Method for recycling cyanobacteria mud and kitchen waste by using hermetia illucens larvae

Abstract

The invention belongs to the technical field of organic waste recycling, and particularly relates to a method for recycling cyanobacteria mud and kitchen waste by using hermetia illucens larvae. And mixing the pretreated cyanobacteria mud with the kitchen waste to obtain a mixed feed, and adding a bacillus inoculant for aerobic fermentation to obtain hermetia illucens larvae and larva sand. By regulating and controlling the matching of the matrix and the synergistic effect of the fungicide, the nutritional value and the utilization efficiency of the mixed feed are increased, and the black soldier flies grow faster in the early stage. The bred hermetia illucens larvae can be used as a high-quality protein feed, and the larva sand can be used as an organic fertilizer raw material. According to the method disclosed by the invention, collaborative recycling of the cyanobacteria and the kitchen waste is realized, and a biological solution capable of being applied on a large scale is provided for treatment and utilization of the water-blooming cyanobacteria mud.

Description

Technical Field

[0001] The invention relates to a method for recycling blue algae mud and kitchen waste by utilizing black soldier fly larvae, and belongs to the field of organic waste recycling. Background Art

[0002] As one of the most challenging environmental problems in the global freshwater ecosystem, cyanobacterial blooms caused by eutrophication have always received widespread attention. For lakes with important drinking, ornamental and ecological values, once a cyanobacterial bloom occurs, the most direct and effective emergency prevention and control measure is to salvage the cyanobacteria. The high water content and sticky extracellular polymers of cyanobacterial slurry pose severe challenges to traditional treatment methods: 1) The natural drying method occupies a large area and is prone to secondary pollution; 2) The efficiency and quality of anaerobic fermentation gas production are low (methane yield <0.3m 3 / ton of fresh algae); 3) Direct landfill leads to secondary pollution, such as the risk of migration of heavy metal ions in the leachate.

[0003] Although cyanobacteria mud is often treated as solid waste, it cannot be ignored that cyanobacteria is also a biomass with great potential utilization value. Algae have a higher efficiency in utilizing the sun than general terrestrial plants. Bloom cyanobacteria contain a large amount of nutrients such as protein, polysaccharides, unsaturated fatty acids, and usable substances such as phycocyanin and carotenoids. Its protein content is as high as 30-65%. The existing commonly used methods for the resource utilization of cyanobacteria are as follows: (1) Preparation of biofuels, there are still major problems such as complex reaction equipment, high operating costs (the economic cost of preparing biodiesel from algae can reach 42,000 to 59,000 yuan / t), and low efficiency; (2) Extraction of useful substances, such as extraction of phycocyanin, the cost of the purification stage accounts for 50-90% of the production cost, the extraction process is complicated and difficult to mass produce; (3) Preparation of organic carbon sources, the key technology is immature, it is very easy to produce secondary pollution, the cost is high, and it does not conform to the concept of energy use under the background of "dual carbon". Biomass carbon sources also have the problem of low denitrification rate. At the same time, the problems of algal toxins and heavy metals in algal mud have also become difficult problems that cannot be ignored in the resource utilization path. Faced with the increasing amount of cyanobacterial biomass, there is an urgent need for methods that are safer, more environmentally friendly and efficient, have large processing capacity and can produce high value-added products.

[0004] The technology of treating organic matter with scavenging insects has the advantages of low cost, low maintenance, simple operation, small land requirements, low ecological footprint, and greater economic potential. Among them, black soldier flies are widely promoted in the treatment of organic wastes such as food waste, animal feces, straw, and sludge because they do not spread diseases, have a wide range of diets, and have high bioconversion efficiency. Among them, food waste is considered to be one of the most suitable feeds for the growth of black soldier flies due to its rich nutrition. It is worth noting that even though food waste is rich in nutrition, the different sources of food waste have a large difference in nutritional components, which has a certain impact on the growth of black soldier flies, especially the carbon-nitrogen ratio in food waste, so it is necessary to develop suitable auxiliary materials to solve the above problems. Summary of the invention

