Resource utilization method of duck breeding waste liquid

Abstract

The invention provides a resource utilization method of duck breeding waste liquid, and belongs to the technical field of biology. The method comprises the following steps: adding a seed solution of saccharomycetes W-1 and W-2 into a sterilized basic culture medium containing the duck breeding waste liquid to carry out primary fermentation on a saccharomycetes group, then carrying out secondary fermentation by using a seed solution of lactobacillus plantarum by using a supernatant obtained after the primary fermentation as a substrate, and carrying out two times of fermentation to obtain the duck breeding waste liquid. The saccharomycetes W-1 and W-2 as well as single-cell protein and fermentation supernate of lactobacillus plantarum are obtained, and the fermentation supernate can be further used for improving saline-alkali soil. According to the method, treatment and resource development and utilization of the breeding waste liquid are combined, reduction, harmlessness and recycling of waste are achieved while environmental pollution is reduced, closed-loop treatment of the breeding waste liquid is achieved, and good economic benefits and social effects are achieved.

Description

Technical Field

[0001] The present invention relates to a method for resource utilization of duck breeding waste liquid, and particularly to a method for obtaining fermentation supernatant for saline-alkali soil improvement by fermenting duck breeding waste liquid. The present invention belongs to the field of biotechnology. Background Art

[0002] With the growth of the global population and the improvement of living standards, the global demand for animal protein is also increasing continuously. The pace of transformation of the livestock and poultry breeding industry towards scale, standardization, industrialization and intensification has been gradually accelerating, providing a large amount of animal protein for humans and playing an important role in meeting the protein needs of humans. Due to its good meat quality and short feeding cycle, meat ducks occupy an important position in the livestock and poultry breeding industry in China. However, with the rapid development of intensive meat duck breeding, a large amount of breeding waste liquid cannot be treated in time, causing serious environmental pollution problems.

[0003] China has a long history of meat duck breeding industry and has maintained the first global meat duck breeding scale for many consecutive years. In 2022, the global meat duck slaughter volume reached 5.59 billion, among which the meat duck slaughter volume in China accounted for about 69% of the world's total, ranking first in the world. However, with the continuous expansion of the meat duck breeding industry chain, the production volume of meat duck feces has increased sharply. According to statistics, the production volume of meat duck feces in China has reached 65 million tons in 2020. Meat ducks are rectum animals with relatively short digestive tracts and low digestion and utilization rates of feed. A large number of undigested nutrients are discharged with feces and urine mixed; in addition, to keep the duck house clean and hygienic, the duck house needs to be flushed regularly. Duck breeding waste liquid mainly comes from various sewage and excreta generated during the meat duck breeding process, which contains a large amount of organic matter, duck manure and feed residues. The ammonia nitrogen content, phosphorus content, suspended solids and organic matter concentration in the wastewater are high and the carbon-nitrogen ratio is seriously unbalanced. If a large amount of untreated wastewater is discharged, it will cause a series of pollution problems such as water eutrophication and soil property change in the environment of lakes, rivers, farmlands, etc. Duck breeding waste liquid has the characteristics of high water content and difficult solid-liquid separation. The commonly used solid manure treatment methods and liquid manure treatment methods both have limitations. Therefore, selecting a treatment method according to the characteristics of duck breeding waste liquid can not only improve the efficiency of resource recovery and utilization, but also be an important method to solve environmental organic pollution.

[0004] Single cell protein (SCP) refers to microbial protein obtained by culturing microorganisms with various substrates under certain conditions. Its production generally involves large-scale culturing of single-celled organisms such as bacteria, yeast, microalgae, and simple multi-celled organisms such as molds using domestic waste, industrial production wastewater, and various industrial and agricultural production wastes and other nutrients. In SCP, 30%-80% is protein, and it also contains other nutrients such as carbohydrates, fats, vitamins, and minerals. Compared with traditional animal and plant proteins, it has the advantages of high production efficiency, wide production raw materials, being unaffected by regions, seasons, and climates, high nutritional value, and high protein content. It is a new type of protein alternative resource very suitable for use as a feed protein additive.

[0005] Soil salinization is one of the main types of soil degradation. Due to the high pH value and rich soil colloid content in saline-alkali soil, its salinization is accompanied by alkalization. The content of soil organic matter and nutrients is low, and trace elements are lacking. The nutrient elements required by crops form precipitates due to the high soil pH and are difficult to be absorbed and utilized by crops, resulting in crop nutrient deficiency and being unfavorable for cultivation, hindering agricultural development. Common methods for improving saline-alkali soil include soil replacement, deep loosening of the soil, planting salt-tolerant crops, subsurface drainage technology, and soil conditioners, etc. Among them, the use of soil conditioners is a new method developed on the basis of modern industry and different from traditional soil improvement. The primary task of saline-alkali soil improvement and utilization is to improve soil quality. Organic fertilizers contain a large amount of organic matter, which plays a buffering role for anions and cations in the soil. Therefore, using organic fertilizers can improve the soil buffering capacity, improve the physical and chemical properties and salt composition of the soil, and reduce soil alkalinity. The fermentation supernatant after removing the thallus has a low pH value and contains residual organic matter, which can be used as a conditioner for saline-alkali soil improvement.

[0006] Although duck-raising waste liquid exerts great pressure on the environment, it itself contains a large amount of organic matter and nutrient elements such as nitrogen, phosphorus, and potassium. However, by reusing its waste resources and using it as a basic medium for the fermentation production of beneficial microorganisms, the production cost of single cell protein can be greatly reduced, and environmental pollution can be reduced through biodegradation. By reusing its organic matter for soil improvement, turning waste water into treasure, and realizing its value.

