Method for producing single-cell protein for feed from livestock and poultry manure biogas slurry

Abstract

The invention discloses a method for producing single-cell protein for feeding from livestock and poultry manure biogas slurry, comprising the following steps: (1) preparation of manure biogas slurry: subjecting livestock and poultry manure to anaerobic fermentation by CSTR, adjusting the pH of the liquid after solid-liquid separation to 5-7, and preparing manure biogas slurry; (2) inoculating fungi: inoculating mixed fungal spores into the biogas slurry for cultivation; (3) recovery of fungi and production of single-cell protein: after the cultivation, separating mycelium and drying at 50-60°C to obtain single-cell protein. The invention ferments edible fungi with livestock and poultry manure biogas slurry as a substrate, generates fungal mycelium from organic matter in the biogas slurry, and recovers the mycelium to produce single-cell protein for feed protein supplement, while reducing the content of pollutants such as ammonia nitrogen in the biogas slurry, and only through a primary mixed fermentation reaction, not only single-cell protein with high economic added value is obtained, but also the purpose of purifying manure biogas slurry is achieved, the overall operating cost is low, low-carbon and environmentally friendly, and has broad application prospects in the field of resource utilization of livestock and poultry manure biogas slurry.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological fermentation and sewage treatment, and more specifically, relates to a method for producing single-cell protein for feed from livestock and poultry manure biogas slurry. Background Art

[0002] Livestock and poultry farming is the main source of animal food raw materials, contributing 18% of global calorie intake and 25% of protein intake, and plays a vital role in world food security. With the continued growth of the global population and the increase in income of people in developing countries, global demand for meat and milk is expected to increase by 57% and 48% respectively between 2020 and 2050, and global livestock production is expected to increase by 21% in 2025 compared with 2010. Based on the current utilization rate of livestock and poultry feed, the above expected expansion will lead to an increase in global demand for feed dry matter from 6.0 to 7.3 billion tons. However, protein supplements have always been one of the most expensive and scarce ingredients in animal feed dry matter. At present, high-quality plant proteins (such as soybean meal) are the main source of protein in livestock and poultry diets worldwide (especially in China). About 85% of soybeans are processed into soybean oil or other foods each year, and almost all (about 97%) of the soybean meal produced is used for animal feed. However, the use of proteins from these edible plant materials (such as soybeans, oilseeds and grains) to feed animals has caused competition between livestock and poultry and humans for food, and is bound to further threaten human food security. In addition, there are 2.5 billion hectares of farmland used to produce livestock and poultry feed worldwide, of which about 700 million hectares (equivalent to 14% of global agricultural land and 50% of arable land) are facing the pressure of land use conversion. The sole use of plant protein as animal feed has become unsustainable. Therefore, seeking alternative feeds and new feed proteins to replace or supplement the current supply of plant protein to meet the growing demand has become an urgent problem for the global animal husbandry industry.

[0003] The development of animal husbandry is not only related to food security, but also has a huge impact on the environment. The pressure of environmental protection has become a bottleneck problem restricting the development of animal husbandry in the world, especially in China, together with the cost of feed. In particular, the harmless treatment and resource utilization of livestock manure wastewater have become the focus that needs to be solved urgently. At present, the use of biological treatment methods can only achieve the primary utilization of livestock manure wastewater through anaerobic digestion. The biogas slurry produced later has high ammonia nitrogen and high CODcr. Direct return to the field not only has low utilization rate, but also pollutes natural water bodies. Although the traditional physicochemical method or biological nitrification-denitrification process can be used to treat the biogas slurry to meet the discharge standards, the biggest defect of these methods is that the high ammonia nitrogen in the biogas slurry is converted into nitrogen gas for direct discharge, which not only has high treatment costs, but also causes great waste of nitrogen sources, which does not meet the goal of resource utilization. Therefore, how to achieve efficient treatment and resource utilization of livestock and poultry manure slurry is still a problem worthy of attention.

[0004] Single cell protein (SCP) is a general term for microbial dry matter with high protein content as well as fat, carbohydrates, vitamins and minerals. Compared with plant-based proteins, the most important advantage of single cell protein is that it can be produced in geographical areas that do not support agricultural farming. It is predicted that by 2050, single cell protein products will replace 10-19% of traditional plant and / or animal proteins worldwide.

[0005] Generally speaking, protein is composed of 16% nitrogen (mass-N / mass-protein). Theoretically, all kinds of organic wastewater, including livestock and poultry manure and biogas slurry, have the potential to produce SCP. At present, existing results have shown that bacteria, fungi and algae can produce SCP using livestock and poultry manure, food waste or industrial organic wastewater residues, but most of them are fermented with a single strain, which not only requires sterilization of the wastewater substrate, but also requires the addition of multiple nutrient substrates; and the fermentation time is long, and the dry mass and protein content of the produced bacteria are not high; in addition, the efficiency of the strain in assimilating ammonia nitrogen in biogas slurry is low, and the amount of ammonia nitrogen after treatment is still higher than the discharge or reuse standard; in particular, the strains used are mostly newly isolated and screened strains, and the safety of their unknown metabolites has great defects. SCP needs to be harmlessly treated before use, which further increases the cost of use. Therefore, there are few reports on results with practical application value.