[0005] In order to solve the above problems, the present invention mixes the cyanobacteria mud obtained by flocculation with kitchen waste and directly feeds the black soldier fly larvae. On the one hand, since kitchen waste is rich in nutrients, it is the most suitable feed source for the growth of black soldier flies, and the process has been maturely applied in production activities. On the one hand, the use of cyanobacteria mixed with kitchen waste can solve the problem of a large amount of cyanobacteria biomass accumulation, and alleviate the adverse effects of algae mud on the growth of black soldier fly larvae during the biological transformation process. On the other hand, the insect protein obtained by breeding has a high economic value and can be used as a protein source in animal feed, giving full play to the resource potential of cyanobacteria biomass. At the same time, by adding probiotics to regulate the fermentation process of the substrate and the absorption of nutrients in the substrate by the insect body, the adverse effects of cyanobacteria on the growth of black soldier flies are reduced, the efficiency of the biological transformation of cyanobacteria by black soldier flies is improved, and the nutritional value of the insect body is improved.

[0006] In order to achieve the above object, the present invention adopts the following technical measures:

[0007] A method for recycling blue algae mud and kitchen waste by using black soldier fly larvae, the specific steps are as follows:

[0008] (1) crushing the food waste after solid-liquid separation, wherein the moisture content of the separated food waste is 65-85%;

[0009] (2) mixing the food waste treated in step (1) and cyanobacteria mud having a moisture content of 65% to 95% to form a black soldier fly breeding matrix, wherein the moisture content of the matrix is ​​controlled to be less than 85%;

[0010] (3) placing black soldier fly larvae in a black soldier fly breeding matrix for breeding, separating the insect bodies from the insect sand after the breeding is completed, using the insect sand as an organic fertilizer raw material and the black soldier fly larvae as an insect protein product to realize the resource processing of cyanobacteria mud and food waste; the fresh weight ratio of the cyanobacteria mud to the food waste is 1: (1-7), preferably 1: 3;

[0011] The ratio of the weight of the black soldier fly larvae to the fresh weight of the black soldier fly breeding substrate is 1:(20-80), preferably 1:50.

[0012] In the step (2), the moisture content of the black soldier fly breeding substrate is 65% to 85%.

[0013] Furthermore, in step (2), 0.1% to 1% of the dry weight of a Bacillus agent is added to the black soldier fly breeding matrix to assist the black soldier fly larvae in the simultaneous bioconversion of cyanobacteria mud and food waste.

[0014] Furthermore, the conditions for completing the breeding in step (3) are: half of the black soldier fly larvae enter the prepupae stage or the black soldier fly breeding matrix is ​​basically converted into granular insect sand. This process can be judged by those skilled in the art based on their professional knowledge.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0016] Black soldier fly larvae have a high bioconversion capacity for organic waste such as food waste, livestock and poultry feces, sludge, straw, etc., and can convert organic matter into larval biomass rich in protein and lipid and treatment residues that can be used as organic fertilizer. Among them, food waste is rich in nutrients and is suitable for the growth and development of black soldier fly larvae. The resource treatment method of mixing algae mud with food waste and adding a fermentation agent has the following advantages:

[0017] (1) It can not only realize the direct bioconversion of algae mud by black soldier fly larvae, but also alleviate the adverse effects of algae mud on black soldier fly larvae;

[0018] (2) Adding fermentation agents can convert macromolecular proteins in the substrate into various metabolites such as small molecular peptides through its own metabolism, thereby improving the nutritional value and utilization efficiency of the mixed feed. The crude polysaccharide content of the mixed feed increases, the black soldier fly grows faster in the early stage (0-4 days), and it can also regulate the intestinal health of the black soldier fly larvae;

[0019] (3) This method not only saves a large amount of cost for further processing of cyanobacteria mud, but also produces valuable products such as larval protein. When the algae mud is mixed with food waste, the harvested black soldier fly larvae have no difference in crude protein and crude fat content from the black soldier fly larvae fed with food waste, which does not affect the use of black soldier fly larvae as insect protein products; and compared with the traditional composting process (processing cycle 30-60 days), the method of the present invention has a significantly shortened processing cycle, and the material reduction rate is more than 50% ( Figure 1 B), especially suitable for emergency treatment of algae mud harvested during the outbreak of blue algae bloom;

