Stepped dairy farm wastewater treatment method based on aspergillus, synechocystis and green algae

Abstract

The invention discloses a method for treating dairy farm wastewater in a stepped manner based on synechocystis, aspergillus and green algae. According to the wastewater treatment method disclosed by the invention, aspergillus is used in the first step, so that organic substances in the wastewater of the dairy farm can be quickly converted; in the second step, synechocystis is used, the synechocystis is screened from wastewater of a biogas digester in a dairy farm, has high treatment capacity on high-concentration wastewater in the dairy farm and is named as synechocystis ZSL-NS1, and rapid increase of biomass can be achieved on the basis of the first two steps. Finally, the removal rate of pollutants in the dairy farm wastewater can reach 80% or above; the biomass yield is increased by 2.3 times. In addition, the synechocystis ZSL-NS1 can also produce biomass such as chlorophyll, protein, polysaccharide, grease and the like, the purpose of recycling is achieved while pollution is removed, and the synechocystis ZSL-NS1 has application value.

Description

Technical Field

[0001] The invention belongs to the field of aquaculture wastewater treatment technology and biotechnology, and specifically relates to a method for treating dairy farm wastewater in a step-by-step manner based on self-screening Aspergillus, Synechocystis and green algae. Background Art

[0002] With the rapid development of my country's economy and the continuous improvement of people's living standards, the proportion of dairy products in daily diet has increased year by year, and the scale of dairy farms has continued to expand and tend to be intensive. Due to the special needs of milking parlors, dairy cow pens, etc., dairy farms have become a high-water-consuming industry. While consuming a large amount of clean water resources, they also produce a large amount of high-concentration wastewater, in which pollutants are complex, high in concentration, and organic matter is difficult to degrade. If not effectively treated, it will cause secondary pollution to the surrounding environment. Up to now, the sewage treatment problem of dairy farms has become a bottleneck problem that restricts the survival and development of enterprises.

[0003] Combining microalgae cultivation with wastewater treatment has become a research hotspot in the field of water treatment. This method utilizes the growth advantages of microalgae to convert the nutrients in wastewater into microalgae biomass and achieve the purification of wastewater resources. At present, this method has gradually been recognized and applied in the field of environmental protection. However, there are still many problems with this technology, which limits its industrial application. For example, dairy farm wastewater has complex components, high concentrations, and poor light transmittance, which inhibit the growth, reproduction, and metabolic activities of microalgae and affect their purification effect on wastewater. In addition, different microalgae have different tolerance to wastewater and different available nutrient components. Therefore, traditional commercially available microalgae are difficult to be directly used for dairy farm wastewater treatment. Summary of the invention

[0004] In view of the problems existing in the existing microalgae wastewater treatment technology, the present invention provides a method for treating dairy farm wastewater in a step-by-step manner based on self-screened Aspergillus, Synechocystis and Chlorella, according to the characteristics of dairy farm wastewater and the physiological characteristics of different microalgae. The self-screened filamentous bacteria are added in the first step to achieve rapid conversion of organic matter in the wastewater, laying the foundation for the growth and utilization of microalgae. The self-screened Synechocystis is added in the second step to achieve rapid purification of high-concentration wastewater. The addition of commercially available Chlorella / Euglenoids in the third step achieves further purification of wastewater and a large amount of biomass value-added. Through the above, efficient purification of dairy farm wastewater and efficient improvement of biomass yield can be achieved.

[0005] The present invention solves the technical problem by adopting the following technical solutions:

[0006] A method for treating dairy farm wastewater in a step-by-step manner based on self-screening Aspergillus, Synechocystis and Chlorella, the specific steps comprising the following:

[0007] S1: Step-by-step wastewater treatment method: Synechocystis ZSL-NS1, Chlorella vulgaris and Euglena were cultured in a light incubator with BG11 culture medium until the logarithmic phase and then centrifuged and concentrated as seed liquid; Aspergillus was cultured to a recombinant amount and stored for later use;

[0008] S2: Add Aspergillus at 0.3 g / L into the wastewater from the oxidation pond of the dairy farm, cultivate it at about 20-30°C, and use natural light for a total of 2-5 days;

