Protein-producing coniothrix sp. And application of coniothrix sp. In wastewater treatment of dairy farm

Abstract

The invention discloses a Trichotoma sp. ZSL-NS2 strain capable of producing protein and application of the Trichotoma sp. ZSL-NS2 strain in wastewater treatment of a dairy farm. The Trichotoma sp. ZSL-NS2 strain named as the Trichotoma sp. ZSL-NS2 strain is preserved in the China Center for Type Culture Collection on November 7, 2024, and the preservation number is CCTCC NO.M20242469. The Trichotoma sp. ZSL-NS2 strain is named as the Trichotoma sp. ZSL-NS2 strain. The algal strain is separated from soil beside a wastewater storage pool of a dairy farm, is purified and enriched under a high-salt condition (the salinity is 2.0-5.0%), has relatively strong wastewater tolerance and pollutant purification capacity, has the removal rates of more than 70% of COD, 85% of ammonia nitrogen, 75% of TN and 95% of conventional pollutants respectively, and particularly has the most obvious removal effect on ammonia nitrogen and total phosphorus. In addition, the protein content of the conneronea is as high as 52%, and the conneronea can be used as a protein raw material for high-value application.

Description

Technical Field

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

[0002] At present, the world economy is developing rapidly, and the scale of agriculture and breeding is expanding. Dairy farming accounts for a large part of livestock and poultry farming. The expansion of the number and scale of dairy farms has led to a large amount of wastewater generated and discharged from dairy farms. The main pollutants in dairy farm wastewater, such as COD, total nitrogen and SS, are very high; however, unlike other livestock and poultry wastewater, the salt content in dairy farm wastewater is high. This is because during the dairy farming process, dairy cows need to supplement salt or lick salt bricks every day. This is a need for the growth and development of ruminants. It can regulate the pH value of green feed and rumen, improve feed digestibility and utilization, and avoid chronic acidosis, anorexia, metabolic abnormalities, decreased milk production, and low-acid alcohol-positive milk production. Damage to dairy cows. According to statistics, the salt content in dairy farm wastewater is 0.6-2.2%. When the salt concentration of sewage reaches 0.5% or more, it is called "high-salt sewage."

[0003] At present, most dairy farms use anaerobic fermentation and oxidative storage treatment. Although this method has low economic cost, the effect of treating wastewater is average and is greatly affected by season and weather. Some researchers have also proposed adding chemical agents or combining them with electrochemistry. Although these methods will improve the treatment effect, they will also greatly increase the economic cost and have no resource regeneration value. In recent years, microalgae have been widely used in wastewater treatment due to their advantages such as fast growth rate, high photosynthesis efficiency, strong adaptability to the environment, high economic feasibility, and strong ability to produce biomass.

[0004] Studies have shown that compared with conventional microbial treatment methods (e.g., anaerobic-anoxic-aerobic, A 2 Compared with conventional wastewater treatment technology, microalgae wastewater treatment technology does not require additional carbon sources, thereby reducing the investment cost of wastewater treatment processes; it does not produce sludge bulking, excess sludge and secondary pollution problems; at the same time, microalgae can also reduce greenhouse gas emissions. Most importantly, the microalgae biomass produced by this technology can be used as raw materials for high-value products such as biofuels, feed, and fertilizers, realizing waste resource utilization. According to estimates, 158.2g of biomass products can be obtained by treating 1 liter of sewage, thereby saving about 0.14-1.19 yuan in microalgae breeding costs, while fixing about 208.4g of CO2, assimilating about 10.09g of nitrogen and absorbing about 1.376g of phosphorus, bringing environmental benefits.