[0006] The production of single-cell protein from organic wastewater can be produced by fermentation of a single microorganism or by fermentation of a mixed microbial community. Compared with single fermentation, SCP based on mixed microbial fermentation has more advantages: first, the mixed fermentation SCP is composed of biomass from different groups of microorganisms. Although the growth of these microorganisms is based on wastewater as the main nutrient source, their protein types are different, and the types of SCP amino acids produced are richer and more diverse; second, unlike single bacteria, the process based on mixed culture does not require pre-sterilization of wastewater. When interference occurs, the mixed bacterial community can show strong resistance and resilience, thereby ensuring the stability of the process at a lower operating cost; finally, the mixed bacterial community has a synergistic metabolic effect and can fully assimilate various carbon and nitrogen sources in the substrate. In particular, the complementary removal of diversified cell proliferation and inhibitory substances has a positive effect on improving the yield and quality of single-cell protein.

[0007] Microorganisms such as fungi, bacteria, and microalgae have fast growth rates and high biological efficiency, and have high application value in the production of SCP using livestock and poultry manure and biogas slurry. However, from the analysis of nutrients, it can be seen that the crude protein content of microalgae and bacterial SCP is the highest, but the ash content of microalgae itself is high, the digestibility is poor, the tolerance of bacteria is insufficient, and the nucleic acid content in SCP is high, so the step of denucleic acid must be added, which increases the cost of use. The SCP content of yeast in fungi is high, but its individual is too small to be easily separated, especially when yeast is used in wastewater treatment, which will greatly increase the cost of recycling. Filamentous fungi have the characteristics of acid resistance, sugar resistance, and high salt resistance, and can adapt to the environment well. In particular, filamentous fungi have been closely related to food fermentation for hundreds of years, and the research on metabolic mechanisms has been very clear, which has great advantages in industrial application. In addition, most filamentous fungal proteins have unique aromatic substances, and the mycelium produced has a good meaty texture. Especially in liquid fermentation, the mycelium can form a spherical floc structure under the action of shear force, which not only does not require centrifugal separation, but also has a biological flocculation effect on suspended matter in water.

[0008] For a long time, the biggest cost of the livestock and poultry farming industry comes from the treatment of feed and manure. Especially with the increasing pressure of the current food crisis and the demand for environmental protection, how to strengthen the pollution prevention and control of the breeding industry, promote the resource utilization of livestock and poultry manure and the water saving and emission reduction of breeding tail water, and develop ecological circular breeding are still urgent problems to be solved. Therefore, the present invention provides a method for producing single-cell protein for feed with low operating cost, simple operation, low carbon and environmental protection, which not only effectively improves the resource utilization of livestock and poultry manure slurry, but also the produced single-cell protein and purified slurry can be directly used as protein supplements for livestock and poultry and production water for breeding farms, so as to achieve the purpose of circular breeding. Summary of the invention

[0009] The present invention provides a method for producing single-cell protein for feed from livestock and poultry manure slurry, wherein mixed fungi are used to ferment organic components in the slurry to prepare single-cell protein, and the single-cell protein is directly used in farms as a feed protein supplement. In addition, pollutants such as ammonia nitrogen and phosphorus in the slurry are effectively removed through assimilation and biological flocculation, and the COD and suspended solids levels are reduced, so that the purified slurry can directly supplement the production water of the farm. The method for treating slurry of the present invention is low-carbon and environmentally friendly, has a simple process, and is low in cost. It can effectively improve the resource utilization efficiency of livestock and poultry manure slurry and realize circular farming.

[0010] The present invention is specifically achieved through the following technical solutions:

[0011] (1) Preparation of manure slurry: The livestock manure is introduced into an anaerobic fermentation tank for CSTR anaerobic fermentation. The liquid after solid-liquid separation is manure slurry. The slurry is introduced into a primary fermentation tank and the pH is adjusted to 5-7 to serve as a fermentation substrate.

[0012] (2) Inoculation of fungi: Mixed fungal spores are evenly added into the manure and biogas slurry and cultured at 25-30°C for 2-3 days;

[0013] (3) Recovery of hyphae and discharge of biogas slurry: The biogas slurry fermented in step (2) is introduced into a flocculation sedimentation tank, and the mycelial flocs are recovered after 2-6 hours. The purified supernatant biogas slurry is used as production water for the farm;

[0014] (4) Preparation of single-cell protein for feed: The mycelial flocs are dried at 50-60°C to obtain single-cell protein, which is then supplied back to the farm as a protein supplement for feed.

[0015] Furthermore, the acidity regulator described in step (1) may include one or a mixture of fumaric acid, tartaric acid, citric acid, lactic acid, malic acid, tartaric acid, acetic acid, phosphoric acid, hydrochloric acid, sodium citrate, potassium citrate, calcium lactate, and sodium acetate, and the preferred regulator is sodium acetate.