[0007] The present invention obtains single cell protein by using duck-raising waste liquid for the propagation of beneficial microorganisms and uses the fermentation supernatant for saline-alkali soil improvement. By combining the treatment of breeding waste liquid with resource development and utilization, while reducing environmental pollution, the reduction, harmlessness, and resource utilization of waste are realized, with economic and social benefits, and the closed-loop treatment of breeding waste liquid is achieved. Summary of the Invention

[0008] The object of the present invention is to provide a method for the resource utilization of duck-raising waste liquid. The method combines the treatment of duck-raising wastewater with the recycling of waste resources. Using duck-raising waste liquid as the basic culture medium, it is used for the production of single-cell protein and the improvement of saline-alkali soil. It can not only effectively degrade COD and NH 4+ -N in duck-raising waste liquid, but also produce single-cell protein. At the same time, the supernatant after fermentation can also be used for the improvement of saline-alkali soil. Overall, a relatively complete resource utilization of duck-raising waste liquid is achieved, avoiding waste of resources.

[0009] To achieve the above object, the present invention adopts the following technical means:

[0010] The present invention uses duck-raising waste liquid as the fermentation culture medium, and ten kinds of yeasts are used to ferment in duck-raising waste liquid under different conditions (sterilized / unsterilized). The fermentation basic strains are screened by the viable count of yeasts after fermentation. Then, the best yeast is used as the fermentation basic strain and combined with four other yeasts with a large number of viable counts. The fermentation strains are re-screened with the viable count after fermentation as the index. After obtaining the optimal yeast flora for fermentation, to increase the yield of single-cell protein, seven factors including carbon source addition amount, nitrogen source addition amount, inoculation amount, rotation speed, dilution ratio, temperature, and culture time that have a significant impact on the viable count after fermentation are screened through Plackett-Burman test. After comprehensive consideration of the results, it is decided to select three factors, namely culture time, liquid-to-material ratio, and carbon source addition amount, for subsequent response surface condition optimization. The best level range of each factor is determined by the climbing experiment, and taking this result as the center of the level, a central composite experiment of 3 factors and 3 levels is carried out through Box-Behnken in Design-Expert 13 to obtain the best fermentation conditions, and the predicted value is 2.30732678×10 8 cfu / mL. Fermentation is carried out under the best conditions optimized by the response surface experiment, which has a good fit with the predicted value, and this model is established. After the fermentation broth is centrifuged to remove single-cell protein, the remaining supernatant is fermented again with Lactobacillus plantarum. Taking the inoculation amount, temperature, and culture time as the optimization conditions, on the basis of the results obtained from the climbing experiment, a central composite experiment of 3 factors and 3 levels is carried out through Box-Behnken to optimize the fermentation conditions and obtain the best values of each condition. This extreme point is OD = 2.247. The actual fermentation is carried out under the conditions optimized by the response surface experiment, and the average OD value of the bacteria is 2.228, which has a good fit with the predicted value, proving the feasibility of the model. The precipitate part after the secondary fermentation is separated into single-cell protein, and the improvement effect of the supernatant on saline-alkali soil is determined by selecting the best addition amount through a climbing experiment with the pH value as the index.

[0011] Based on the above research, the present invention proposes a method for the resource utilization of duck-raising waste liquid. The method includes adding the seed solutions of yeasts W-1 and W-2 to the sterilized basal medium for the first fermentation of yeast flora, and then using the supernatant obtained after the first fermentation as a substrate and the seed solution of Lactiplantibacillus plantarum for the secondary fermentation. After two fermentations, single-cell proteins of yeasts W-1 and W-2 and Lactiplantibacillus plantarum and the fermentation supernatant are obtained, and the fermentation supernatant is used for the improvement of saline-alkali soil.

[0012] Among them, the basal medium includes duck-raising waste liquid, maltose and water.

[0013] Among them, the yeast W-1 is Candida tropicalis W-1, which is preserved in the China Center for Type Culture Collection, and its strain preservation number is CCTCC NO: M 20242182.

[0014] Among them, the yeast W-2 is Candida tropicalis W-2, taxonomically named Candida tropicalis W-2, which is preserved in the China Center for Type Culture Collection, and its strain preservation number is CCTCC NO: M 20242183.

[0015] Among them, the Lactiplantibacillus plantarum is Lactiplantibacillussp. WX-1, which is preserved in the China Center for Type Culture Collection, and its strain preservation number is CCTCC NO: M 20242501.

[0016] Among them, preferably, the basal medium is obtained by diluting the duck-raising waste liquid and water in a volume ratio of 1:2, and then adding 3-5% w / v maltose to the diluted duck-raising waste liquid. More preferably, 4% w / v maltose is added to the diluted duck-raising waste liquid.

[0017] Among them, preferably, the duck-raising waste liquid is a mixture of duck manure and urine and water obtained by leaching in a duck farm.

[0018] Among them, preferably, the pH value of the duck-raising waste liquid is 7.93 ± 0.06; the chemical oxygen demand (COD) is 70931 ± 720 mg / L; NH 4+ -N is 84.47 ± 1.73 mg / L.

[0019] Among them, preferably, the single-cell protein is obtained by drying the precipitate obtained by centrifugation after the basic medium is fermented by yeast strains W-1 and W-2 and the precipitate obtained by centrifugation after fermentation by Lactobacillus plantarum WX-1 at a low temperature of 35°C.

[0020] Among them, preferably, for the first fermentation, the seed solutions of yeast strains W-1 and W-2 are added to the sterilized basic medium, and fermented at a temperature of 30-35°C at 140-160 r / min for 37 h. The ratio of yeast strains W-1 and W-2 is 1:1, and the inoculation amount is 5-10% v / v; for the second fermentation, the supernatant obtained by removing the cells from the fermentation broth fermented by yeast strains W-1 and W-2 by centrifugation is used for the fermentation of Lactobacillus plantarum WX-1, and cultured at 30.9°C and 130 r / min for 48 h. The inoculation amount of Lactobacillus plantarum WX-1 is 1-5% v / v.

[0021] Among them, preferably, the fermentation supernatant obtained after the two fermentations is applied to the saline-alkali soil at 0.4 mL / g to improve it.

