A method for resource utilization of blue-green algae sludge and kitchen waste by using black soldier fly larvae

Abstract

The present application belongs to the technical field of organic waste resource utilization, and particularly relates to a method for resource utilization of blue-green algae sludge and kitchen waste by using black soldier fly larvae. The pretreated blue-green algae sludge and kitchen waste are mixed as mixed feed, and a bacillus agent is added for aerobic fermentation to obtain black soldier fly larvae and worm sand. By regulating the substrate ratio and the synergistic effect of the agent, the nutritional value and utilization efficiency of the mixed feed are increased, and the growth of the black soldier fly in the early stage is faster. The obtained black soldier fly larvae can be used as a high-quality protein feed, and the worm sand can be used as an organic fertilizer raw material. The present application realizes the collaborative resource utilization of blue-green algae and kitchen waste, and provides a scalable biological solution for the disposal and utilization of water bloom blue-green algae sludge.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for resource utilization of blue-green algae sludge and kitchen waste by using black soldier fly larvae, belonging to the field of organic waste resource utilization. BACKGROUND

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

[0003] Although blue-green algae sludge is often treated as solid waste, it cannot be ignored that blue-green algae is also a biomass with great potential utilization value. Algae have higher solar utilization efficiency than general terrestrial plants, and water bloom blue-green algae contains a large amount of nutrients such as protein, polysaccharide, unsaturated fatty acid, and usable substances such as phycocyanin and carotenoid. The protein content is as high as 30-65%. The existing common blue-green algae resource utilization methods are as follows: (1) preparation of biofuels, which still has major problems such as complex reaction device, high operating cost (algal bio-diesel production economic cost can reach 42-59 thousand yuan / t), and low efficiency; (2) extraction of useful substances, such as extraction of phycocyanin, which has a purification stage cost accounting for 50-90% of the production cost, a complex extraction process, and difficulty in large-scale production; (3) preparation of organic carbon sources, which has immature key technologies, is prone to secondary pollution, has high cost, and does not meet the energy use concept under the background of "double carbon", and the biomass carbon source also has the problem of low denitrification rate. At the same time, the problems of algal toxins and heavy metals in the sludge also become difficult problems that cannot be ignored in resource utilization approaches. In the face of increasing blue-green algae biomass, there is an urgent need for a method that is safer, more environmentally friendly and efficient, has a large treatment capacity, and can produce high-value products.

[0004] The technology of using saprophagous insects to treat organic matters has the advantages of low cost, less maintenance, simple operation, small land requirement, low ecological footprint and greater economic potential. Among them, black soldier fly is widely promoted in the treatment of kitchen waste, animal manure, straw, sludge and other organic waste because of its characteristics of not spreading diseases, wide food habits and high biological conversion efficiency. Kitchen waste is considered as one of the most suitable feed for black soldier fly growth due to its rich nutrients. It is worth noting that even though kitchen waste is rich in nutrients, the difference in nutrient composition of kitchen waste from different sources has a certain impact on the growth of black soldier fly, especially the carbon-nitrogen ratio in kitchen waste, so it is necessary to develop suitable auxiliary materials to solve the above problems. SUMMARY

[0005] To solve the above problems, the present application mixes the flocculated blue-green algae slurry with kitchen waste and directly feeds black soldier fly larvae. On the one hand, kitchen waste is the most suitable feed for black soldier fly growth due to its rich nutrients, and the process has been maturely used in production activities. Mixing blue-green algae with kitchen waste can solve the problem of large amount of blue-green algae biomass accumulation and alleviate the adverse effects of algae slurry on the growth of black soldier fly larvae during biological conversion. On the other hand, the insect protein obtained by feeding has high economic value and can be used as a protein source in animal feed, fully realizing the resource potential of blue-green algae biomass. At the same time, the addition of probiotics can regulate the fermentation process of the substrate and the absorption of nutrients by the insects, reduce the adverse effects of blue-green algae on the growth of black soldier fly, improve the efficiency of black soldier fly in biological conversion of blue-green algae, and improve the nutritional value of the insects.

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

[0007] A method for resource utilization of blue-green algae slurry and kitchen waste by black soldier fly larvae, the specific steps are as follows:

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

[0009] (2) mixing the kitchen waste treated in step (1) and blue-green algae slurry with a moisture content of 65-95% to form a black soldier fly breeding substrate, and controlling the moisture content to be less than 85%;

[0010] (3) placing black soldier fly larvae in the black soldier fly breeding substrate for breeding, separating the insects from the sand after breeding, using the sand as organic fertilizer raw material, and using the black soldier fly larvae as insect protein product to realize the resource utilization of blue-green algae slurry and kitchen waste; the fresh weight ratio of blue-green algae slurry to kitchen 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] The water content of the black soldier fly breeding substrate in step (2) is 65%-85%.

[0013] Further, in step (2), 0.1%-1% of Bacillus agent based on the dry weight of the black soldier fly breeding substrate is added to assist the black soldier fly larvae in simultaneously bioconverting the cyanobacterial sludge and kitchen waste.