[0016] Furthermore, the pH of the biogas slurry in step (1) may be 4-7, preferably 5-6.

[0017] Furthermore, the mixed fungi in step (2) are selected from saprophytic fungi rather than parasitic fungi. Saprophytic fungi are preferred because they are more suitable for mycelial growth and liquid fermentation is more likely to produce mycelial flocs.

[0018] Furthermore, the fungi in step (2) are selected from edible fungi for fermented foods and / or food processing, are non-pathogenic filamentous fungi, and do not produce pigments and / or metabolites that are detrimental to animal health. In addition, for centuries, several fungal species have been used in the production of fermented soy products around the world, especially in Asia, so their edible safety is certain, which also means that the single-cell protein produced by the present invention can be directly added to the feed as a protein supplement.

[0019] Further, the fungus described in step (2) is selected from the genera Rhizopus, Rhizomucor, Mucor, Mortierella, Fusarium and Penicillium, including Rhizopus stolonifer, Rhizopus arrhizus, Rhizopus oligosporus, Rhizopus delemar, Aspergillus oryzae, etc., and preferred fungi are Rhizopus oryzae, Aspergillus niger, Rhizomucor miehei, Mucor racemosus and Fusarium venenatum.

[0020] Furthermore, the preferred fungi in step (2) are those that have been found to have high growth rates in liquid culture, can grow on many different carbon and nitrogen sources, and have broad substrate flexibility.

[0021] Furthermore, the preferred fungus described in step (2) has a low carbon-nitrogen ratio, and the dry matter of protein in the cell accounts for at least 60%, or even more, so the carbon-nitrogen ratio of the biogas slurry during the fermentation process is not particularly critical. In particular, the carbon-nitrogen ratio of chicken manure is generally 10:1, pig manure is 13:1, cattle manure is 22:1, and dairy cow manure is 20:1, while the biogas slurry from livestock and poultry manure also contains organic carbon components such as feed, plant straw, and wheat bran left over from the flushing site. Therefore, an external carbon source can be added to adjust the carbon-nitrogen ratio, but preferably not added.

[0022] Furthermore, the temperature of the biogas slurry during the fermentation process in step (2) is not particularly critical and can be 10-40°C, preferably 25 to 30°C.

[0023] Furthermore, the fermentation time in step (2) can be 2-7 days, preferably 2-3 days.

[0024] Furthermore, the fermentation process described in step (2) is preferably aerobic fermentation, so the dissolved oxygen content of the biogas slurry and the dispersion degree of the spores may be important. Oxygen can be provided for the fermentation by stirring and / or aeration, and the process can also achieve sufficient mixing of the biogas slurry and fungal spores.

[0025] Further, the mixing method described in step (2) can be selected to stir, such as using an impeller (including an axial flow agitator or a radial flow agitator). Although stirring can improve the liquid mass transfer and heat transfer efficiency, such as: faster stirring can promote the mass transfer of oxygen and nutrients, and make the distribution of substances and heat in the fermentation system more uniform. However, higher stirring also produces higher shear force, which will cut off the outer area of ​​the spherical mycelium flocs or destroy the entire sphere. Not only does it affect the growth rate of the fungus, it will also greatly increase the difficulty of subsequent recovery. Therefore, it is preferred to use forced aeration.

[0026] Furthermore, the oxygen content in the biogas slurry fermentation process in step (2) is one of the limiting factors for the growth rate of fungi, especially the spore germination stage requires a large amount of oxygen. In addition, the solubility of oxygen in biogas slurry is low, especially the diffusion in the spherical fungal flocs is also low, which requires that the oxygen content in the fermentation process should be kept at a relatively high level, at least 20% in terms of air saturation. Therefore, the ventilation rate can be 0.2-2.0 vvm, preferably 0.8-1.0 vvm.

[0027] Furthermore, the residence time of the fermented biogas slurry in step (3) in the flocculation sedimentation tank can be 2-6 hours, and considering the flocculation and purification effects, it is preferably 3-5 hours.

[0028] Furthermore, the mycelial flocs in step (3) can be recovered by solid-liquid separation methods such as filtration, centrifugation, gravity sedimentation, heating, etc. Considering the cost and recovery effect, gravity sedimentation is preferred.

[0029] Furthermore, the average water content of the single cell protein in step (4) may be 20-80%, and is preferably 30-40% in consideration of drying cost, usage requirements, and storage conditions.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] (1) The present invention provides a group of mixed fungi that can grow well in livestock and poultry manure slurry without adding an external carbon source, and efficiently produce single-cell protein in a short period of time (2 to 3 days). The DCW concentration is finally as high as 18g / L or more, which is much better than the general level (4-8g / L).

[0032] (2) Compared with other methods of producing single-cell protein by fungal fermentation, the fungi involved in the present invention are all derived from edible fungi, and the protein content accounts for 34% to 52% of the dry weight of mycelium, and contains 9 essential amino acids for livestock and poultry. Therefore, it can be used as a safe and high-quality feed protein supplement.