[0022] Among them, preferably, the method includes the following steps:

[0023] (1) Strain activation: Take out the yeast strains W-1 and W-2 and Lactobacillus plantarum WX-1 preserved at -80°C. Under aseptic operation conditions, use an inoculation loop to pick out each strain from the centrifuge tube and inoculate them into the sterilized medium respectively; Yeast strains W-1 and W-2 are respectively inoculated into YPD medium, and Lactobacillus plantarum WX-1 is cultured in MRS medium. Each bottle of medium is 100 mL, and cultured at 30°C at 130 r / min for 24 h for activation to obtain the activated bacterial liquid of each strain for standby;

[0024] (2) Seed solution preparation: Adjust the activated bacterial liquids of yeast strains W-1, W-2 and Lactobacillus plantarum WX-1 to seed solutions with OD = 1 using YPD and MRS media respectively;

[0025] (3) First fermentation of yeast flora: Mix the seed solutions of yeast strains W-1 and W-2 in a volume ratio of 1:1 and inoculate them into the sterilized basic medium according to an inoculation amount of 5%-10% v / v. Then, culture them in a shaker at 30°C at 140-160 r / min for 37 h. After the culture is completed, obtain the fermentation broth; Centrifuge the fermentation broth at 5000 r / min. The precipitate obtained after centrifugation is dried at 35-40°C to obtain the single-cell protein of yeast strains W-1 and W-2, and the separated supernatant is used for the next fermentation;

[0026] (4) Second fermentation of Lactiplantibacillus plantarum: Using the fermentation supernatant obtained after the first fermentation as the substrate, Lactiplantibacillus plantarum WX-1 was used for secondary fermentation; the Lactiplantibacillus plantarum seed liquid was inoculated into the supernatant at an inoculation amount of 1-5% v / v, and cultured in a shaker at 30.9 °C and 130 r / min for 48 h. After the culture was completed, the precipitate was obtained by centrifugation at 5000 r / min. The precipitate obtained after centrifugation was dried at 35-40 °C to obtain single-cell protein, and the supernatant was used for saline-alkali soil improvement;

[0027] (5) Saline-alkali soil improvement: After two fermentations, the finally obtained supernatant was applied to the soil at an addition amount of 40% of the weight of the saline-alkali soil, and it was stirred evenly to achieve the improvement of the saline-alkali soil.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] The main purpose of the present invention is to reuse waste resources. By using a yeast strain flora composed of Candida tropicalis W-1 and W-2 and Lactiplantibacillus plantarum WX-1 to perform gradient fermentation on duck-raising waste liquid. Yeast is a commonly used strain in wastewater treatment and has many achievements in wastewater treatment; Lactiplantibacillus plantarum WX-1 is a strain isolated from duck-raising waste liquid in the early stage and also has good practicability in reducing the pH of the fermentation broth and producing single-cell protein as a protein substitute resource. Therefore, compared with the traditional method, using this method to treat duck-raising waste liquid has a relatively complete resource utilization of duck-raising waste liquid, combines the treatment of wastewater with the resource development and utilization, while reducing the COD, pH and ammonia nitrogen in duck-raising waste liquid, a relatively high yield of single-cell protein can also be obtained after the fermentation is completed, and its fermentation supernatant also achieves a certain effect in improving saline-alkali soil. By using the method of the present invention, economic benefits can be obtained while reducing the cost of pollutants. In addition, the present invention optimizes the fermentation conditions of duck-raising waste liquid, enabling microorganisms to grow rapidly therein, shortening the fermentation cycle and reducing the fermentation cost.

[0030] The present invention combines the treatment of breeding waste liquid with the resource development and utilization, while reducing environmental pollution, realizes the reduction, harmlessness and resource utilization of waste, realizes the closed-loop treatment of breeding waste liquid, and has good economic benefits and social effects. Description of the Drawings

[0031] Figure 1 is the biomass of different strain combinations in the fermentation broth;

[0032] Among them, A is the biomass of yeast under different fermentation conditions; B is the biomass of yeast combination in the fermentation broth;

[0033] Figure 2 Effect of different factors on biomass

[0034] Among them, a is carbon source, b is carbon source addition amount, c is material-liquid ratio, d is nitrogen source, e is nitrogen source addition amount, f is rotation speed, g is culture time, h is temperature, and I is inoculum size;

[0035] Figure 3 Response surface analysis diagram;

[0036] Among them, A: maltose, B: material-liquid ratio, C: culture time;

[0037] Figure 4 Effect of different factors on cell density;

[0038] Among them, a is culture time; b is inoculum size, c is temperature;

[0039] Figure 5 Response surface analysis diagram;

[0040] Among them, A: culture time, B: inoculum size, C: temperature.

[0041] Strain preservation information:

[0042] The isolated Candida tropicalis was named Candida tropicalis W-1, and its taxonomic name was Candida tropicalis W-1. It was preserved in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. Its strain preservation number was CCTCC NO: M 20242182, and the preservation time was October 14, 2024.

[0043] Another isolated Candida tropicalis was named Candida tropicalis W-2, and its taxonomic name was Candida tropicalis W-2. It was preserved in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. Its strain preservation number was CCTCC NO: M20242183, and the preservation time was October 14, 2024.

[0044] The isolated Lactiplantibacillus plantarum was named Lactiplantibacillus sp. WX-1, and its taxonomic name was Lactiplantibacillus sp. WX-1. It was preserved in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. Its strain preservation number was CCTCC NO: M 20242501, and the preservation time was November 11, 2024. Detailed implementation manners

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0046] Example 1: Optimization of fermentation conditions

[0047] 1. Materials and methods

[0048] 1.1 Source of experimental strains

[0049] The strains used in the experiment were ten yeast strains W-1 to W-10 isolated and screened by the research group. It was found through research that each strain was a highly efficient strain with good potential for industrial application in aspects such as plant growth promotion, biological control, and pollutant control. Moreover, each strain could grow rapidly in duck manure wastewater and had good removal effects on ammonia nitrogen, phosphorus, organic matter, etc.

[0050] 1.2 Physicochemical properties of aquaculture wastewater

[0051] The duck manure wastewater used in the experiment was from a duck farm in Chifeng, Inner Mongolia and was stored at room temperature before use. The physicochemical properties of the duck manure wastewater are shown in Table 1.