[0020] (4) The cyanobacteria mud can be directly processed on a large scale in the breeding factory, which can meet the seasonal characteristics of cyanobacteria production and has high feasibility, and can effectively solve the problem of large amounts of cyanobacteria mud accumulation and difficulty in handling in emergency treatment;

[0021] (5) Generate economic benefits. This method not only saves a large amount of cost for further processing of cyanobacteria mud, but also produces valuable products such as larval protein. The black soldier fly larvae can biotransform 4 tons of mixed feed (including 1 ton of algae mud) to obtain 431 kg of fresh black soldier flies and 454 kg of fresh insect sand. The income of insect sand is 0 yuan. Based on the fresh insect value of about 2,400 yuan / t and the breeding cost of 1,920 yuan / t, the black soldier flies harvested from processing 1 ton of algae mud can be converted into 207 yuan of income. Compared with the anaerobic fermentation of algae mud to produce biogas (government subsidy of 280 yuan / ton), the benefits of converting algae mud with black soldier flies are significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the effect of adding cyanobacteria mud in Test Example 1 on the biotransformation capacity of black soldier fly larvae in each treatment group.

[0023] Figure 2 The growth of black soldier fly larvae during the breeding process in Test Example 2.

[0024] Figure 3 and Figure 4 They are the PCoA of the bacterial community corresponding to β diversity in the breeding process of test example 3 ( Figure 3 (A)), NMDS( Figure 3 (B)) and a heat map of the abundance of the top 50 genera of microorganisms in each treatment group ( Figure 4 ). DETAILED DESCRIPTION

[0025] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0026] The raw materials used in the embodiment:

[0027] 3-day-old black soldier fly larvae: provided by Yuanchuang Environmental Technology Co., Ltd., obtained by hatching eggs for 3 days in a black soldier fly breeding factory. The breeding environment temperature is 30°C and the relative humidity is about 70%.

[0028] The cyanobacteria mud (the dominant species is Microcystis) was obtained from the algae-water separation station of Taihu Lake. In the experiment, the initial water content of the cyanobacteria mud was 90.72%, the concentration of microcystin per gram dry weight of the mud was 6.63 μg (the content is low and negligible), the crude protein content was 35.93%, the crude fat was 2.09%, and the crude polysaccharide was 6.56%.

[0029] Kitchen waste: The three-phase solid residue of kitchen waste from the black soldier fly breeding factory (located in Jiangxia District, Wuhan City) is stably processed. The kitchen waste is crushed and fed, and the moisture content is 76.7%.

[0030] Bacillus agent: a fermentation agent produced by Hubei Qiming Bioengineering Co., Ltd.

[0031] Example 1

[0032] A resource utilization method for bioconverting a mixture of cyanobacteria mud and kitchen utensils by using black soldier flies comprises the following steps:

[0033] 25 kg of cyanobacteria mud and 75 kg of crushed kitchen waste were mixed evenly in a breeding box (1.2 m × 2.4 m × 0.3 m), and a box of 3-day-old black soldier fly larvae (about 2 kg) was put into the breeding box. The breeding box was moved into a breeding warehouse (temperature of about 30°C and relative humidity of about 70%) for breeding; it was named T-25 group.

[0034] Example 2

[0035] 0.06 kg of fermentation agent suspension (based on the weight of the fermentation agent, the fermentation agent was purchased from Hubei Qiming Bioengineering Co., Ltd., the main components: Bacillus, actinomycetes, yeast, filamentous fungi and other beneficial microorganisms and their various extracellular enzymes, the effective viable count is ≥ 20 billion / g, and the relative abundance of Bacillus is 99.4% by measuring 16SrRNA, the same below) was added to the matrix of Example 1, and then subsequent operations were carried out, and it was named TF-25 group.

[0036] Example 3

[0037] The fresh weight of the cyanobacteria mud in Example 1 was increased to 50 kg, the kitchen waste was reduced to 50 kg, and the other subsequent operations were carried out in accordance with Example 1, and the group was named T-50.

[0038] Example 4

[0039] After adding 0.06 kg of fermentation agent to the substrate of Example 3, the subsequent operation was carried out and the group was named TF-50.