[0033] (3) Compared with other single-cell protein production methods, the fermentation nutrient sources involved in the present invention are all from the "waste" in the biogas slurry, such as organic carbon, ammonia nitrogen, phosphorus and other elements. These nutrients are usually treated and discarded as harmful substances in the livestock and poultry manure biogas slurry, which means that the nutrient source (i.e. biogas slurry) used to cultivate fungi is actually "free". Therefore, the actual production cost of the present invention is lower, and the obtained single-cell protein is directly fed back to the animals, without additionally increasing the level of net CO2 in the environment, which is a "carbon neutral" method.

[0034] (4) The present invention also provides a method for rapidly removing high ammonia nitrogen from biogas slurry, which can effectively remove ammonia nitrogen from biogas slurry with a removal rate of more than 90%. Compared with the two-stage biological method of nitrification and denitrification used in traditional biogas slurry treatment, the present invention only requires one-stage biological fermentation reaction, and the treatment time is the same as that of the traditional method. The overall operating cost is low and the process is simple. It not only has a good treatment effect on biogas slurry, but also can produce protein products with high economic added value.

[0035] (5) Compared with other methods of purifying biogas slurry using microorganisms, the microorganisms involved in the present invention are all filamentous fungi. During the fermentation of biogas slurry, the mycelium can form a spherical floc structure, which not only does not require centrifugal recovery, but also has a biological flocculation effect on suspended matter in the water body. Without adding additional flocculants, "purified" biogas slurry with low suspended matter and COD levels can be obtained, which can be directly used for production water in the farm. Therefore, the present invention provides a more low-carbon and environmentally friendly solution for the recycling of biogas slurry resources.

[0036] The "fermented foods and / or foods for food processing" referred to in the present invention refer to foods prepared by fungal culture at home and abroad, such as natto, distiller's grains, hairy tofu, vinegar, soy sauce, bean paste, noodle sauce, pickled vegetables, fermented glutinous rice, tempeh, vegetarian meat, plant meat, etc.

[0037] The "metabolites that are detrimental to animal health" involved in the present invention refer to major fungal toxins such as aflatoxins, zearalenone, vomitoxin, T2 toxin, ochratoxin, fumonisin, etc.

[0038] The "mycelium", "flocs", "hyphal flocs", "spherical mycelial flocs", and "floc particles" mentioned in the present invention can be used interchangeably to refer to mycelial pellets. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The present invention provides a flow chart of a method for producing single-cell protein for feed using livestock and poultry manure and biogas slurry.

[0040] Figure 2 This is a schematic diagram comparing the dry matter content of each strain after being cultured in manure and biogas slurry.

[0041] Figure 3 This is a comparative diagram of the ammonia nitrogen content in the manure slurry after each strain was cultured in the manure slurry. DETAILED DESCRIPTION

[0042] First, the frozen fungus is activated (the activation method is a conventional technique and will not be described in detail), sterile water is added to the slant culture medium full of mature spores, and the washing liquid is poured into a sterile conical flask filled with glass beads and fully shaken for 20 minutes to obtain a spore suspension or prepare spore powder by centrifugal drying, and then subsequent operations are carried out.

[0043] The specific implementation is as follows:

[0044] (1) Preparation of manure slurry: The livestock manure is introduced into an anaerobic fermentation tank for CSTR anaerobic fermentation. The liquid after solid-liquid separation is manure slurry. The slurry is introduced into a primary fermentation tank and the pH is adjusted to 5-7 to prepare a fermentation substrate;

[0045] (2) Inoculating fungi: Mixed fungal spores are evenly added into the manure slurry, with the inoculation amount being 2% to 10% of the volume of the fermentation substrate, and cultured at 25 to 30° C. for 2 to 3 days;

[0046] (3) Recovery of hyphae and discharge of biogas slurry: The biogas slurry fermented in step (2) is introduced into a flocculation sedimentation tank, and the mycelial flocs are recovered after 2-6 hours. The purified supernatant biogas slurry is transported back to the farm as production water;

[0047] (4) Preparation of single-cell protein for feed: The mycelial flocs are dried at 50-60°C to obtain single-cell protein, which is then supplied back to the farm as a protein supplement for feed.

[0048] In order to better understand the content of the present invention, the present invention will be further described below in conjunction with the examples and drawings. This example is implemented based on the technology of the present invention, and provides detailed implementation methods and operating steps, but the protection scope of the present invention is not limited to the following examples.

[0049] Example 1: Screening of strains

[0050] The candidate fungi need to be edible fungi with high protein content, and the SCP produced can be directly added to the feed as a complete protein supplement.

[0051] The advantages of edible filamentous fungi include: 1. They produce no or very little harmful substances such as toxins; 2. They can grow on a variety of different carbon and nitrogen sources, have a low carbon-nitrogen ratio, and can use simple and cheap fermentation substrates; 3. They grow fast in liquid culture and have the form of spherical hyphae flocs; 4. The hyphae have a flocculation effect, which facilitates separation and recovery.