[0052] Table 1 Physicochemical properties of the original duck manure wastewater

[0053]

[0054] 1.3 Culture medium

[0055] YPD medium: 10 g of yeast extract powder, 20 g of peptone, 20 g of glucose, 1000 mL of water, and 20 g of agar was added for solid medium.

[0056] MRS medium: 10 g of peptone, 10 g of beef extract powder, 5 g of yeast extract powder, 5 g of glucose, 5 g of sodium acetate, 2 g of diammonium citrate, 1 g of Tween 80, 2 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate heptahydrate, 20 g of calcium carbonate, 1000 mL of water, and 20 g of agar was added for solid medium.

[0057] 1.4 First fermentation of duck manure wastewater

[0058] 1.4.1 Batch fermentation to determine the culture medium conditions and the best yeast flora

[0059] First, different treatments of the culture medium were carried out, namely the selection between substrate sterilization (high-temperature and high-pressure steam sterilization, temperature 121 °C, time 20 min) and non-sterilization. After activating the ten existing yeast strains W-1 to W-10 in the laboratory, they were inoculated into the fermentation medium. After fermentation, the viable yeast count in the medium was calculated, and this was used as an indicator to select the culture medium conditions. All fermentation tests were inoculated with 10% (volume fraction, the same below) of the inoculum and cultured with shaking at 30 °C and 130 r / min for 24 h (the same below).

[0060] Under the selected culture medium conditions, the yeast flora was gradually optimized, and the fermentation strain flora was selected. According to the results of the culture medium treatment condition experiments, they were arranged in ascending order, and 4 yeast strains with higher viable counts were selected for the best yeast flora selection test. The strain with the largest number of viable counts was used as the primary fermentation bacterium for subsequent dual-bacterium and triple-bacterium combined fermentation tests. The four selected yeasts were arranged and combined based on the primary yeast for the dual-yeast fermentation test to select the best dual-yeast combination with the best fermentation performance under the determined favorable yeast W-1. Following the same rules, triple-yeast and quadruple-yeast mixing tests were carried out. Each group of tests was set up with 3 replicates. After that, the best yeast flora was determined by evaluating the maximum biomass of the preferred yeast and the combination during fermentation as the evaluation index.

[0061] 1.4.2 Single-factor experiment

[0062] The initial fermentation medium and fermentation conditions were as follows: duck-raising waste liquid: water = 1.5, rotation speed 130 r / min, temperature 30 °C, fermentation time 48 h. The types of carbon sources, carbon source addition amounts, nitrogen source types, nitrogen source addition amounts, inoculation amounts, rotation speeds, liquid-to-material ratios, temperatures, and culture times were set as variables, and the optimal range of each factor was determined through the biomass of the strain. Each treatment had 3 parallels.

[0063] 1.4.3 Plackett-Burman experimental design

[0064] The results of the single-factor experiment were analyzed, and the Plackett-Burman experiment designed by Design-Expert 13.0 software was used to determine the significant factors affecting the viable count after fermentation. The carbon source addition amount (A), nitrogen source addition amount (B), inoculation amount (C), rotation speed (D), dilution ratio (E), temperature (F), and culture time (G) were used as factors to be optimized. Each factor had two levels, +1 and -1, for the high and low factors respectively. The viable count after the fermentation of duck-raising waste liquid was used as the response value for screening, and the level settings of each component are shown in Table 2.

[0065] Table 2 Level design of each group in the Plackett-Burman experiment

[0066]

[0067] 1.4.4 Optimization of the Fermentation Conditions of Duck Waste Liquid by Yeast Flora Using Response Surface Methodology

[0068] Using the central composite design model of Box-Behnken and combining the significant factors screened by the Plackett-Burman test: the addition amount of carbon source (maltose) (A), dilution ratio (B), and culture time (C), these three factors were used as the factors to be optimized, and the viable cell count (Y) was used as the response value. A three-factor and three-level response surface analysis optimization experiment was carried out to optimize the liquid fermentation process of duck waste liquid. Each group was subjected to 3 parallel tests. The experimental factor levels of Box-Behnken are shown in Table 3.

[0069] Table 3 Response Surface Test and Level Design

[0070]

[0071] 1.5 Secondary Fermentation of Fermentation Supernatant

[0072] 1.5.1 Single-Factor Experiments

[0073] Taking three factors such as A inoculation amount (2%, 4%, 6%, 8%, 10%), B temperature (27 °C, 30 °C, 33 °C, 36 °C, 39 °C), and C culture time (12 h, 24 h, 36 h, 48 h, 60 h) as the single-factor experimental levels, and using the absorbance of the fermentation broth after fermentation as the evaluation index, the effects of different fermentation conditions on the growth ability of Lactobacillus plantarum in the secondary fermentation medium were investigated. The inoculation amount of 10%, rotation speed of 130 r / min, temperature of 30 °C, and culture time of 48 h were set as the single-factor basic conditions. When studying a certain factor, it was determined that other factors remained unchanged. Each treatment had 3 parallels.

[0074] 1.5.2 Optimization of the Secondary Fermentation Conditions of Duck Waste Liquid by Response Surface

[0075] Based on the results of the single-factor experiments, a response surface test model with inoculation amount, temperature, and culture time as variables. According to the experimental design principle of Box-BenhnkenDesign, the experimental data was processed by Design-Expert 13.0 software. The experimental factors and levels are shown in Table 4.

[0076] Table 4 Response Surface Test Design

[0077]

[0078] 1.6 Test Measurement Indexes

[0079] 1.6.1 Water Quality Indexes

[0080] Measure the pH, total nitrogen, total carbon, and ammonia nitrogen content of the finally obtained sample after fermentation.

[0081] 1.6.2 Microbiological indicators

[0082] Use a hemocytometer to count the number of viable bacteria after fermentation. The formula for calculating the number of viable bacteria is: Number of viable bacteria = N × 16 × 25 × dilution factor. Where N is the average number of viable bacteria in the small squares.