[0005] However, there are still many problems in using microalgae to treat livestock and poultry wastewater. On the one hand, microalgae cultivated by traditional methods have poor tolerance and insufficient wastewater purification capabilities, making it difficult to adapt to the complex environment of dairy farm wastewater; therefore, we should continue to look for new microalgae varieties that can adapt to the characteristics of dairy farm wastewater. On the other hand, the subsequent utilization value of microalgae biomass is very important. Traditionally cultivated or commercially available microalgae have insufficient protein content, and methods of increasing the protein content of microalgae by adding nutrients usually have high economic costs. Therefore, screening out new microalgae varieties with higher protein content will be of practical significance to meet the market demand for protein and enhance the subsequent utilization value of microalgae. Summary of the invention

[0006] The present invention selects a new microalgae variety that can improve tolerance, water purification ability and biomass protein content according to the characteristics of dairy farm wastewater. The present invention provides a strain of Nodosiline asp. ZSL-NS2 and a method for treating dairy farm wastewater, providing technical support for microalgae treatment of aquaculture wastewater.

[0007] The present invention solves the technical problem by adopting the following technical solutions:

[0008] A salt-tolerant, protein-producing filamentous algae, named Nodosilineas ZSL-NS2, with a Latin name of Nodosilineas p., a deposit date of November 7, 2024, a deposit institution of China Center for Type Culture Collection, a deposit number of CCTCC NO.M20242469, and a deposit address of Wuhan University, Wuhan, China.

[0009] Moreover, the growth medium of the filamentous algae is: 1.5 g of sodium nitrate, 0.04 g of dipotassium hydrogen phosphate, 0.075 g of magnesium sulfate heptahydrate, 0.036 g of calcium chloride dihydrate, 0.006 g of ammonium ferric citrate, 0.006 g of citric acid, 0.001 g of disodium ethylenediaminetetraacetic acid, 0.02 g of 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 at 121°C under high pressure for 20 min.

[0010] Moreover, the formula of the trace element mother solution is: 2.86 g / L boric acid, 1.86 g / L manganese chloride tetrahydrate, 0.22 g / L zinc sulfate heptahydrate, 0.08 g / L copper sulfate pentahydrate, 0.39 g / L sodium molybdate dihydrate; 0.05 g / L cobalt nitrate hexahydrate, and water is added to make the volume to 1000 mL.

[0011] Moreover, the filamentous algae produces protein, polysaccharide and carotene.

[0012] Moreover, the dry weight of the knot algae can reach up to 2.167 g / L, and the protein, polysaccharide and oil yields are 155, 128.5 and 86.8 μg / mL respectively; the dry weight and chlorophyll yields are 2.14 g / L and 5.28 mg / L respectively.

[0013] Application of salt-tolerant protein-producing filamentous algae in dairy wastewater treatment.

[0014] Application of salt-tolerant protein-producing filamentous algae in the preparation of dairy wastewater preparations.

[0015] The method for producing protein by fermentation of salt-tolerant protein-producing filamentous algae is characterized in that: filamentous algae ZSL-NS2 is cultured in a culture medium to obtain protein. The culture medium is: 1.5g sodium nitrate, 0.04g dipotassium hydrogen phosphate, 0.075g magnesium sulfate heptahydrate, 0.036g calcium chloride dihydrate, 0.006g ammonium ferric citrate, 0.006g citric acid, 0.001g disodium ethylenediaminetetraacetic acid, 0.02g sodium carbonate, 1mL trace element mother solution, water is added to make the volume 1000mL, and the pH is adjusted to about 7.1.

[0016] A method for treating dairy farm wastewater using salt-tolerant protein-producing filamentous algae, wherein the dairy farm wastewater is taken, and filamentous algae liquid in the logarithmic growth period of the salt-tolerant protein-producing filamentous algae is taken, wherein the volume ratio of the liquid to the wastewater is 1:5, and 50 mL of the liquid is added to 250 mL of the wastewater, corresponding to an algae dry weight of 0.2 g / L;

[0017] The algae liquid is centrifuged at 7000-8000r / min for 10 minutes, the upper culture medium is removed after centrifugation, and the filamentous algae balls are added to the wastewater, and the balls are shaken or stirred evenly to prevent the filamentous algae from agglomerating or settling;

[0018] The wastewater dilution is 25-75%, the light intensity is set to 4000-5000 Lux, the light-dark ratio is 12:12 hours, and the temperature is 20-28 ° C. Use air circulation or shaking to prevent microalgae from settling or sticking to the wall.