[0052] The examples demonstrate that it is feasible to produce single-cell protein by fermenting livestock and poultry manure slurry using edible filamentous fungi, and some potential strain resources have been reported in the filamentous fungi of the genera Rhizopus, Rhizomucor, Mucor, Mortierella, Fusarium and Penicillium.

[0053] Edible filamentous fungi belonging to the genera Rhizopus, Rhizomucor, Mucor, Mortierella, Fusarium and Penicillium were tested using shake flask culture experiments to analyze their growth performance in livestock and poultry manure biogas slurry.

[0054] Each candidate strain (partially shown in Table 1 ) was tested three times in the following culture media, and those with good growth performance were selected as positive.

[0055] Table 1

[0056]

[0057] Biogas slurry culture medium: Take the manure biogas slurry after anaerobic fermentation and solid-liquid separation, mix and measure the initial ammonia nitrogen content to be 2500 mg / L, COD to be 15000 mg / L, and pH to be 7-8. Use 60% sodium acetate (food grade) to adjust the pH and set aside.

[0058] Shake flask fermentation: Take 100 mL of biogas slurry culture medium in a 500 mL Erlenmeyer flask, adjust the pH to 6, and inoculate the freshly prepared spore suspension to a final concentration of 1×10 6-7 CFU / ml, cultured at 28°C, 150r / min shaking for 72h, with 3 parallels in each group.

[0059] For some strains, specific culture conditions are required to force them to grow in the form of spherical flocs rather than free hyphae or dispersed clumps in liquid. Take appropriate measures (such as adjusting the size of the flask and / or the shaking rate) to increase the number of floc particles and avoid the dispersed state of hyphae.

[0060] The culture bottles were taken out every 24 hours, the growth form of the fungi was visually observed, and the state of the hyphae was examined microscopically. The results are shown in Table 2. Subsequently, the cells were filtered with pre-dried and weighed filter paper, which was placed in a drying oven and dried at 105°C to constant weight, and the cell dry matter DCW was calculated. At the same time, the filtrate was used to determine the ammonia nitrogen content according to the salicylic acid spectrophotometric method.

[0061] Table 2

[0062]

[0063] +: means hyphae can be observed

[0064] -: means hyphae cannot be observed

[0065] *: Refers to the compactness of the spherical hyphae flocs

[0066] As shown in Table 2, except for No. 5, the remaining strains all became turbid at 24h, and hyphae growth could be clearly seen under the microscope, indicating that most candidate strains can adapt to the high ammonia nitrogen environment. The results at 48h showed that spherical or granular flocs appeared in each group, among which the hyphae flocs of No. 2, 4, 6, and 7 were relatively compact, and the culture medium was basically clear. After 72h, no pigment was produced in each group, but No. 3 and No. 9 showed the combination of flocs, showing loose clusters, and the culture medium became turbid.

[0067] according to Figure 2 As shown, the DCW of each group at 48h was significantly higher than that at 24h, and the DCW of No. 2, 4, 6, and 7 was above 10g / L, which was consistent with their growth conditions. Figure 3 It showed that all strains could assimilate ammonia nitrogen in the substrate to a certain extent. Among them, strains 2, 4, 6, and 7 had the highest ammonia nitrogen removal rates, all above 80%.

[0068] Example 2: Amplification and culture

[0069] In this embodiment, the working volume is 2m 3 All candidate strains were subjected to expansion culture experiments in micro-aeration tanks, and the mycelial growth form, the composition of the biogas slurry, and the content of fungal biomass during further expansion culture were tested.

[0070] The same biogas slurry as in Example 1 was introduced into the aeration tank, and the pH value was adjusted to 6 by sodium acetate for standby use;

[0071] Evenly add fungal spore powder into the manure slurry, with the inoculation amount being 2% to 5% of the volume of the fermentation substrate, and culture at 25 to 30°C for 2 to 3 days;

[0072] The fermented biogas slurry is introduced into the flocculation sedimentation tank, and the mycelium flocs are recovered after 5 hours;

[0073] The single cell protein can be obtained by drying the mycelial flocs at 50-60°C.

[0074] Samples were taken three times every 24 hours for visual and microscopic observation, as well as analysis of biogas slurry composition, cell dry matter, and protein content.

[0075] In the samples of the fermentation tank, the microscopic observation results of 24h of culture showed that all fungi were growing as hyphae. Visual observation showed that spherical floc particles visible to the naked eye appeared in No. 2, 4, 6, and 7, but the sizes were different. After 48h of culture, the diameter of the mycelial flocs of these four strains was 3-8mm, and the cumulative concentration of cell dry matter exceeded 13g / L (Table 3). After the fermentation was completed, the mycelial flocs of the four strains naturally settled to the bottom, and the upper layer of biogas slurry was clear, and the removal rate of ammonia nitrogen was above 82%. The protein content of the mycelial flocs was further tested. The results are shown in Table 3. The protein content of No. 8 was the highest at 52.3%, followed by No. 2, 4, 6, and 7, but they were all above 40%.