[0083] 1.6.3 Determination of single-cell protein indicators

[0084] In the following specific embodiments of the present invention, the test methods for dry weight, yield, and crude protein content are as follows:

[0085] (1) Dry weight of single-cell protein: Unit is g / L

[0086] Weigh 1.00 L of the fermentation broth, centrifuge at 8000 r / min for 10 min to obtain the bacterial cells, add 1 L of water to resuspend evenly, and centrifuge again at 8000 r / min for 10 min to obtain the wet bacterial cells. Weigh its mass as m 1 ; Take 5.000 g from m1 and dry it at 103 ± 2 °C according to the method of GB / T 6435-2014 "Determination of Moisture in Feed" to obtain the dry bacterial cells, and weigh its weight m 2 (Accurate to 4 decimal places).

[0087] Dry cell weight DCW = m 2 / 5.000 × m 1 / 1.00

[0088] (2) Yield: Unit is g / (L·h)

[0089] Yield = Dry weight of single-cell protein ÷ Fermentation time (fermentation cycle)

[0090] (3) Crude protein content

[0091] According to GB / T 6432-2018 "Determination of Crude Protein in Feed - Kjeldahl Method", after digestion, ammonia distillation, and titration, obtain the crude protein content (mass fraction).

[0092] (4) Hydrolyzed amino acid content

[0093] Ferment under the optimal conditions. Centrifuge the fermentation product to obtain the precipitate, dry it at 30 - 35 °C to obtain the single-cell protein product, and measure the amino acid content in accordance with GB 5009.124.

[0094] 2 Results and analysis

[0095] 2.1 First fermentation of duck waste liquid

[0096] 2.1.1 Determination of culture medium treatment conditions, identification and screening of the best fermentation strains

[0097] A single-yeast fermentation test of duck waste liquid was carried out in batch fermentation. As Figure 1 shown in A, the biomass of ten yeasts after 24 h of culture under sterilized and non-sterilized conditions is as Figure 1 shown in B. The biomass of each strain under sterilized conditions is significantly higher than that under non-sterilized conditions. Under sterilized conditions, after the culture ended, the biomass of strains W-1, W-2, W-6, and W-8 was relatively high, and the biomass of W-1 was the highest. Therefore, W-1 was selected as the preferred strain for subsequent selection of strain flora. The selection of strain flora is as Figure 1 shown in B. The biomass of the strain flora AC (combination of W-1 and W-2) after fermentation is higher than that of other strain combinations and single strains. Finally, the strain flora of W-1 and W-2 was selected as the fermentation strain for this experiment for subsequent optimization of fermentation conditions. The strains were all identified by strain morphology and 18S rDNA classification, and the identification list is shown in Table 5

[0098] Table 5 Test strains

[0099]

[0100] 2.1.2 Single-factor experiments

[0101] The ranges of factors such as carbon source addition amount, nitrogen source addition amount, inoculation amount, rotation speed, liquid-to-material ratio, temperature, and culture time were optimized to determine the optimal value points of each factor. The factors and levels of the single-factor experiments are shown in Table 6, and the results are as Figure 2 shown

[0102] Table 6 Factor levels of single-factor experiments

[0103]

[0104] 2.1.3 Plackett-Burman experiments

[0105] The Plackett-Burman experiment is a method that can obtain the most significant factors in the experiment with the fewest experiments and can identify the main influencing factors from various influencing factors. Through the Plackett-Burman experiment, which of the seven factors of carbon source addition amount (A), nitrogen source addition amount (B), inoculation amount (C), rotation speed (D), dilution ratio (E), temperature (F), and culture time (G) affects the viable cell count after fermentation was screened, and subsequent experiments were carried out. The results of the Plackett-Burman experiment and the variance analysis are shown in Tables 7 and 8

[0106] Table 7 Plackett-Burman experimental design and response values

[0107]

[0108]

[0109] Table 8 Results of variance analysis of Plackett-Burman test

[0110]

[0111] Note: "*" indicates significant influence (P<0.05)

[0112] The results of the Plackett-Burman test and variance analysis on improving the comprehensive score of yeast biomass by fermenting duck waste liquid with yeast are shown in Table 7 and Table 8. The model analysis results show that the P value of the model is 0.0105 (<0.05), indicating that the model is significant. The coefficient of determination R 2 = 0.9624, indicating that the model has a good correlation.

[0113] Taking the response value Y as the dependent variable and each fermentation condition as the independent variable, the Design-Expert 13.0 software was used to fit the P-B experimental data, and the first-order regression equation obtained was Y = 1.691×10 8 +3.691×10 7 A + 3.374×10 7 B + 1.707×10 6 C - 4.576×10 6 D - 4.601×10 7 E + 5.505×10 7 F + 8.248×10 7 G; Among all the influencing factors, (A) carbon source addition amount, (B) rotation speed, (E) temperature, (F) liquid-to-material ratio, (G) culture time are significant influencing factors. Among them, (E) temperature is negative, indicating that it has a negative effect on the response value Y (yeast biomass), and (A) carbon source addition amount, (B) rotation speed, (F) liquid-to-material ratio, (G) culture time are positive, and they have a positive effect on the response value Y (yeast biomass). Sort the four significant influencing factors in descending order: (G) culture time > (F) liquid-to-material ratio > (A) carbon source addition amount > (B) rotation speed. Therefore, (G) culture time, (F) liquid-to-material ratio, and (A) carbon source addition amount are selected as the main effects that have a significant impact on the fermentation result, and a response surface test is carried out.

[0114] 2.1.4 Optimization of fermentation conditions by response surface analysis method

[0115] (1) Fitting and variance analysis of the second-order regression model

[0116] The results of the response surface test and the variance analysis of the regression model are shown in Tables 9 and 10. Using Design Expert 13.0 software, the comprehensive score data in Table 9 was fitted by quadratic multiple regression, and the regression equation is as follows: Y = 1.720×10 8 + 3.993×10 7 A + 7.072×10 7 B + 2.651×10 6 C - 1.121×10 7 AB - 7.781×10 5 AC + 4.895×10 6 BC - 3.168×10 7 A 2 - 1.867×10 7 B 2 - 7.291×10 7 C 2 . The F value of the comprehensive score model is 15.82, and P = 0.0007 (<0.01), indicating a significant difference; the result of the lack-of-fit term is P = 0.7959 (>0.05), indicating no significant difference, which means that the proportion of abnormal errors in the actual fitting of the obtained equation is very small, and the residuals are all caused by random errors, with little interference to the experiment. The correlation coefficient R 2 = 0.9531, indicating a significant linear relationship between the investigated variables and the response surface. The adjusted coefficient R 2 adj = 0.8929, indicating a good fit between the predicted value of the model and the actual measured value, indicating that the model can be used for the optimization of the fermentation conditions of yeast in duck manure wastewater.

