Talaromyces sp. producing polysaccharide and its application in treatment of wastewater with high nitrogen and phosphorus

Abstract

The application discloses a polysaccharide-producing desertifilum sp., and application of the polysaccharide-producing desertifilum sp. in high-nitrogen and high-phosphorus wastewater treatment, and the algae strain named desertifilum sp. ZSL-NS5 has been preserved in China Center for Type Culture Collection on November 7, 2024, and the preservation number is CCTCC NO. M 20242470. The algae species is separated from wastewater in an oxygen pond of a dairy farm, has strong wastewater tolerance and pollutant purification capacity, the removal rate of total phosphorus is more than 95%, the removal rate of ammonia nitrogen is close to 100%, the removal rate of TN is more than 85%, and the removal rate of COD is more than 70%. In addition, the desertifilum sp. has the ability to produce microalgae EPS and polysaccharides, the polysaccharide content reaches 63%, which is significantly higher than the yield of ordinary microalgae, and has application values in preparation of chemical raw materials and preparation of medicines.

Description

Technical Field

[0001] The invention belongs to the field of aquaculture wastewater treatment technology and biotechnology, and in particular relates to a polysaccharide-producing Talsa algae and application thereof in dairy farm wastewater treatment. Background Art

[0002] Microalgae are tiny microorganisms whose cells can only be seen under a microscope. They use sunlight to convert carbon dioxide and water in water into oxygen and organic matter. This process consumes inorganic carbon sources in the water, helping to reduce eutrophication. Microalgae efficiently absorb nitrogen (such as ammonia nitrogen and nitrates) and phosphorus (such as phosphates) from wastewater, converting them into nutrients for their own growth, thereby reducing the nutrient load in the water. Furthermore, microalgae remove organic pollutants such as surfactants and oils from wastewater through adsorption on their cell surfaces and internal metabolic processes. Certain microalgae have the ability to accumulate heavy metals, effectively removing heavy metal ions such as cadmium, chromium, mercury, and lead from water through biosorption and bioaccumulation. Microalgae have a rapid growth rate, enabling them to respond quickly in wastewater treatment and effectively remove pollutants. Microalgae biomass is rich in protein, polysaccharides, and other nutrients, and can be used as raw materials for high-value products such as biofuels, pharmaceuticals, and fertilizers, achieving resource conversion in wastewater. Furthermore, microalgae have a high carbon sequestration capacity, helping to mitigate greenhouse gas emissions. In summary, microalgae technology has been widely used in various wastewater treatment fields due to its advantages of "pollution reduction and carbon reduction, resource transformation, and turning waste into treasure".

[0003] Dairy farm wastewater is characterized by high wastewater production, complex composition, and difficulty in treatment. The water quality of dairy farm wastewater varies greatly, and its ammonia and nitrogen content is high, resulting in an imbalance in the carbon-nitrogen ratio. This makes wastewater treatment complex and poses a challenge. At present, dairy farm wastewater treatment technology is mainly based on biological combination processes, but there are still high concentrations of organic matter and high concentrations of ammonia and nitrogen in the effluent. In addition, existing processes usually produce large amounts of residual sludge, which still requires further treatment. However, microalgae biological treatment technology does not require the addition of an additional carbon source, thereby reducing the investment cost of the wastewater treatment process; it also does not produce residual sludge and its secondary treatment problems; and microalgae biomass can be used as chemical raw materials to generate economic value. Therefore, microalgae biotechnology has gradually gained favor among scholars in the field of livestock and poultry wastewater.

[0004] While research on microalgae for treating dairy wastewater is increasing, limited research is underway on the selection of native microalgae from wastewater habitats and their application in wastewater treatment and biomass recovery. Traditional commercially available microalgae struggle to adapt to the high concentrations of organic matter, ammonia nitrogen, and toxic and hazardous components found in dairy wastewater. Therefore, identifying new microalgae varieties that can adapt to the characteristics of dairy wastewater is crucial. Furthermore, the subsequent utilization of microalgae biomass is crucial. The polysaccharides found in microalgae have diverse applications, including as gel materials for soft materials and sensors; in medicine, for the preparation of drug carriers such as injections and tablets; and in the preparation of polymer scaffolds and composite materials for the repair and replacement of human tissue. However, cultivating microalgae with high polysaccharide yields remains a pressing challenge. Summary of the Invention

[0005] In response to the current problems of complex composition of dairy farm wastewater, low pollutant removal efficiency, and difficulty in returning it to farmland, the present invention provides a self-isolated Tarsha filamentous algae (Desertifilum sp.) ZSL-NS5, which is used to treat dairy farm wastewater and produce polysaccharides, providing technical support for aquaculture wastewater treatment.