[0076] Table 3

[0077]

[0078]

[0079] * Refers to the protein content of fungal cell dry matter

[0080] Therefore, based on the above results, it can be seen that most strains can adapt well to further expanded cultivation and have the potential to produce single-cell protein. Combined with the ammonia nitrogen removal rate, mycelial floc state, dry matter mass and protein content, strains 2, 4, 6, 7 and 8 are preferred for mixed fermentation.

[0081] Example 3: Mixed fermentation

[0082] In this embodiment, the working volume is 2m 3 The mixed fermentation was carried out in a micro-aeration tank using strains 2, 4, 6, 7 and 8, and the mycelial growth form, the composition of the biogas slurry and the content of the overall fungal biomass in the further mixed culture process were detected.

[0083] The same biogas slurry as in Example 2 was introduced into the aeration tank, and the pH value was adjusted to 6 by sodium acetate for standby use;

[0084] Mixed fungal spore powder is evenly added into the manure and biogas slurry, with the inoculation amount being 2% to 5% of the volume of the fermentation substrate, and cultured at 25 to 30°C for 2 to 3 days;

[0085] The fermented biogas slurry is introduced into the flocculation sedimentation tank, and the mycelium flocs are recovered after 5 hours;

[0086] The single cell protein can be obtained by drying the mycelial flocs at 50-60°C.

[0087] Samples were taken three times every 24 hours for visual and microscopic observation, as well as analysis of biogas slurry composition, cell dry matter, and protein content.

[0088] In the samples of the fermentation tank, the microscopic observation results of 24h culture showed that all fungi were growing in hyphae. Visual observation showed that there were spherical floc particles visible to the naked eye in the mixed fermentation samples, but they were of different sizes. After 48h of culture, the diameter of the mycelial flocs of these five strains was 4-10mm, and the cumulative concentration of cell dry matter exceeded 18g / L. After the fermentation was completed, the mixed mycelial flocs naturally settled to the bottom, and the upper layer of biogas was clear, and the removal rate of ammonia nitrogen was 90%. The protein content of the mycelial flocs was further tested, and the results showed that the protein content in the dry matter of the mixed fermentation mycelial flocs was 53.8%.

[0089] Therefore, mixed fermentation was superior to single-cell fermentation in terms of ammonia nitrogen removal rate, mycelial floc state, dry matter mass and protein content of biogas slurry, proving that the strain can adapt to further mixed culture and achieve the purpose of purifying biogas slurry and producing single-cell protein.

[0090] Example 4: Treatment effect of dairy farm manure slurry

[0091] Preparation of manure slurry: introduce the livestock manure into the anaerobic fermentation tank for CSTR anaerobic fermentation, and then the liquid after solid-liquid separation is manure slurry. The slurry is introduced into the primary fermentation tank, and sodium acetate is added to adjust the pH to 6 for standby use;

[0092] Inoculation of fungi: evenly add mixed fungal spores into the manure slurry, with the inoculation amount being 2% to 5% of the volume of the fermentation substrate, and culture at 25 to 30°C for 2 to 3 days;

[0093] The fermented biogas slurry is introduced into the flocculation sedimentation tank, and the mycelium flocs are recovered after 2-6 hours;

[0094] The mycelial flocs are dried at 50-60°C to obtain single-cell protein, which is supplied back to the farm as a protein supplement for feed.

[0095] It should be noted that after anaerobic fermentation and solid-liquid separation, the initial ammonia nitrogen content of the dairy farm manure slurry was 1500 mg / L and the COD content was 13000 mg / L, both lower than that of Example 3. Therefore, the spore inoculation amount was adjusted to 3% and the pH was 6.5.

[0096] After 2 days of fermentation, the ammonia nitrogen content and COD of the supernatant in the flocculation tank, as well as the DCW and SCP contents of the mycelial flocs were measured. The results are shown in Table 4. The ammonia nitrogen content in the treated biogas slurry dropped to 136 mg / L, and the CODcr dropped to 920 mg / L.

[0097] Table 4

[0098]

[0099] * Refers to the protein content of fungal cell dry matter

[0100] Example 5: Treatment effect of pig farm manure slurry

[0101] Preparation of manure slurry: introduce the livestock manure into the anaerobic fermentation tank for CSTR anaerobic fermentation, and then the liquid after solid-liquid separation is manure slurry. The slurry is introduced into the primary fermentation tank, and sodium acetate is added to adjust the pH to 6 for standby use;

[0102] Inoculation of fungi: evenly add mixed fungal spores into the manure slurry, with the inoculation amount being 2% to 5% of the volume of the fermentation substrate, and culture at 25 to 30°C for 2 to 3 days;

[0103] The fermented biogas slurry is introduced into the flocculation sedimentation tank, and the mycelium flocs are recovered after 2-6 hours;

[0104] The mycelial flocs are dried at 50-60°C to obtain single-cell protein, which is supplied back to the farm as a protein supplement for feed.