[0019] The advantages and positive effects of the present invention are:

[0020] 1. The present invention screened out a strain of ZSL-NS2 with strong pollution resistance and purification ability from the habitat near dairy farm wastewater. Its growth performance in wastewater is better than that in culture medium, and its growth rate in wastewater is faster. It can adapt to dairy farm wastewater with high concentrations of ammonia nitrogen and COD (COD is 4633.3±50.2mg / L; ammonia nitrogen is 451.5±26.7mg / L; total nitrogen is 695.8±40.9mg / L).

[0021] 2. The filamentous algae in the present invention has a strong ability to purify wastewater. Due to its rich extracellular polymers and its own filamentous characteristics, the filamentous algae has obvious effects on removing chromaticity, COD, ammonia nitrogen, TN and TP in wastewater, which is better than traditional pure cultured microalgae varieties.

[0022] 3. The protein content of the filamentous algae provided by the present invention reaches more than 50%, which is higher than that of traditional legumes and conventional microalgae, and can provide high-quality protein resources for replenishing aquaculture bait or animal feed. Microalgae can also produce other products, such as polysaccharides, carotene, etc., which can achieve the purpose of biomass recovery while treating wastewater.

[0023] 4. The present invention provides a wastewater treatment method that does not require additional carbon source supply and energy consumption, and can significantly reduce the economic cost of dairy farm wastewater treatment. In addition, the treated wastewater contains microalgae cells and active substances secreted by them, which can irrigate farmland or lawns in the field, promote crop yields, and prevent plant diseases. Compared with other methods for treating dairy farm wastewater, the treatment method of the present invention is more efficient, more stable in performance, economically beneficial, and meets the requirements of clean production. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Microscopic photograph of microalgae cell morphology.

[0025] Figure 2 Maximum Parsimony tree constructed based on 16S rDNA fragment sequences (microalgae of the present invention marked with a red frame).

[0026] Figure 3 Growth indicators of filamentous algae (a: dry weight; b: chlorophyll; c: component content; d: component ratio).

[0027] Figure 4 Purification effect of filamentous algae on dairy wastewater with different concentrations.

[0028] Figure 5 This is a schematic diagram of the weight change of grass carp in Example 4. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below through specific examples. The following examples are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto.

[0030] 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.

[0031] The dairy farm wastewater of the present invention is taken from the anaerobic fermentation effluent of Tianjin Shenchi Dairy Farm. The filamentous algae of the present invention is separated from the soil beside the wastewater storage tank of the above-mentioned manufacturer. Specifically, a protein-producing microalgae isolated from dairy farm wastewater has better tolerance and treatment effect on wastewater, and has been preserved (the preservation number is CCTCCNO.M20242469), and the preservation address is: Wuhan University, Wuhan, China, and has very good application prospects.

[0032] The salt-tolerant, protein-producing filamentous algae is named Nodosilineas ZSL-NS2, with the Latin name: Nodosilineas p. The preservation date is November 7, 2024, the preservation institution is China Center for Type Culture Collection, the preservation number is CCTCCNO.M20242469, and the preservation address is Wuhan University, Wuhan, China.