[0014] Further, the breeding completion condition in step (3) is that half of the black soldier fly larvae enter the pre-pupa stage or the black soldier fly breeding substrate is basically converted into granular worm sand, which can be determined by professionals in the art.

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

[0016] The black soldier fly larvae have high bioconversion capacity for organic waste such as kitchen waste, livestock and poultry manure, sludge, and straw, 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, kitchen waste is suitable for the growth and development of black soldier fly larvae due to its rich nutrients. The resource utilization treatment method of mixing algal sludge with kitchen waste and adding a fermenting agent has the following advantages:

[0017] (1) Not only can the black soldier fly larvae directly bioconvert the algal sludge, but also can alleviate the adverse effects of the algal sludge on the black soldier fly larvae;

[0018] (2) The addition of the fermenting agent can convert large molecular proteins and other substances in the substrate into small molecular polypeptides and other metabolic products through its own metabolism, thereby improving the nutritional value and utilization efficiency of the mixed feed, increasing the crude polysaccharide content of the mixed feed, and making the black soldier fly grow more rapidly in the early stage (0-4d), and also regulating the intestinal health of the black soldier fly larvae;

[0019] (3) This method not only saves a lot of cost for further treatment of the cyanobacterial sludge, but also produces valuable products such as larval protein. When the algal sludge is mixed with kitchen waste for breeding, there is no difference in the crude protein and crude fat content between the harvested black soldier fly larvae and the kitchen-raised black soldier fly larvae, which does not affect the black soldier fly larvae as insect protein products. Compared with the traditional composting process (treatment period of 30-60 days), the treatment period of the method of the present application is significantly shortened, and the material reduction rate is more than 50% (B), which is particularly suitable for emergency treatment of harvested algal sludge during the outbreak of cyanobacterial blooms. Figure 1 B), which is particularly suitable for emergency treatment of harvested algal sludge during the outbreak of cyanobacterial blooms.

[0020] (4) Large-scale processing of cyanobacteria mud can be directly carried out in aquaculture factories, which can meet the seasonal characteristics of cyanobacteria production, has high feasibility, and effectively solves the problem of large-scale accumulation of cyanobacteria mud that is difficult to handle in emergency treatment.

[0021] (5) Economic benefits: This method not only saves a significant amount of cost for further processing of cyanobacteria sludge, but also produces valuable products such as larval protein. Bioconversion of 4 tons of mixed feed (including 1 ton of cyanobacteria sludge) by black soldier fly larvae yields 431 kg of fresh black soldier flies and 454 kg of fresh insect sand. The revenue from the insect sand is calculated as 0 yuan. Based on a fresh insect value of approximately 2400 yuan / ton and a breeding cost of 1920 yuan / ton, the black soldier flies harvested from processing 1 ton of cyanobacteria sludge can be converted into a revenue of 207 yuan. Compared to the anaerobic fermentation of cyanobacteria sludge to produce biogas (with a government subsidy of 280 yuan / ton), the benefits of converting cyanobacteria sludge with black soldier flies are significantly increased. Attached Figure Description

[0022] Figure 1 To test the effect of adding cyanobacteria to the biotransformation capacity of black soldier fly larvae in each treatment group in Example 1.

[0023] Figure 2 To test the growth of black soldier fly larvae during the rearing process in Example 2.

[0024] Figure 3 and Figure 4 These represent the PCoA values ​​of the bacterial communities corresponding to β-diversity during the culture process in Test Example 3. Figure 3 (A) and NMDS Figure 3 A combination diagram of the top 50 genera of microorganisms in each treatment group (B), and a heatmap showing the abundance of each treatment group. Figure 4 ). Detailed Implementation

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

[0026] Raw materials used in the examples:

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

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

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

[0030] Bacillus agent: fermentation agent produced by Hubei Qiming Biological Engineering Co., Ltd.

[0031] Example 1

[0032] A resource utilization method for biological conversion of mixed blue-green algae sludge and kitchen waste by black soldier fly, comprising the following steps:

[0033] Mix 25 Kg of blue-green algae sludge with 75 Kg of crushed kitchen waste in a feeding box (1.2 m x 2.4 m x 0.3 m), put 1 box of 3-day-old black soldier fly larvae (about 2 kg) into the feeding box, and move the feeding box into a breeding warehouse (temperature at about 30℃, relative humidity at about 70%) for breeding; named as T-25 group.

[0034] Example 2

[0035] Add 0.06 Kg of fermentation agent suspension (fermentation agent purchased from Hubei Qiming Biological Engineering Co., Ltd., main components: Bacillus, Actinomyces, yeast, filamentous fungi and other beneficial microorganisms and various extracellular enzymes, effective viable count ≥200 billion / g, by determining 16S rRNA, the relative abundance of Bacillus is 99.4%, same below) to the substrate in Example 1, and then perform subsequent operations, named as TF-25 group.

[0036] Example 3

[0037] Increase the fresh weight of blue-green algae sludge in Example 1 to 50 Kg, and reduce the kitchen waste to 50 Kg, and perform subsequent operations in accordance with Example 1, named as T-50 group.