[0117] Table 9 Response surface test design scheme and results

[0118]

[0119]

[0120] Through the significance analysis of the test results, among the first-order terms, B has a highly significant effect on the biomass of yeast (P<0.001), and A has a relatively significant effect on the biomass of yeast (P<0.01). Among the second-order terms, C 2 has a highly significant effect (P<0.001), and A 2 has a significant effect (P<0.05). It can be seen from the F value test that the order of the influence of each factor on the comprehensive score is: B > A > C, that is, the ratio of liquid to material > the addition amount of maltose > the culture time. In summary, the obtained regression model equation can be well used for the analysis and prediction of the biomass of yeast cells in duck manure wastewater.

[0121] Table 10 Variance analysis of the regression equation

[0122]

[0123]

[0124] (2) Response surface analysis and verification of the best prediction value

[0125] Use Design-Expert to analyze the fitted regression equation and draw the response surface analysis diagrams corresponding to the main factors, as Figure 2 shown. Each response surface can reflect a certain interaction between two main factors. The highest point of the surface is the highest point of the viable bacteria count after flocculation, which is the optimal fermentation condition.

[0126] Analyze the equation through Design-Expert software. The results show that when the addition amount of carbon source (maltose) is 3.906% (about 4.0%), the ratio of material to liquid is 2, and the culture time is 37.192 h, there is a maximum viable bacteria count of 2.30732678×10 8 cfu / mL in the regression equation. Then, under the conditions optimized by the response surface experiment, the actual fermentation is carried out, and the viable bacteria counts are 2.345×10 8 , 2.375×10 8 , 2.175×10 8 cfu / mL respectively, and the average value is 2.29×10 8 cfu / mL, which has a good fit with the predicted value, proving the feasibility of the model.

[0127] 2.2 Secondary fermentation of the fermentation supernatant

[0128] 2.2.1 Single-factor experiment

[0129] Optimize the inoculation amount, temperature, and culture time of Lactiplantibacillus plantarum WX-1 (Lactiplantibacillus sp. WX-1, the strain preservation number is CCTCC NO: M 20242501) during the fermentation process through single-factor experiments to determine the optimal range. The factors and levels of each single factor are shown in Table 11.

[0130] Table 11 Factors and levels of the single-factor experiment

[0131]

[0132] 2.2.2 Optimization of fermentation conditions by response surface analysis method

[0133] (1) Fitting of the quadratic regression model and variance analysis

[0134] According to the results of single-factor experiments, the inoculation amount, fermentation temperature, and culture time were used as the three factors for the response surface experiment. Using the Design-Expert 13.0 response surface design software, a Box-Behnken Design experiment was designed with three factors and three levels. The fermentation experiment was carried out according to the design results. The response surface design scheme and results are shown in Table 12. Multiple quadratic regression fitting was performed on the data presented in Table 12, and the regression equation of cell density and each factor was obtained as follows: Y = 2.12 + 0.1426A - 0.0088B + 0.0326C + 0.0358AB + 0.0349AC - 0.0131BC - 0.0489A 2 - 0.0027B 2 - 0.0844C 2 。

[0135] Table 12 Response surface experiment design factor levels

[0136]

[0137] The results of the variance analysis of the regression equation and partial regression coefficients are shown in Table 13. It can be seen from Table 13 that this model (P < 0.01) is extremely significant. The P value of the lack-of-fit term of the model is 0.7236 (P > 0.05), and the difference is not significant, that is, the equation fits the experiment well. Therefore, the selection and establishment of this model are reasonable. From the significance test of the regression equation coefficients, it can be seen that the first-order term A culture time (P < 0.01) has an extremely significant difference in the effect on the cell density after fermentation; the B inoculation amount (P > 0.05) and C temperature (P > 0.05) have no significant difference in the effect on the cell density after fermentation; the second-order terms A 2 culture time (P < 0.05) and C 2 temperature (P < 0.05) have a significant effect; the interaction terms AC, AB, and BC (P > 0.05) have no significant effect. According to the different F values, the factors affecting the cell density after the secondary fermentation of duck manure wastewater are arranged in ascending order as follows: fermentation time > temperature > inoculation amount.

[0138] Table 13 Box-Behnken test regression model variance analysis

[0139]

[0140] (2) Response surface analysis and verification of the best prediction value

[0141] Design-Expert was used to analyze the fitted regression equation, and the response surface analysis diagrams corresponding to the main factors were drawn, as Figure 2 shown. Each response surface can reflect a certain interaction between two main factors. The highest point of the surface is the highest point of the viable bacteria count after flocculation, which is the best fermentation condition.

[0142] The equation was analyzed using Design-Expert software. The results showed that when the temperature was 30.963 °C, the inoculum size was 3%, and the culture time was 48 h, there was a maximum OD value in the regression equation, and this extreme value was OD 600 = 2.247. Then, fermentation was carried out under the conditions optimized by the response surface test. The OD values were 2.215, 2.237, and 2.233 respectively, and the average value was 2.228, showing good fitting with the predicted value, which proved the feasibility of the model.

[0143] 2.3 Changes in each index of the fermentation broth after removing the bacterial cells from the duck-raising waste liquid after fermentation

[0144] The changes in each index before and after fermentation are shown in Table 14.