[0006] The present invention solves the technical problem by adopting the following technical solutions:

[0007] A polysaccharide-producing strain of Tarsa algae, named Tarsa algae ZSL-NS5, classified as Desertifilum sp, deposited on November 7, 2024, deposited in the China Center for Type Culture Collection, with the deposit number CCTCC M 20242470, and deposited at Wuhan University, Wuhan, China.

[0008] Moreover, the formula of the culture medium of Tarsa algae ZSL-NS5 is as follows: 1.5 g sodium nitrate, 0.04 g potassium hydrogen phosphate, 0.075 g magnesium sulfate heptahydrate, 0.036 g calcium chloride dihydrate, 0.006 g ammonium ferric citrate, 0.006 g citric acid, 0.001 g disodium ethylenediaminetetraacetic acid, 0.02 g sodium carbonate, 1 mL of trace element mother solution, add water to make up to 1000 mL, adjust the pH to about 7.1, and sterilize by high pressure at 121°C for 20 min.

[0009] Moreover, the formula of the trace element mother solution is: 2.86g / L boric acid, 1.86g / L manganese chloride tetrahydrate, 0.22g / L zinc sulfate heptahydrate, 0.08g / L copper sulfate pentahydrate, 0.39g / L sodium molybdate dihydrate; 0.05g / L cobalt nitrate hexahydrate, add water to make up to 1000mL.

[0010] Application of polysaccharide-producing Talsa filamentosa ZSL-NS5 in dairy farm wastewater.

[0011] A method for treating dairy farm wastewater using polysaccharide-producing Tarsa algae. The wastewater needs to undergo four treatment steps: coarse filtration, centrifugation, refiltration, and dilution. The inoculation ratio of ZSL-NS5 algae liquid to wastewater is 1:5 by volume. The algae liquid is centrifuged at 7000-8000 r / min for 10 minutes. After centrifugation, the upper culture medium is discarded, and Tarsa algae mud is added to the dairy farm wastewater and shaken or stirred evenly.

[0012] Moreover, the treatment conditions are: light intensity 4000-5000 Lux, light-dark ratio 12:12 hours, temperature 25°C, treatment cycle can be 8-12 days, and air circulation or shaking is used to prevent microalgae from settling or sticking to the wall.

[0013] Moreover, in the treatment of dairy farm wastewater by Tarsa algae ZSL-NS5, the wastewater can be sterilized or not, the dilution ratio of anaerobic tank effluent and oxidation pond wastewater is 1-5 times, and the dilution ratio of stabilization pond wastewater is 1-2 times.

[0014] Application of polysaccharide-producing Tarsa algae ZSL-NS5 as biofertilizer.

[0015] The advantages and positive effects of the present invention are:

[0016] 1. The present invention screened out a strain of Tarsa algae with strong pollution tolerance and purification ability from the wastewater of aerobic ponds in dairy farms. Its growth performance in wastewater is better than that in culture medium, and its growth rate in wastewater is faster, and it can adapt to high-concentration dairy farm wastewater.

[0017] 2. The Talsa microalgae of the present invention has a strong ability to purify wastewater. Due to its rich extracellular polymers and its own filamentous characteristics, the microalgae has a significant effect on removing color, COD, ammonia nitrogen, TN and TP in wastewater, which is better than traditional pure cultured microalgae varieties.

[0018] 3. The sugar content of the Talsa microalgae provided by the present invention reaches more than 60%, which is higher than that of conventional microalgae, and can provide high-quality sugar raw materials. The microalgae also produces other products, such as proteins, oils, etc., which can achieve the purpose of biomass recovery while treating wastewater.

[0019] 4. The present invention provides a wastewater treatment method that does not require additional material and energy consumption, and can significantly reduce the economic cost of dairy farm wastewater treatment. Compared with other methods of treating dairy farm wastewater, the treatment method of the present invention is more efficient, has more stable performance, is economical, and meets the requirements of clean production. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Micrographs of microalgae cell morphology.

[0021] Figure 2 Maximum parsimony tree constructed based on 16s rDNA fragment sequences (microalgae of the present invention marked with red boxes).

[0022] Figure 3 Growth indicators of Talsa algae (a: dry weight; b: chlorophyll; c: component content; d: component ratio).

[0023] Figure 4 Purification effect of Talsa algae on dairy farm wastewater with different concentrations. DETAILED DESCRIPTION

[0024] The present invention will be further described in detail below through specific examples. The following examples are only illustrative and not restrictive, and the scope of protection of the present invention cannot be limited thereto.

