g of calcium chloride dihydrate, 1 mL of trace element mother solution, add water to 1000 mL, adjust the pH to 7.0 with NaOH or HCl. Dispense in 250 mL Erlenmeyer flasks and autoclave at 121°C for 20 minutes. The formula of trace element mother solution includes 3.58 g of Al2(SO4)3·18H2O, 12.98 g of MnCl2·4H2O, 1.83 g of CuSO4·5H2O, 3.20 g of ZnSO4·7H2O, add water to 1000 mL, and autoclave at 121°C for 20 minutes.

[0059] Under sterile conditions, the Acetobacter pasteurianus cells were picked from the solid culture medium 1 with an inoculation loop and inoculated into the growth medium 2. The cells were placed in a shaking incubator at 30° C. and 120 rpm for 72 hours to obtain an acetic acid bacteria solution.

[0060] The formula of solid culture medium 1 is 10.0 g of yeast extract, 2.0 g of calcium carbonate, 100.0 g of glucose, and 15.0 g of agar. Water is added to make the volume 1000 mL, and the pH is adjusted to 7.0. After the above ingredients are mixed evenly, they are sterilized at 121° C. for 20 minutes for use.

[0061] The formula of growth medium 2 is 10 g glucose, 10 g yeast extract, 20 mL anhydrous ethanol, and water is added to make up to 1000 mL, and then sterilized by high pressure at 121°C for 20 minutes before use.

[0062] The method for preparing the composite bacterial liquid comprises the following steps:

[0063] 1) Centrifuge a certain amount of acetic acid bacteria liquid, discard the supernatant, resuspend with sterile water, repeat 3 times, and adjust the OD600 to 1.0 with sterile experimental culture medium to obtain acetic acid bacteria inoculum.

[0064] 2) Centrifuge a certain amount of Chlorella algae liquid, discard the supernatant, resuspend with sterile water, repeat 3 times, and adjust the OD750 to 1.0 with sterile experimental culture medium to obtain Chlorella inoculum liquid.

[0065] 3) Mixing the inoculum solutions prepared in step 1) and step 2) at a volume ratio of 1:10, and mixing them evenly to obtain a composite bacterial solution.

[0066] The formula of the experimental culture medium includes: 5 mL of anhydrous ethanol, 1 g of ammonium sulfate, 0.288 g of dipotassium hydrogen phosphate, 0.127 g of sodium dihydrogen phosphate, 0.1 g of magnesium sulfate heptahydrate, 0.05 g of calcium chloride dihydrate, 1 mL of trace element stock solution, add water to make up to 1000 mL, and sterilize by high pressure at 121°C for 20 minutes.

[0067] The pretreated composite bacterial liquid in the above steps was inoculated into 1000 mL of experimental culture medium and cultured at 30°C. The pH value was controlled at 6.5 by an automatic liquid addition pH adjustment device. The light cycle was 12 hours of light and 12 hours of darkness, and the light intensity was 5000 Lux. The experiment was repeated three times, and after 9 days of culture, the ethanol removal rate, Chlorella biomass and lipid accumulation were determined.

[0068] The experimental results showed that on the 9th day of cultivation, the ethanol removal rate of the composite bacterial liquid reached 97.6%, the maximum biomass yield reached 416.7 mg / L, and the lipid yield reached 30.5% dry weight.

Claims

1. A method for promoting the recovery of lipids from ethanol wastewater by adding acetic acid bacteria to microalgae, characterized in that: The steps include: Step 1: inoculating Acetobacter pasteurianus and Chlorella sorokinensis into a seed culture medium respectively, until the optical density OD600 of the Acetobacter pasteurianus bacterial solution reaches 1, and the optical density OD750 of the Chlorella sorokinensis algae solution reaches 1, to obtain an acetic acid bacteria inoculation solution and a Chlorella sorokinensis inoculation solution; Step 2: mixing the acetic acid bacteria inoculum solution with the Chlorella inoculum solution in a volume ratio of 1:5-1:10 to prepare a composite bacteria-algae system; Step 3: inoculating the composite bacteria-algae system into a transparent reactor containing ethanol wastewater at a volume fraction of 5%-20%, wherein the concentration of the ethanol wastewater is 0.5%-2.0% anhydrous ethanol per liter of wastewater; Step 4: Use a pH automatic liquid adding machine to control the pH value in the reactor in the range of 6.0-7.0, control the light intensity at 3000-8000 lux by LED light, the light cycle is 12 hours of light and 12 hours of darkness, the reaction temperature is controlled at 25-30°C, and the culture time is 7-9 days; Step 5: After the culture is completed, the microalgae in the reactor are harvested by centrifugation and lipid extraction is performed.

2. The method of claim 1, wherein: The ethanol wastewater is derived from alcohol-containing wastewater produced in the brewing, pharmaceutical or chemical industries, or ethanol waste liquid formed after ethanol volatile gas is sprayed, absorbed and captured.

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