[0105] It should be noted that after anaerobic fermentation and solid-liquid separation, the initial ammonia nitrogen content of the pig farm manure slurry was 950 mg / L and the CODcr content was 10000 mg / L, both lower than that of Example 3. Therefore, the spore inoculation amount was adjusted to 2% and the pH was 6.5.

[0106] After 2 days of fermentation, the ammonia nitrogen content and COD of the supernatant in the flocculation tank, as well as the DCW and SCP contents of the mycelial flocs were measured. The results are shown in Table 5. The ammonia nitrogen content in the treated biogas slurry dropped to 95 mg / L, and the CODcr dropped to 385 mg / L.

[0107] Table 5

[0108]

[0109] * Refers to the protein content of fungal cell dry matter

[0110] Example 6: Treatment effect of chicken manure slurry

[0111] Preparation of manure slurry: introduce the livestock manure into the anaerobic fermentation tank for CSTR anaerobic fermentation, and then the liquid after solid-liquid separation is manure slurry. The slurry is introduced into the primary fermentation tank, and sodium acetate is added to adjust the pH to 6 for standby use;

[0112] Inoculation of fungi: evenly add mixed fungal spores into the manure slurry, with the inoculation amount being 2% to 5% of the volume of the fermentation substrate, and culture at 25 to 30°C for 2 to 3 days;

[0113] The fermented biogas slurry is introduced into the flocculation sedimentation tank, and the mycelium flocs are recovered after 2-6 hours;

[0114] The mycelial flocs are dried at 50-60°C to obtain single-cell protein, which is supplied back to the farm as a protein supplement for feed.

[0115] It should be noted that after anaerobic fermentation and solid-liquid separation, the initial ammonia nitrogen content of the chicken manure slurry was 2200 mg / L and the CODcr content was 14000 mg / L. Therefore, the spore inoculation amount was adjusted to 4% and the pH was 6.

[0116] After 2 days of fermentation, the ammonia nitrogen content and COD of the supernatant in the flocculation tank, as well as the DCW and SCP contents of the mycelial flocs were measured. The results are shown in Table 7. The ammonia nitrogen content in the treated biogas slurry dropped to 210 mg / L, and the CODcr dropped to 980 mg / L.

[0117] Table 7

[0118]

[0119]

[0120] * Refers to the protein content of fungal cell dry matter

[0121] After 3 days of fermentation, the ammonia nitrogen content in the treated sludge dropped to 110 mg / L, and CODcr dropped to 390 mg / L.

[0122] Example 7: Preparation of single cell protein for feeding and purification of biogas slurry

[0123] After the fermentation is completed, the fungi grow into spherical mycelium flocs with a diameter of 3-10 mm. After gravity sedimentation in the flocculation tank, the pellets can usually be captured using a simple sieve or a sieve with appropriate openings, and then dried at 50-60°C to obtain SCP. The SCP produced by mixed fungal fermentation not only has the unique aroma of fermented food, but also has a richer variety of amino acids. The single-cell protein prepared by the present invention has 9 essential amino acids for livestock and poultry (Table 9), and is particularly suitable as a protein supplement for feed. The supernatant of the flocculation tank is the purified biogas slurry. Through the assimilation of the mixed fungi, pollutants such as ammonia nitrogen, phosphorus, and organic carbon in the biogas slurry are removed. The suspended matter and part of the COD level in the biogas slurry are reduced through the biological flocculation of the hyphae, and purified biogas slurry that meets the production water standards is obtained.

[0124] Example 8: Resource Utilization of Slurry from Manure from a Thousand-head Dairy Farm

[0125] A dairy farm with thousands of cows produces about 100 tons of manure and wastewater every day.

[0126] The livestock manure is introduced into the anaerobic fermentation tank for CSTR anaerobic fermentation, and the liquid after solid-liquid separation is manure slurry, which is introduced into the primary fermentation tank, and sodium acetate is added to adjust the pH to 6 for standby use;

[0127] Mixed fungal spores are evenly added into the manure biogas slurry, with the inoculation amount being 2% to 5% of the volume of the fermentation substrate, and cultured at 25 to 30°C for 2 to 3 days;

[0128] The fermented biogas slurry is introduced into the flocculation sedimentation tank, and the mycelium flocs are recovered after 2-6 hours;

[0129] The mycelial flocs are dried at 50-60°C to obtain single-cell protein, which is supplied back to the farm as a protein supplement for feed.

[0130] It should be noted that if the temperature is low in winter, Figure 1 -K route uses biogas from anaerobic fermentation for heating to maintain the temperature of the fermentation tank.

[0131] It should be noted that after the first fermentation is completed, the ammonia nitrogen content, CODcr and other indicators in the supernatant in the flocculation tank are measured. If they are still higher than the standard for production water in the farm, they can be treated according to Figure 1 -H returns the biogas slurry in the flocculation tank to the fermentation tank for further treatment until the supernatant biogas slurry in the flocculation tank reaches the use standard and is transported back to the farm to supplement production water.