[0033] Example 1

[0034] Screening and identification of filamentous algae

[0035] (1) Isolation and cultivation of filamentous algae

[0036] Take the soil next to the wastewater storage tank of the dairy farm, put 10g of soil sample into 20mL of high salt solution, with a salt content of 0.5%, NaCl:Na2HCO3=1:1 (mass ratio), stir well and let it stand for 10min. Take an appropriate amount for coating plate operation, use BG11 solid culture medium (salt content of 0.2%, NaCl:Na2HCO3 mass ratio of 1:1), after a single algae grows on the plate, pick a single algae to the growth medium. Culture for 15 days at a temperature of 25℃, 3000Lux of light, and a light-dark ratio of 16:8 hours to obtain the algae solution of the knotted filamentous algae. After obtaining the algae solution of the single algae species knotted filamentous algae ZSL-NS2, expand the culture until there is enough algae solution (volume ratio during expansion: algae solution: culture medium = 1:5).

[0037] 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.

[0038] 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.

[0039] (2) Morphological identification of microalgae

[0040] A small amount of sample was observed under an Olympus BX53 microscope and the image was acquired using an Olympus DP80 ( Figure 1 ). The filaments are solitary, unbranched, blue-green, sometimes forming nodules, usually with a colorless thin sheath. The filaments are immobile, and the cell walls are slightly or obviously constricted. The cell length is usually greater than the width, sometimes the length is equal to the width, 2-4μm long, 1-2.5μm wide, with peritrichous thylakoids, and no air sacs.

[0041] (3) Phylogenetic analysis

[0042] Phylogenetic tree based on 16S rDNA sequences of cells ( Figure 2 ) showed that the microalgae sample Contig 1 was closely related to the genus Nodosilinea and was located on the same evolutionary branch.

[0043] Example 2

[0044] Growth performance of filamentous algae in dairy wastewater

[0045] (1) The separation and expansion of filamentous algae were the same as in Example 1.

[0046] (2) Wastewater (COD: 4633.3±50.2mg / L; ammonia nitrogen: 451.5±26.7mg / L; total nitrogen: 695.8±40.9mg / L, salinity: 2.1‰, pH=7.9) needs to be treated in four steps: coarse filtration, centrifugation, refiltration, and dilution.

[0047] Take the algae liquid of the logarithmic growth period of the filamentous algae. The volume ratio of the algae liquid to the wastewater is 1:5. Add 50mL of the algae liquid to 250mL of the wastewater, and the corresponding algae dry weight is 0.2g / L. The algae liquid is centrifuged at 7000-8000r / min for 10 minutes. After centrifugation, the upper culture medium is removed, and the filamentous algae balls are added to the wastewater, and shake or stir evenly to avoid the filamentous algae agglomeration or precipitation. The wastewater dilution is 25%, 50% and 75%. Set the light intensity to 4000-5000Lux, the light-dark ratio to 12:12 hours, and the temperature to 25℃. Treat for a total of 12 days. Use air circulation or shaking to prevent the microalgae from settling or sticking to the wall. Take samples to measure dry weight, chlorophyll, biomass yield and composition, etc.

[0048] On the 12th day, the dry weight of filamentous algae could reach a maximum of 2.167 g / L, and the protein, polysaccharide and oil yields were 155, 128.5 and 86.8 μg / mL, respectively; the dry weight and chlorophyll yields were 2.14 g / L and 5.28 mg / L, respectively.

[0049] Example 3

[0050] Effect of filamentous algae on treatment of dairy wastewater

[0051] (1) The separation and expansion of filamentous algae were the same as in Example 1.

[0052] (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 content.

[0053] Its total phosphorus removal rate can reach 99.48%; the ammonia nitrogen removal rate is as high as 93.83%; the COD removal rate is as high as 82.3% and the TN removal rate is as high as 88.7% ( Figure 4 ).

[0054] Example 4

[0055] BG11 was used to expand the culture of filamentous algae, and then dried at low temperature of 60℃ and ground into algae powder. Algae powder was used to replace 0% (T0), 10% (T10), 20% (T20), 30% (T30) and 50% (T50) of the basic feed of grass carp. The experimental fish were purchased from an aquaculture farm. Feeding was stopped 24 hours before the formal experiment. 150 healthy grass carps of uniform size (initial body weight was about 100.±2.3g) were selected and randomly assigned to 15 fish tanks. There were 5 treatment groups in the experiment, 3 parallel fish tanks in each treatment group, and 10 fish in each fish tank.