[0009] S3: centrifuge the treated liquid in S1 at 7000-8000r / min, collect the supernatant; add 0.2g / L of Synechocystis to the supernatant, shake evenly, light intensity 4000-5000Lux, light-dark ratio 12h:12h, temperature 20-25℃, treat for 4 days;

[0010] S4: centrifuge the treated liquid in S2 at 7000-8000r / min, collect the supernatant; add 0.2-0.5g / L of Chlorella or Euglena to the supernatant, shake evenly, light intensity 3000-4000Lux, light-dark ratio 12h:12h, temperature 25°C, treat for 6 days in total, use a shaker to prevent microalgae from settling or sticking to the wall, 100-200rpm, 30-60min, 3-5 times a day;

[0011] S4: Simultaneous wastewater treatment method: The three microorganisms in S1 are added to the wastewater at the same time and react for 12-15 days, with a light intensity of 3000-4000 Lux, a light-dark ratio of 12h:12h, a temperature of 25°C, and a total treatment period of 12 days, including 0-2 days of natural light.

[0012] Furthermore, the treatment in step S3 is carried out in a shaking table to prevent the microalgae from settling or adhering to the wall, at 100 rpm, 30 min, 3 times a day.

[0013] Moreover, the algae strain of the Synechocystis sp. ZSL-NS1 (Svnechocvstis sp. ZSL-NS1) has been deposited in the China Center for Type Culture Collection on November 7, 2024, with the deposit number being CCTCC M 20242468.

[0014] Furthermore, the Chlorella is Chlorella vulgaris FACHB-2338 and the Euglena is Euglena fachibians FACHB-848.

[0015] Moreover, the deposit number of Aspergillus is CGMCC No3.15297.

[0016] The advantages and positive effects of the present invention are:

[0017] The present invention selects a strain of Synechocystis ZSL-NS1 with strong pollution resistance from the wastewater of a dairy farm methane tank, and then combines it with Aspergillus and Chlorella / Euglenoids with good commercial effects, and uses "filamentous fungi"-"cyanobacteria"-"green algae" in stages to treat the dairy farm wastewater: in the first step, by adding self-screened Aspergillus, the organic matter in the wastewater can be converted into small molecules, providing a nutritional basis for the subsequent steps; in the second step, by utilizing the high tolerance of the self-screened Synechocystis ZSL-NS1, the growth inhibition problem caused by high-concentration wastewater can be overcome, and the reduction of wastewater concentration and the conversion of organic matter can be further achieved; in the third step, the use of Chlorella / Euglenoids can further purify the wastewater and achieve a large amount of accumulation of microalgae biomass.

[0018] Through the three-stage treatment method of the present invention, dairy cow wastewater can be effectively purified, and the removal rate of pollutants therein reaches more than 80%. In addition, high-value-added microalgae biomass can be obtained, the cumulative amount of biomass increases by 2.3 times, and the protein content is more than 50%.

[0019] The method of the present invention can effectively overcome the problems of poor tolerance of microorganisms / microalgae, growth inhibition, low biomass yield, etc. in traditional methods, and achieve the dual goals of reducing pollution and carbon emissions, and reducing costs and increasing efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a microscope morphological photograph of Synechocystis.

[0021] Figure 2 This is the Neighbor-Joining phylogenetic tree of strain NS1 constructed based on the 16S rRNA gene sequence alignment results with Pseudomonas aeruginosa DSMZ50071 (X06684) as the outer branch.

[0022] Figure 3 This is a microscope photo of the Aspergillus morphology.

[0023] Figure 4 This is the treatment effect of the step-by-step treatment method on wastewater.

[0024] Figure 5 The changes in dry weight of microalgae biomass in different treatments (T1: Synechocystis ZSL-NS1; T2: Chlorella; T3: Euglena; T4: Synechocystis + Chlorella; T5: Synechocystis + Euglena).