[0025] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.

[0026] The dairy farm wastewater of the present invention is obtained from the anaerobic fermentation effluent of a dairy farm in Tianjin. The present invention's Talsa algae was isolated from the aerobic pond wastewater of the aforementioned dairy farm. This polysaccharide-producing Talsa algae isolated from dairy farm wastewater exhibits improved wastewater tolerance and treatment effectiveness. The strain has been deposited (with the deposit number CCTCC No. M20242470) and has excellent application prospects.

[0027] The Tarsa algae of the present invention is separated from the aerobic pond wastewater of the dairy farm.

[0028] Example 1

[0029] Isolation and identification of Talsa algae

[0030] 1. Isolation of Tarsa algae

[0031] Take the aerobic pond wastewater from the dairy farm (COD is 4562.3 mg / L, total nitrogen is 560.8 mg / L, ammonia nitrogen is 507.5 mg / L, total phosphorus is 64.1 mg / L, salinity is 1.9‰, pH is 7.7), perform gradient dilution and spread on the plate. After single algae grow on the plate, pick the single algae and transfer it to the growth medium.

[0032] The culture was carried out for 15 days at 25°C under a light intensity ratio of 6000 Lux, 3000 Lux, and darkness of 8:8:8 hours to obtain a Tarsa algae liquid. After obtaining a single algae liquid, it was identified as Tarsa algae ZSL-NS5. The culture was then expanded until sufficient liquid was obtained (volume ratio of liquid to culture medium = 1:5).

[0033] The formula of the growth medium (BG11) is as follows: sodium nitrate 1.5 g, potassium phosphate dibasic 0.04 g, magnesium sulfate heptahydrate 0.075 g, calcium chloride dihydrate 0.036 g, ferric ammonium citrate 0.006 g, citric acid 0.006 g, disodium ethylenediaminetetraacetate 0.001 g, sodium carbonate 0.02 g, trace element stock solution 1 mL, water to 1000 mL, and pH adjusted to about 7.1, high-pressure sterilization at 121°C for 20 min.

[0034] The formula of the trace element stock solution is as follows: boric acid 2.86 g / L, manganese chloride tetrahydrate 1.86 g / L, zinc sulfate heptahydrate 0.22 g / L, copper sulfate pentahydrate 0.08 g / L, sodium molybdate dihydrate 0.39 g / L, and cobalt nitrate hexahydrate 0.05 g / L, water to 1000 mL.

[0035] 2. Morphological identification of microalgae

[0036] A small amount of sample was observed under an Olympus BX53 microscope, and images were collected using an Olympus DP80. Figure 1 The filaments were single or intertwined with each other, often curved, unbranched, with thin sheaths, and blue-green in color. The transverse walls were slightly constricted, and the filament tips were thin and lacked cap-shaped structures, heteromorphic cells, and chlamydospores. The cells were columnar, 3.0-8.0 μm long and 2.2-4.0 μm wide.

[0037] The above-mentioned Desertifilum sp. ZSL-NS5 was screened and classified, and the preservation date was November 07, 2024, the preservation unit was China Center for Type Culture Collection, and the preservation number was CCTCC NO. M20242470.

[0038] Example 2

[0039] Growth performance of Desertifilum sp. in dairy farm wastewater

[0040] (1) Isolation and expansion of Desertifilum sp.

[0041] (2) The wastewater needs to be coarsely filtered through gauze to remove large impurities, centrifuged at 4000 rpm for 10 minutes, and then filtered through ordinary filter paper and diluted according to different gradients. Take the algae liquid of Tarsa microalgae ZSL-NS5 in the logarithmic growth period. The volume ratio of algae liquid to wastewater is 1:5. Add 50 mL of algae liquid to 250 mL of wastewater, corresponding to a dry weight of 0.2 g / L. The algae liquid is centrifuged at 7000-8000 r / min for 10 minutes. After centrifugation, remove the upper culture medium and add Tarsa microalgae balls to the wastewater. Shake or stir evenly to prevent Tarsa microalgae from agglomerating or precipitating. The wastewater dilution is 25%, 50% and 75% V / V. Set the light intensity to 4000-5000 Lux, the light-dark ratio to 12:12 hours, and the temperature to 25°C. Treat for a total of 12 days. Use air circulation or shaking to prevent microalgae from settling or sticking to the wall. Samples were taken to measure dry weight, chlorophyll, biomass yield and composition, etc.