[0040] Comparative Example 1

[0041] The 25 kg of cyanobacteria mud and 75 kg of crushed kitchen waste in Example 1 were adjusted to 100 kg of crushed kitchen waste, and the rest was the same as in Example 1, and was named the Control group.

[0042] Performance tests were performed on Examples 1-4 and Comparative Example 1, specifically: when half of the prepupae appeared in each group or the matrix had obviously turned into granular insect litter, the culture time of each group was recorded, the insect litter and larvae (including prepupae and other larvae) were separated and weighed, and the body length and weight of the black soldier fly larvae were measured. The obtained black soldier fly larvae can be used as protein feed, and the insect litter can be used as a raw material for organic fertilizer.

[0043] Test Example 1:

[0044] 1. Determination of the quality of harvested black soldier fly larvae and insect litter:

[0045] The worm sand and black soldier fly larvae were separated and weighed.

[0046] 2. Nutritional composition determination:

[0047] The initial substrate, insect sand and the larvae harvested at last were placed in a freeze dryer for freeze drying and the following nutritional components were determined.

[0048] Crude fat was determined by Soxhlet extraction: 1 g of the ground sample was placed in a filter paper cylinder and then in an extraction cylinder, and then extracted using anhydrous ether as a solvent using a Soxhlet extractor. The extraction cylinder was dried in an oven to constant weight and the crude fat content was calculated;

[0049] The phenol-sulfuric acid method was used to determine polysaccharides, and the Kjeldahl method was used to determine crude protein in the matrix and insect sand:

[0050] First, 0.5 g of dry sample was accurately weighed into a digestion tube, and after adding 0.2 g of copper sulfate, 3 g of potassium sulfate and 10 mL of concentrated sulfuric acid, the sample was digested in a digestion furnace. No sample was added to the blank sample. The digestion temperature was set to 120 ° C for 30 min, 240 ° C for 40 min, 320 ° C for 40 min, 380 ° C for 30 min, and then 420 ° C for 1.5 h, and then measured using a Kjeldahl nitrogen analyzer. Crude protein was determined as TN (total nitrogen) × 6.25 (Chinese standard GB 5009.5-2016).

[0051] The crude protein content in black soldier fly larvae was calculated using the above method, except that the conversion factor of 6.25 was replaced by 4.76.

[0052] 3. Conversion rate determination:

[0053] The black soldier fly larvae screened out were microwave-dried to obtain dried worms, which were stored in a -20°C refrigerator. The worm sand was dried at 105°C for 24 hours, and their weights were weighed and recorded ( Figure 1 In the middle (A), the material reduction rate, bioconversion rate, larval growth rate, substrate reduction index and other indicators were calculated.

[0054] The material reduction rate (dry weight) is calculated as follows (1):

[0055]

[0056] The bioconversion rate (all indicators in the formula are dry weight) is calculated as follows (2), where the dry weight of the initially added larvae is very small and is ignored in the calculation of this result:

[0057]

[0058] The larval growth rate (fresh weight) was calculated as follows (3):

[0059]

[0060] The matrix reduction index (dry weight) was calculated as follows (4):

[0061]

[0062] The results are shown in Tables 1, 2 and Figure 1 , 2 :

[0063] Table 1 Chemical composition of the initial matrix of the five groups

[0064]

[0065] Note: The values ​​with different superscript letters (a, b, c) in the same row are significantly different (p < 0.05). The values ​​are expressed as mean ± standard deviation (n = 3), and crude polysaccharides, crude protein, and crude fat all refer to the mass percentage of the initial matrix dry weight.

[0066] Table 1 shows that:

[0067] (1) In Examples 1-4, as the proportion of algae mud increases, the water content increases. The highest water content is 84.35% in Example 4, which is still lower than 85%, meeting the requirements of relevant black soldier fly growth and development research.

[0068] (2) In Examples 1-4, as the proportion of algae mud increased, the crude polysaccharide content in the matrix increased, the crude fat content decreased, and the crude polysaccharide content in the group with added starter increased. The crude protein content in Examples 1 and 3 was not significantly different from that in Comparative Example 1.

[0069] from Figure 1 It can be seen that:

[0070] (1) Figure 1 As shown in A, the total production of larvae in Examples 3-4 decreased significantly, and the production of larvae in Examples 1-2 was not significantly different from that in Comparative Example 1.