[0025] Figure 6-1 Paper Figure 6-3 The composition of microalgae and the production of extracellular polymeric substances (EPS). DETAILED DESCRIPTION

[0026] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0027] In a specific example, the dairy farm wastewater used in the treatment effect experiment of the present invention is wastewater from an oxidation pond, an anaerobic biogas tank or a stabilization pond of a dairy farm in Tianjin; the dairy farm wastewater used for screening blue algae is effluent from a biogas tank of a dairy farm in Tianjin.

[0028] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0029] Example 1

[0030] In 2023, the present invention isolated a strain of Synechocystis sp. from the soil beside a manure storage tank of a dairy farm in Tianjin. The strain survived well in the wastewater of the dairy farm and had an obvious decontamination effect. The strain was screened from the wastewater of the biogas digester of the dairy farm and had a strong treatment capacity for high-concentration wastewater from the dairy farm. The strain named Synechocystis sp. ZSL-NS1 (Svnechocvstis sp.) was deposited in the China Center for Type Culture Collection on November 7, 2024, with a deposit number of CCTCC NO.M 20242468. The following method for isolating and cultivating Synechocystis sp. ZSL-NS1 is specifically described as follows:

[0031] The wastewater sample was diluted in a gradient manner and then plated to grow a single algae colony on the plate. The single algae colony was picked and placed on the growth medium. The culture was carried out for 15 days at a temperature of 25°C, a light of 3000 Lux, and a light-dark ratio of 16:8 hours to obtain a cyanobacterial algae solution. The growth status and morphology were observed under a microscope, and purification culture was performed multiple times. After identification, it was identified as Synechocystis ZSL-NS1. After obtaining a single algae solution, it was expanded until there was enough algae solution (volume ratio during expansion: algae solution: culture medium = 1:5).

[0032] The formula of growth medium (BG11): sodium nitrate 1.5g, potassium dihydrogen phosphate 0.04g, magnesium sulfate heptahydrate 0.075g, calcium chloride dihydrate 0.036g, ammonium ferric citrate 0.006g, citric acid 0.006g, disodium ethylenediaminetetraacetic acid 0.001g, sodium carbonate 0.02g, trace element mother solution 1mL, add water to make up to 1000mL, adjust the pH to about 7.1, and sterilize at 121℃ for 20min.

[0033] The formula of trace element mother solution: boric acid 2.86g / L, manganese chloride tetrahydrate 1.86g / L, zinc sulfate heptahydrate 0.22g / L, copper sulfate pentahydrate 0.08g / L, sodium molybdate dihydrate 0.39g / L; cobalt nitrate hexahydrate 0.05g / L, add water to make up to 1000mL.

[0034] The results showed that Synechocystis ZSL-NS1 was blue-green in color, with spherical or elliptical cells. The cells were small, generally about 1-5 microns in diameter. The cell surface was relatively smooth, without obvious protrusions or appendages. In some cases, Synechocystis cells could aggregate to form colonies with diverse morphologies, and the cells in the colonies were arranged relatively tightly.

[0035] Example 2

[0036] Comparative test on treatment effect of dairy wastewater

[0037] S1: Step-by-step wastewater treatment method: Synechocystis ZSL-NS1, Chlorella (FACHB-2338) and Euglena (FACHB-848) were cultured in a light incubator using BG11 culture medium to the logarithmic phase and then centrifuged and concentrated as seed liquid; Aspergillus was cultured to the recombinant amount and stored for later use.

[0038] S2: Aspergillus sp. (CGMCC No. 3.15297) (disclosed in CN109170279A) was added into the wastewater from the oxidation pond of the dairy farm at a rate of 0.3 g / L. The culture conditions were about 25°C and natural light source, and the treatment lasted for 2 days.

[0039] S3: Centrifuge the treated liquid in S1 at 7000-8000r / min for 10 minutes, collect the supernatant; add 0.2g / L of Synechocystis to the supernatant, shake evenly, light intensity 4000-5000Lux, light-dark ratio 12h:12h, temperature 25°C, and treat for 4 days in total.