[0145] Table 14 Changes in each index before and after fermentation

[0146]

[0147] Example 2 Experiment on the improvement of saline-alkali soil by the fermentation supernatant

[0148] 1 Materials and methods

[0149] 1.1 Test materials

[0150] The fermentation broth of the duck-raising waste liquid after two fermentations in Example 1 was centrifuged. The supernatant obtained after centrifuging to remove the bacterial cells was used as the modifier for improving saline-alkali soil. The main physical and chemical properties of this reagent are shown in Table 15.

[0151] Table 15 Basic physical and chemical properties of the fermentation supernatant of duck-raising waste liquid

[0152]

[0153] Tested soil: Daqing soda saline-alkali soil in Heilongjiang Province and soil from a certain place were selected, and their basic physical and chemical indexes are shown in Table 16.

[0154] Table 16 Basic physical and chemical properties of the tested soil

[0155]

[0156] Tested variety: The tested rice seeds were Longjing 31, which were sourced from the Heilongjiang Academy of Agricultural Sciences.

[0157] 1.2 Test methods

[0158] 1.2.1 Improvement of saline-alkali soil by duck-raising waste liquid

[0159] A total of 6 treatment groups were set up in the experiment. The treatments were applying 40%, 80%, 120%, 160%, 200% of the weight of saline-alkali soil and the control without application, and the codes were 0.4, 0.8, 1.2, 1.6, 2.0, CK respectively. Each treatment had 3 replicates. Each treatment group was mixed evenly with duck-raising waste liquid according to the application amount, dried at a low temperature of 35°C, and the pH value was measured after drying.

[0160] 1.2.2 Pot experiment

[0161] Through pot experiments, using rice as an indicator plant, the improvement effect of fermented duck-raising waste liquid on saline-alkali soil was verified.

[0162] The saline-alkali soil was treated with duck-raising waste liquid according to the treatment groups set in 1.2.1. The duck-raising waste liquid was mixed evenly with the saline-alkali soil and dried at a low temperature, then filled into seedling trays. Select plump rice seeds, disinfect them in 75% alcohol for 5 minutes, and rinse them with deionized water; then soak the rice seeds in 5% NaClO for 3 minutes, rinse them with sterile distilled water, and finally place them in a petri dish containing filter paper moistened with sterile water, and place them in a constant temperature incubator at 30°C in the dark for germination. After the seeds germinated, select rice sprouts with consistent growth for sowing. Sow 3 seeds in each pot, and each treatment had 10 replicates. After culturing for 30 days, samples were taken to measure and analyze the basic growth indexes such as the plant height and root length of rice seedlings.

[0163] 1.2.3 Index determination

[0164] The root length and plant height were measured with a ruler and a digital display vernier caliper. The dry mass of the plants was measured with a balance after drying in an oven. The chlorophyll content was determined by the anhydrous ethanol extraction method. The root system was scanned with a ScanMaker i800 Plus root system scanning and analysis system.

[0165] 2 Results and analysis

[0166] 2.1 Improvement effect of the fermentation supernatant of duck-raising waste liquid on saline-alkali soil

[0167] The pH value of the saline-alkali soil treated with duck-raising waste liquid was measured, and the results are shown in Table 17. The experiment proved that there was a linear relationship between the application amount of duck-raising waste liquid and the pH of saline-alkali soil. The more the application amount, the lower the pH value of saline-alkali soil.

[0168] Table 17 Effects of different application amounts of duck-raising waste liquid on the pH value of saline-alkali soil

[0169]

[0170] 2.2 Effects of different treatment groups on the main traits of rice

[0171] The results are shown in Table 18. The experiments prove that rice grows differently in saline-alkali soils under different treatments. Compared with CK, when the application rate is 40%, each index has increased to varying degrees. The plant height, total root length, dry weight, root surface area, and root tip number have increased by 39.76%, 3.39%, 39.29%, 6.64%, and 11.11% respectively. This shows that when cultivating rice in saline-alkali soil, the rice grows best under the treatment condition with an application rate of 40%. While reducing the pH value of the saline-alkali soil, it promotes the growth of rice.

[0172] Table 18 Effects of Different Treatments on the Main Traits of Rice

[0173]

[0174] 3 Experimental Conclusions

[0175] The effect of using the fermentation supernatant of duck-raising waste liquid to improve saline-alkali soil is significant. Considering the impact on the growth of rice, when the application rate of duck-raising waste liquid is 40% of the weight of saline-alkali soil, while reducing the pH value of the saline-alkali soil, it promotes the growth of rice. Therefore, considering comprehensively, the application rate of 40% is selected as the optimal usage amount.

[0176] Example 3 Resource Utilization of Duck-Raising Waste Liquid

[0177] (1) Strain activation: Take out the yeast strains W-1 and W-2 and Lactobacillus plantarum WX-1 preserved at -80°C. Under aseptic operation conditions, use an inoculation loop to pick out each strain from the centrifuge tube and inoculate them into the sterilized culture medium respectively; Yeast strains W-1 and W-2 are respectively inoculated into YPD medium, and Lactobacillus plantarum WX-1 is cultured in MRS medium. Each bottle of culture medium is 100 mL, and cultured at 30°C at 130 r / min for 24 h for activation to obtain the activated bacterial liquid of each strain for standby;

[0178] The basic culture medium is obtained by diluting the duck-raising waste liquid and water at a volume ratio of 1:2, and then adding 4% w / v maltose to the diluted duck-raising waste liquid;

[0179] (2) Seed liquid preparation: Adjust the activated bacterial liquid of yeast strains W-1, W-2 and Lactobacillus plantarum WX-1 to seed liquid with OD = 1 using YPD and MRS media respectively;

[0180] (3) First fermentation of yeast flora: The seed solutions of yeast W-1 and W-2 were mixed at a volume ratio of 1:1 and inoculated into the sterilized basic medium at an inoculation amount of 5%-10% v / v. Then, it was cultured on a shaker at 140-160 r / min at 30 °C for 37 h. After the culture, the fermentation broth was obtained. The fermentation broth was centrifuged at 5000 r / min. The precipitated part after centrifugation was dried at 35-40 °C to obtain the single-cell protein of yeast W-1 and W-2. The separated supernatant was used for the next fermentation;

[0181] (4) Second fermentation of Lactobacillus plantarum: Using the fermentation supernatant obtained after the first fermentation as the substrate, Lactobacillus plantarum WX-1 was used for the second fermentation. The seed solution of Lactobacillus plantarum was inoculated into the supernatant at an inoculation amount of 1-5% v / v and cultured on a shaker at 30.9 °C and 130 r / min for 48 h. After the culture, it was centrifuged at 5000 r / min to obtain the precipitate. The precipitated part after centrifugation was dried at 35-40 °C to obtain the single-cell protein, and the supernatant was used for saline-alkali soil improvement;

[0182] (5) Saline-alkali soil improvement: After two fermentations, the finally obtained supernatant was applied to the soil at an addition amount of 40% of the weight of the saline-alkali soil, and it was stirred evenly to achieve the improvement of the saline-alkali soil.