[0042] On the 12th day, the dry weight and chlorophyll production of Tarsa algae were 2.307 g / L and 5.46 mg / L, respectively, and the protein, polysaccharide and oil production were 99.1 μg / mL, 176.2 μg / mL and 25.6 μg / mL, respectively.

[0043] Example 3

[0044] Effect of Talsa algae on the treatment of dairy wastewater

[0045] 1. The isolation and culture of Talsa algae were the same as those in Example 1.

[0046] 2. Treatment of dairy farm wastewater: The treatment steps were similar to those in Example 2. After 12 days of treatment, samples were taken to determine the chemical oxygen demand, total nitrogen, total phosphorus, and ammonia nitrogen contents.

[0047] Its total phosphorus removal rate can reach 99.9%; the ammonia nitrogen removal rate is as high as 95.6%; the COD removal rate is as high as 75.8% and the TN removal rate is as high as 85.6% ( Figure 4 ).

[0048] Table 1 Variation of various parameters of dairy farm wastewater

[0049]

[0050]

[0051] Example 4

[0052] Experimental study on the effect of returning the liquid of Tarsha silk algae ZSL-NS5 to the fields

[0053] The algae liquid after harvest can be used as biofertilizer and returned to the field. In order to verify the promoting effect of Tarsa silk algae on crops, four groups of fertilization experiments were set up: CK was the control group without fertilizer, T1 was the group with chemical fertilizer (25 kg / mu), T2 was the group with Tarsa silk algae fertilizer (40 L (algae concentration 1.35×10 7 T3 was a combination of chemical fertilizer and Talsa algae fertilizer (25 kg chemical fertilizer / mu + 40 L organic fertilizer / mu). The crop was rapeseed (Brassica chinensis L.).

[0054] The experimental results showed that compared to CK, algae fertilizer significantly increased alkaline nitrogen, but reduced total nitrogen content in the soil. However, total nitrogen content in rapeseed increased, indicating that algae fertilizer can promote nutrient absorption and utilization by crops. The organic fertilizer plus algae fertilizer group showed increases in both soil nutrients and rapeseed nutrient base. For example, compared to CK and chemical fertilizer group T1, soil alkaline nitrogen in group T3 increased by 20.5% and 6.9%, respectively.

[0055] Table 1 Effects of algae fertilizer on soil nutrients and rapeseed basic nutrients

[0056]

[0057] As demonstrated in the above examples, the provided strain of Talsa algae, with excellent growth properties, exhibits exceptional pollution tolerance and pollutant purification capabilities in dairy wastewater at varying dilutions. Furthermore, the Talsa algae can produce high levels of carbohydrates and extracellular polymeric substances while purifying pollutants. This present invention provides an excellent strain for the production of microalgal carbohydrates and its application in aquaculture wastewater treatment.

[0058] The above description is only a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any simple modification, equivalent replacement and improvement made by any technician familiar with the profession to the above embodiment without departing from the scope of the technical solution of the present invention and based on the technical essence of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

[0059] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand 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 polysaccharide-producing strain of Talsa algae, characterized by: The name is Tarshas algae ZSL-NS5, and the classification name is: Desertifilum sp., deposited on November 7, 2024, deposited with China Center for Type Culture Collection, with the deposit number CCTCC NO. M 20242470, and deposited at Wuhan University, Wuhan, China.

2. Use of the polysaccharide-producing Tarsa algae ZSL-NS5 according to claim 1 in the treatment of dairy farm wastewater.

3. A method for treating dairy farm wastewater using the polysaccharide-producing Talsa algae according to claim 1, characterized in that: Wastewater treatment requires four steps: coarse filtration, centrifugation, refiltration, and dilution. The inoculation ratio of ZSL-NS5 algae liquid to wastewater is 1:5 by volume. The algae liquid is centrifuged at 7,000-8,000 rpm for 10 minutes. After centrifugation, the upper culture medium is discarded and the Talsa silk algae mud is added to the dairy farm wastewater and shaken or stirred evenly. Treatment conditions are: light intensity 4000-5000 Lux, light-dark ratio 12:12 hours, temperature 25°C, treatment cycle can be 8-12 days, use air circulation or shaking to prevent microalgae from settling or sticking to the wall; In the treatment of dairy farm wastewater by Tarsa Silk Algae ZSL-NS5, the wastewater can be sterilized or not. The dilution ratio of anaerobic tank effluent and oxidation pond wastewater is 1-5 times, and the dilution ratio of stabilization pond wastewater is 1-2 times.

4. Use of the polysaccharide-producing Talsa algae according to claim 1 as a biofertilizer.

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