[0071] (2) Figure 1 There is no significant difference in the material reduction rate between Examples 1-4 and Comparative Example 1 in B; Figure 1As shown in C, the bioconversion rates in Examples 1-2 are significantly higher than that in Comparative Example 1, while Examples 3-4 are significantly lower than that in Comparative Example 1; Figure 1 The substrate reduction index in Example 3-4 shown in D is significantly lower than that in Example 1-2 and Comparative Example 1. This indicates that the substrates in Example 1-2 are more palatable to the larvae, while the substrates in Example 3-4 are less palatable to the larvae.

[0072] Table 2 Growth and chemical composition of black soldier fly larvae

[0073]

[0074] Note: The same index with different superscript letters (a, b, c, d) indicates significant difference (p < 0.05). The values ​​are expressed as mean ± standard deviation (n = 3).

[0075] From Table 2 we can see that:

[0076] (1) Compared with the 6-day culture time of the control group 1, the development time was extended to 7 days and 9 days respectively as the proportion of algae mud increased. The water content in the substrate increased with the increase of the proportion of algae mud, and the crude fat content decreased with the increase of the proportion of algae mud, which may lead to the extension of the larval development time.

[0077] (2) As the proportion of algae mud increased, the weight of the larvae in Example 1-2 group decreased significantly, because the growth rate of the larvae decreased significantly after the addition of algae mud, which indicated that the growth of the larvae was inhibited. However, there was no significant difference in the chemical composition of the larvae harvested from Example 1-2 group and the control group, while the crude polysaccharide and crude fat content of the larvae in Example 3-4 group decreased significantly, which indicated that Example 3-4 may be detrimental to the growth of the larvae.

[0078] (3) Compared with Examples 1 and 3, the addition of fermentation agent in Examples 2 and 4 increased the crude protein content in the larvae and decreased the crude polysaccharide content.

[0079] Test Example 2:

[0080] During the breeding process, samples were taken every two days to monitor the body length and weight of black soldier flies until 50% of the prepupae appeared or the substrate was obviously changed into granular insect sand. The body length and weight of black soldier flies changed as follows: Figure 2 (“6d or 7d” means: except for the T-25 and TF-25 groups, which are the monitoring results on the 7th day, the other groups are the monitoring results on the 6th day). Figure 2 It can be seen that:

[0081] (1) In the early stage of culture (0-4 days), the growth of black soldier flies in Examples 1-4 with the addition of algae mud was significantly inhibited, and the growth of black soldier flies was improved in the group with the addition of fermentation agent.

[0082] (2) The body lengths of the black soldier flies finally obtained in Examples 1-4 and Comparative Example 1 were relatively close, with no significant difference. However, the body weight of the black soldier flies in Example 1-2 decreased significantly. As the breeding time increased, the body weight of the black soldier flies in Example 1-4 was not significantly different from that in the comparative example. This may be because the black soldier flies in Example 1-2 failed to adapt to the mixed feed environment in the early stage of breeding, and the black soldier flies were not fully grown due to the short time in the later stage.

[0083] Test Example 3: Effects on intestinal microbiota:

[0084] The structure and composition of the larval intestinal bacterial community were analyzed using high-throughput sequencing of the bacterial 16S rRNA gene:

[0085] (1) The collected larvae were starved for 12 h to empty their digestive contents. The larvae were picked out and rinsed with pure water. Ten larvae were randomly selected, and the surface was wiped with 70% alcohol for 30 seconds, soaked in 0.25% sodium hypochlorite for 1 minute, and then rinsed with sterile water three times to remove external contaminants;

[0086] (2) Finally, under a sterile environment, use sterilized fine-tipped forceps to cut open the abdomen of the larvae, remove the entire intestine, and immediately rinse it twice with 0.9% sterile NaCl solution. Then, slowly separate the intestine and place it in a microcentrifuge tube. The collected samples are quickly frozen in liquid nitrogen and stored at -80°C.

[0087] (3) The preserved samples were subjected to PCR amplification and sequencing (Meiji Biotechnology, Shanghai, China). The primers for 16S rRNA gene amplification were 515F: 5'-GTGCCAGCMGCCGCGG-3' and 907R: 5'-CCGTCAATTCMTTTRAGTTT-3'.