[0056] The results showed that adding algae powder could increase the weight of grass carp by an average of 0%-10.5%, among which replacing 20% ​​of the original feed had the best effect, and replacing 0%-50% was beneficial to the growth and development of grass carp.

[0057] From the above examples, it can be seen that the provided filamentous algae with excellent growth can show excellent pollution resistance and pollutant purification ability in dairy wastewater of different dilutions. In addition, the filamentous algae can produce biomass while purifying pollutants. The present invention provides an excellent algae strain for the production of microalgae-related products and in the process of aquaculture wastewater treatment.

[0058] Table 1 Variation of dairy farm sewage indicators with treatment time

[0059]

[0060]

[0061] 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.

[0062] 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 salt-tolerant protein-producing filamentous algae, characterized in that: The name is Nodosilinea ZSL-NS2, the Latin name is Nodosilinea sp., the preservation date is November 7, 2024, the preservation institution is China Center for Type Culture Collection, the preservation number is CCTCC NO.M20242469, and the preservation address is Wuhan University, Wuhan, China.

2. The salt-tolerant protein-producing filamentous algae according to claim 1, characterized in that: The growth medium of ZSL-NS2 is: 1.5 g sodium nitrate, 0.04 g dipotassium 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 at 121°C under high pressure for 20 min.

3. The salt-tolerant protein-producing filamentous algae according to claim 2, characterized in that: The formula of the trace element mother solution is: 2.86 g / L boric acid, 1.86 g / L manganese chloride tetrahydrate, 0.22 g / L zinc sulfate heptahydrate, 0.08 g / L copper sulfate pentahydrate, 0.39 g / L sodium molybdate dihydrate; 0.05 g / L cobalt nitrate hexahydrate, and water is added to make the volume to 1000 mL.

4. The salt-tolerant protein-producing filamentous algae according to claim 1, characterized in that: The filamentous algae produces protein, polysaccharide and carotene.

5. The salt-tolerant protein-producing filamentous algae according to claim 1, characterized in that: The dry weight of the knot algae can reach up to 2.167 g / L, and the protein, polysaccharide and oil yields are 155 μg / mL, 128.5 μg / mL and 86.8 μg / mL respectively; the dry weight and chlorophyll yields are 2.14 g / L and 5.28 mg / L respectively.

6. Use of the salt-tolerant protein-producing filamentous algae according to claim 1 in the treatment of dairy farm wastewater.

7. Use of the salt-tolerant protein-producing filamentous algae according to claim 1 in the preparation of dairy farm wastewater preparations.

8. The method for producing protein by fermentation of salt-tolerant protein-producing filamentous algae according to claim 1, characterized in that: The protein was obtained by culturing ZSL-NS2 in a culture medium containing: 1.5 g sodium nitrate, 0.04 g dipotassium 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, and water was added to make the volume to 1000 mL.

9. A method for treating dairy farm wastewater using the salt-tolerant protein-producing filamentous algae according to claim 1, characterized in that: Take dairy farm wastewater, take salt-tolerant protein-producing filamentous algae, take filamentous algae liquid in the logarithmic growth period, the volume ratio of algae liquid to wastewater is 1:5, add 50mL algae liquid to 250mL wastewater, corresponding to the algae dry weight of 0.2g / L; The algae liquid is centrifuged at 7000-8000r / min for 10 minutes, the upper culture medium is removed after centrifugation, and the filamentous algae balls are added to the wastewater, and the balls are shaken or stirred evenly to prevent the filamentous algae from agglomerating or settling; The wastewater dilution is 25-75%, the light intensity is set to 4000-5000Lux, the light-dark ratio is 12:12 hours, the temperature is 20-28℃, and air circulation or shaking is used to prevent microalgae from settling or sticking to the wall.

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