[0132] It should be noted that after anaerobic fermentation and solid-liquid separation, the initial ammonia nitrogen content of the dairy farm manure slurry was 1300 mg / L, the total nitrogen was 1500 mg / L, the total phosphorus was 300 mg / L, the CODcr was 12000 mg / L, and the suspended solids were 18000 mg / L. Therefore, the spore inoculation amount was adjusted to 4-5%, and other conditions remained unchanged. After 2 days of fermentation, all the slurry in the fermentation tank was introduced into the flocculation tank, and the results of the supernatant slurry were measured 4 hours later as shown in Table 8.

[0133] Table 8

[0134]

[0135] After 100 tons of manure wastewater is purified, approximately 86 tons remain to supplement production water.

[0136] After drying the spherical mycelium flocs at 60°C for about 4-6 hours (the time can be appropriately extended in winter), they are bagged to prevent moisture resurgence. The analysis results of the single-cell protein prepared by the above process are shown in Table 9. The proportion of SCP in the dry matter of mycelium is 52.63%, of which the proportion of essential amino acids is 51%.

[0137]

[0138]

[0139] The single-cell protein produced from 100 tons of biogas slurry was added to the ground feed formula at a rate of 45% as a protein supplement and then fed to dairy cows, which meant that the fungal protein ultimately contributed about 22% of the total protein in the feed.

[0140] Therefore, using fungi to ferment livestock and poultry manure slurry to prepare single-cell protein can effectively reduce the costs of production water, environmental protection, pollutant treatment, protein feed, etc., and bring significant benefits to the breeding industry.

[0141] The above is only an embodiment of the present invention, and does not limit the present invention in any form. The present invention can also have other forms of embodiments according to the above structures and functions. Therefore, any simple modification, equivalent change and modification made by any technician familiar with the profession to the above embodiment according to the technical essence of the present invention without departing from the scope of the technical solution of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. A method for preparing single cell protein for feed, characterized in that: The single-cell protein is prepared by fermenting the organic components in the livestock and poultry manure and biogas slurry with mixed fungi. The preparation method includes the following steps: (1) Preparation of manure slurry: The livestock manure is introduced into the anaerobic fermentation tank for CSTR anaerobic fermentation. The liquid after solid-liquid separation is manure slurry. The slurry is introduced into the primary fermentation tank to adjust the pH value of the slurry. (2) Inoculation: evenly adding mixed fungal spores to the fermentation substrate prepared in step (1), and culturing at 25-30° C. for 2-3 days for fermentation; (3) Recovery and discharge: The biogas slurry after fermentation in step (2) is introduced into a flocculation sedimentation tank, where the mycelial flocs are recovered after 2-6 hours of residence, and the purified supernatant biogas slurry is transported back to the farm as production water; (4) Drying: drying the mycelial flocs obtained in step (3) at 50-60° C. to obtain the single cell protein for feed; In step (2), the inoculated mixed fungi are selected from saprophytic fungi rather than parasitic fungi, and the inoculation amount is 2% to 10% of the volume of the fermentation substrate; The fungi are selected from edible fungi for fermented foods and / or used in food processing, including Rhizopus stolonifer, Rhizopus arrhizus, Rhizopus oligosporus, Rhizopus delemar, Aspergillus oryzae, Rhizopus oryzae, Aspergillus niger, Rhizomucor miehei, Mucorracemosus and Fusarium venenatum.

2. The method for preparing single cell protein for feed according to claim 1, characterized in that: In step (1), the pH value of the prepared manure slurry is adjusted to 4-7 using an acidity regulator.

3. The method for preparing a single cell protein for feed according to claim 2, characterized in that: The pH value of the biogas slurry is adjusted to 5-7, and the acidity regulator includes one or a mixture of fumaric acid, metatartaric acid, citric acid, lactic acid, malic acid, tartaric acid, acetic acid, phosphoric acid, hydrochloric acid, sodium citrate, potassium citrate, calcium lactate, and sodium acetate.

4. The method for preparing a single cell protein for feed according to claim 1, characterized in that: In step (2), the culture condition is aerobic fermentation, and oxygen is provided to the fermentation by stirring and / or aeration during the fermentation process, and the ventilation rate is 0.2-2.0 vvm.

5. The method for preparing a single cell protein for feed according to claim 1, characterized in that: The fungi were Rhizopus oryzae, Aspergillus niger, Rhizomucor miehei, Mucor racemosus, and Fusarium venenatum.

6. The method for preparing a single cell protein for feed according to claim 4, characterized in that: Aerobic fermentation uses forced aeration with a ventilation rate of 0.8-1.0 vvm.

7. The method for preparing a single cell protein for feed according to claim 1, characterized in that: In step (3), the mycelial flocs are recovered by filtering, centrifuging, gravity settling and heating.

8. The method for preparing single cell protein for feed according to claim 7, characterized in that: In step (3), considering the flocculation and purification effects, the residence time is 3-5 hours; the mycelial flocs are recovered by gravity sedimentation.

9. The method for preparing single cell protein for feed according to claim 1, characterized in that: In step (4), the average water content of the single cell protein for feeding is 20-80%.

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