[0040] S4: The treated liquid in S2 was centrifuged at 7000-8000r / min for 10 minutes, and the supernatant was collected; Chlorella or Euglena was added to the supernatant at an amount of 0.2g / L, and the mixture was shaken evenly. The illumination intensity was 3000-4000Lux, the light-dark ratio was 12h:12h, and the temperature was 25°C. The treatment lasted for 6 days. The treatment was carried out in a shaking table to prevent the microalgae from settling or sticking to the wall, at 100rpm, for 30min, and 3 times a day.

[0041] S4: Simultaneous wastewater treatment method: The three microorganisms in (1) are added to the wastewater at the same time and reacted for 12 days, with a light intensity of 3000-4000 Lux, a light-dark ratio of 12h:12h, a temperature of 25°C, and a total treatment period of 12 days, including 0-2 days of natural light.

[0042] Example 3

[0043] Microalgae biomass production experiment

[0044] The specific method is the same as (1) in Example 3. The microalgae samples are collected and their dry weight is analyzed by dry weighing method ( Figure 5 ).

[0045] Example 4

[0046] Microalgae ingredient test

[0047] The specific method is the same as that in Example 3 (1), wherein microalgae samples are collected, and the composition of the microalgae and the production of extracellular polymeric substances (EPS) are determined ( FIG. 6 ).

[0048] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, according to the technical essence of the present invention, any simple modification, equivalent replacement and improvement made to the above embodiment still falls within the protection scope of the technical solution of the present invention.

[0049] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for treating dairy farm wastewater in a step-by-step manner based on Synechocystis, Aspergillus and Chlorella, characterized in that: The specific steps include the following: S1: Step-by-step wastewater treatment method: Synechocystis ZSL-NS1, Chlorella vulgaris and Euglena were cultured in a light incubator with BG11 culture medium until the logarithmic phase and then centrifuged and concentrated as seed liquid; Aspergillus was cultured to a recombinant amount and stored for later use; S2: Add Aspergillus at 0.3 g / L into the wastewater from the oxidation pond of the dairy farm, cultivate it at about 20-30°C, and use natural light for a total of 2-5 days; S3: centrifuge the treated liquid in S1 at 7000-8000r / min, collect the supernatant; add 0.2g / L of Synechocystis to the supernatant, shake evenly, light intensity 4000-5000Lux, light-dark ratio 12h:12h, temperature 20-25℃, treat for 4 days; S4: centrifuge the treated liquid in S2 at 7000-8000r / min, collect the supernatant; add 0.2-0.5g / L of Chlorella or Euglena to the supernatant, shake evenly, light intensity 3000-4000Lux, light-dark ratio 12h:12h, temperature 25°C, treat for 6 days in total, use a shaker to prevent microalgae from settling or sticking to the wall, 100-200rpm, 30-60min, 3-5 times a day; S4: Simultaneous wastewater treatment method: The three microorganisms in S1 are added to the wastewater at the same time and react for 12-15 days, with a light intensity of 3000-4000 Lux, a light-dark ratio of 12h:12h, a temperature of 25°C, and a total treatment period of 12 days, including 0-2 days of natural light.

2. The method for treating dairy wastewater based on Aspergillus, Synechocystis and Chlorella in a stepwise manner according to claim 1, characterized in that: In step S3, the treatment is carried out in a shaking table to prevent the microalgae from settling or adhering to the wall, at 100 rpm, for 30 minutes, three times a day.

3. The method for treating dairy wastewater based on Aspergillus, Synechocystis and Chlorella in a stepwise manner according to claim 1, characterized in that: The algae strain of Synechocystis ZSL-NS1 was deposited in the China Center for Type Culture Collection on November 7, 2024, with the deposit number CCTCC NO.M 20242468, and the deposit address is: Wuhan University, Wuhan, China.

4. The method for treating dairy farm wastewater in a stepwise manner based on Aspergillus, Synechocystis and Chlorella according to claim 1, characterized in that: The chlorella is chlorella FACHB-2338 and the euglena is euglena FACHB-848.

5. The method for treating dairy farm wastewater in a stepwise manner based on Aspergillus, Synechocystis and Chlorella according to claim 1, characterized in that: The deposit number of Aspergillus is CGMCC No.3.15297.

Images