[0088] The combined PCoA (A) and NMDS (B) diagrams of the bacterial communities corresponding to β diversity are shown in Figure 2. Figure 3 The abundance of the top 50 genera of microorganisms in each treatment group was plotted as a heat map. Figure 4 .

[0089] from Figure 3 and Figure 4 It can be seen that:

[0090] (1) Based on the dimensionality reduction analysis of β diversity, the results showed that the intestinal microorganisms in Example 1-2 were similar to those in the control group, but were significantly different from those in Example 3-4. After the fermentation agent was added, the intestinal microorganisms changed significantly compared with the group without the addition of the fermentation agent.

[0091] (2) As the proportion of algae mud increases, the diversity and abundance of intestinal microorganisms of black soldier fly larvae increase. At the phylum level, among the intestinal microorganisms of larvae in different groups, as the proportion of algae mud increases, the relative abundance of Firmicutes microorganisms decreases, and the relative abundance of Proteobacteria increases. The increase in crude polysaccharide content in the matrix will limit the utilization of protein by Firmicutes microorganisms, which may be the reason for the decrease in the relative abundance of Firmicutes. At the genus level, the dominant bacteria Enterococcus in Examples 1-4 decreased, and the conditional pathogenic bacteria Peptostreptococcus increased, especially in Example 3-4, from 0.034% (Comparative Example 1) to 8.61%-12.48%. At the same time, in Example 3-4, the abundance of Morganella and Providencia, which can protect the larvae from pathogens and promote the growth of black soldier flies, increased. This shows that black soldier fly larvae will respond accordingly to adverse environments to maintain their growth.

[0092] (3) The relative abundance of Bacillus in the intestines of black soldier flies in Examples 2 and 4 with the addition of fermentation agents increased, which can regulate intestinal health and protect the larval intestines to a certain extent. Combining the above transformation effect and nutritional components of black soldier flies, it can be seen that if cyanobacteria and kitchen utensils are used as substrates, as the proportion of cyanobacteria increases, the transformation effect decreases, and the best effect is achieved when the fresh weight of algae mud accounts for 25%. Too high a proportion of cyanobacteria has a certain limiting effect on the transformation of black soldier flies and their nutritional components.

[0093] In summary, the present invention respectively measured and analyzed the growth of black soldier flies, nutritional components, and the structure of the intestinal microbial community of black soldier flies, and found that the black soldier fly larvae had a good conversion effect on the kitchen waste added with algae mud. By using the mature black soldier fly breeding factory process, each breeding box can process 25 kg of cyanobacteria mud, which can be used as one of the effective resource utilization methods for large-scale treatment of cyanobacteria mud and can be promoted and applied in real life.

[0094] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for recycling blue algae mud and kitchen waste by using black soldier fly larvae, the specific steps are as follows: (1) The kitchen waste is crushed after solid-liquid separation, and the moisture content of the separated kitchen waste is 65-85%; (2) mixing the kitchen waste treated in step (1) and cyanobacteria mud with a moisture content of 65-95% to form a black soldier fly breeding matrix, wherein the moisture content is controlled to be less than 85%; (3) The black soldier fly larvae are placed in a black soldier fly breeding matrix for breeding. After the breeding is completed, the insect bodies are separated from the insect sand, and the insect sand is used as an organic fertilizer raw material, and the black soldier fly larvae are used as an insect protein product; The fresh weight ratio of the blue algae mud to the kitchen waste is 1:(1-7); The ratio of the weight of the black soldier fly larvae to the fresh weight of the black soldier fly breeding substrate is 1: (20-80).

2. The method according to claim 1, characterized in that In the step (2), 0.1% to 1% of the dry weight of a Bacillus agent is added to the black soldier fly breeding substrate to assist the black soldier fly larvae in the simultaneous bioconversion of cyanobacteria mud and food waste.

3. The method according to claim 1, characterized in that The conditions for completing the breeding in step (3) are: half of the black soldier fly larvae enter the prepupa stage or the black soldier fly breeding matrix is ​​basically converted into granular insect sand.

Images