Claims

1. A method for resource utilization of duck farming wastewater, characterized in that: The method comprises the steps of adding the seed liquid of yeasts W-1 and W-2 to the sterilized basic culture medium to carry out the first fermentation of the yeast flora, and then using the supernatant obtained after the first fermentation as a substrate and the seed liquid of Lactobacillus plantarum to carry out the second fermentation, and after the two fermentations, obtaining the single cell protein of yeasts W-1 and W-2 and Lactobacillus plantarum and the fermentation supernatant, and the fermentation supernatant is used for the improvement of saline-alkali soil; Wherein, the basic culture medium comprises duck waste liquid, maltose and water; Wherein, the yeast W-1 is Candida tropicalis W-1, which is deposited in China Center for Type Culture Collection, and its strain deposit number is CCTCC NO:M 20242182; Wherein, the yeast W-2 is Candida tropicalis W-2, which is classified and named as Candida tropicalis W-2, and is deposited in the China Center for Type Culture Collection, and its strain collection number is CCTCC NO: M 20242183; Wherein, the plant lactobacillus is plant lactobacillus WX-1 (Lactiplantibacillus sp. WX-1), which is preserved in China Center for Type Culture Collection, and its strain preservation number is CCTCC NO: M 20242501.

2. The method according to claim 1, characterized in that The basic culture medium is obtained by diluting duck waste liquid and water in a volume ratio of 1:2, and then adding 3-5% w / v maltose to the diluted duck waste liquid. Preferably, 4% w / v maltose is added to the diluted duck waste liquid.

3. The method according to claim 1 or 2, characterized in that The duck farming waste liquid is a mixture of duck excrement and urine obtained by leaching in a duck farm and water.

4. The method according to claim 3, characterized in that The pH value of the duck waste liquid is 7.93±0.06; the chemical oxygen demand (COD) is 70931±720 mg / L; NH 4+ -N is 84.47±1.73mg / L.

5. The method according to claim 1, characterized in that The single cell protein is obtained by drying at 35°C the precipitate obtained by centrifugation after fermentation of yeast W-1 and W-2 and the precipitate obtained by centrifugation after fermentation of Lactobacillus plantarum WX-1.

6. The method according to claim 1, characterized in that The first fermentation is to add the seed liquid of yeast W-1 and W-2 to the sterilized basic culture medium, and ferment at 30-35°C and 140-160r / min for 37 hours, the ratio of yeast W-1 and W-2 is 1:1, and the inoculation amount is 5-10% v / v; the second fermentation is to use the supernatant obtained after the fermentation liquid obtained by yeast W-1 and W-2 fermentation is centrifuged to remove the bacteria for Lactobacillus plantarum WX-1 fermentation, and culture at 30.9°C and 130r / min for 48 hours, and the inoculation amount of Lactobacillus plantarum WX-1 is 1-5% v / v.

7. The method according to claim 1, characterized in that The fermentation supernatant obtained after the two fermentations is applied to the saline-alkali soil at 40% of the weight of the saline-alkali soil to improve it.

8. The method according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) Activation of strains: The yeast strains W-1 and W-2 and Lactobacillus plantarum WX-1 stored at -80°C were taken out, and each strain was picked out from the centrifuge tube using an inoculation loop under aseptic operation conditions, and inoculated into sterilized culture media respectively; yeasts W-1 and W-2 were inoculated into YPD culture media respectively, and Lactobacillus plantarum WX-1 was cultured in MRS culture media, with 100 mL of culture media per bottle, and cultured at 30°C and 130 r / min for 24 h for activation, to obtain the activated bacterial solution of each strain for use; (2) Seed solution preparation: The activated bacterial solutions of yeast W-1, W-2 and Lactobacillus plantarum WX-1 were adjusted to a seed solution with OD=1 using YPD and MRS medium, respectively; (3) First fermentation of yeast flora: yeast W-1 and W-2 seed liquids were mixed in a volume ratio of 1:1 and inoculated into a sterilized basal culture medium at a rate of 5%-10% v / v, and then cultured in a shaking incubator at 30° C. and 140-160 r / min for 37 h, and fermentation liquid was obtained after the culture was completed; the fermentation liquid was centrifuged at 5000 r / min, and the precipitate obtained after centrifugation was dried at 35-40° C. to obtain single cell protein of yeast W-1 and W-2, and the separated supernatant was used for the next fermentation; (4) Second fermentation with Lactobacillus plantarum: The fermentation supernatant obtained after the first fermentation is used as a substrate, and Lactobacillus plantarum WX-1 is used for secondary fermentation; Lactobacillus plantarum seed liquid is inoculated into the supernatant at a bacterial inoculation amount of 1-5% v / v, and cultured in a shaking incubator at 30.9° C. and 130 r / min for 48 h. After the culture is completed, centrifugation is performed at 5000 r / min to obtain a precipitate, and the precipitate obtained after centrifugation is dried at 35-40° C. to obtain single cell protein, and the supernatant is used for saline-alkali soil improvement; (5) Improvement of saline-alkali soil: After two fermentations, the supernatant obtained is applied to the soil at a rate of 40% of the weight of the saline-alkali soil and stirred evenly to improve the saline-alkali soil.

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