[0038] Example 4

[0039] Add 0.06 Kg of fermentation agent to the substrate in Example 3, and then perform subsequent operations, named as TF-50 group.

[0040] Comparative Example 1

[0041] Adjust 25 Kg of blue-green algae sludge and 75 Kg of crushed kitchen waste in Example 1 to 100 Kg of crushed kitchen waste, and perform subsequent operations in accordance with Example 1, named as Control group.

[0042] The performance tests of Examples 1-4 and Comparative Example 1 were carried out, specifically, when half of the pre-pupae appeared in each group or the substrate was obviously changed into granular frass, the culture time of each group was recorded, the frass and larvae (including pre-pupae and other larvae) were separated and weighed, the body length and weight of black soldier fly larvae were measured, and the obtained black soldier fly larvae could be used as protein feed and the frass could be used as organic fertilizer raw material.

[0043] Test Example 1:

[0044] 1. Measurement of the mass of harvested black soldier fly larvae and frass:

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

[0046] 2. Nutrient component determination:

[0047] The initial substrate, frass and finally obtained larvae were placed in a freeze dryer for freeze-drying, and the following nutrient components were determined.

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

[0049] Polysaccharides were determined by phenol-sulfuric acid method, and crude protein in the substrate and frass was determined by Kjeldahl method:

[0050] First, 0.5 g of dried sample was accurately weighed into a digestion tube, 0.2 g of copper sulfate, 3 g of potassium sulfate and 10 mL of concentrated sulfuric acid were added, and the sample was digested in a digestion furnace. The blank sample was not added with 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 determined using a Kjeldahl nitrogen determination instrument. The crude protein determination is TN (total nitrogen) x 6.25 (Chinese standard GB 5009.5-2016).

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

[0052] 3. Conversion rate determination:

[0053] The screened black soldier fly larvae were microwave-dried to obtain dried insects, which were stored in a -20°C refrigerator, and the frass was dried at 105°C for 24 hours, and they were weighed and recorded Figure 1 A), and the material reduction rate, biological conversion rate, larval growth rate, substrate reduction index and other indicators were calculated.

[0054] The material reduction rate (dry weight) was calculated according to the following formula (1):

[0055]

[0056] The bioconversion rate (all indexes in the formula are dry weight) is calculated as follows (2), wherein the initial dry weight of the larvae is very small and is ignored in the calculation of the results this time:

[0057]

[0058] The growth rate of the larvae (fresh weight) is calculated as follows (3):

[0059]

[0060] The substrate reduction index (dry weight) is 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 substrate in the five groups

[0064]

[0065] Note: The values in the same row with different superscripts (a, b, c) are significantly different (p<0.05). The values are represented by mean ± standard deviation (n=3), and the crude polysaccharide, crude protein and crude fat refer to the mass percentage of the initial substrate dry weight.

[0066] As can be seen from Table 1:

[0067] (1) In Examples 1-4, the moisture content increases with the increase of the proportion of algal sludge. The highest moisture content is 84.35% in Example 4, which is still lower than 85%, meeting the requirements of related studies on the growth and development of black soldier flies.

[0068] (2) In Examples 1-4, the crude polysaccharide content in the substrate increases with the increase of the proportion of algal sludge, and the crude fat content decreases. The crude polysaccharide content increases in the group with the addition of the ferment. The crude protein content in Examples 1 and 3 has no significant difference with that in Comparative Example 1.

[0069] As can be seen from Figure 1 :

[0070] (1) Figure 1 The total yield of larvae in Examples 3-4 in Figure A significantly decreases, and the yield of larvae in Examples 1-2 has no significant difference with 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 Figure B; Figure 1The bioconversion rates in Examples 1-2 were significantly higher than those in Comparative Example 1, while Examples 3-4 were significantly lower than those in Comparative Example 1. Figure 1 The substrate reduction indices in Examples 3-4 were significantly lower than those in Examples 1-2 and Comparative Example 1. This indicates that the substrates in Examples 1-2 were more palatable to the larvae than those in Examples 3-4.

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

[0073]

[0074] Note: Different letters (a, b, c, d) on the same index represent significant differences (p < 0.05). Values are expressed as mean ± standard deviation (n = 3).

[0075] From Table 2, we can see that:

[0076] (1) Compared to the 6d cultivation time of Comparative Example 1, the development time was extended to 7d, 9d, respectively, as the proportion of algal sludge increased. The moisture content in the substrate increased with the increase of the proportion of algal sludge, and the crude fat content decreased with the increase of the proportion of algal sludge, which may be the factors leading to the extension of the development time of the larvae.

[0077] (2) As the proportion of algal sludge increased, the weight of larvae in Examples 1-2 group decreased significantly, because the growth rate of larvae decreased significantly after the addition of algal sludge, which indicated that the growth of larvae was inhibited. However, there was no significant difference in the chemical composition of the harvested larvae between Examples 1-2 group and the control group, while the crude polysaccharide and crude fat content of larvae in Examples 3-4 group decreased significantly, which indicated that Examples 3-4 may not be conducive to the growth of larvae.

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

[0079] Test Example 2:

[0080] The body length and weight of black soldier fly were monitored every two days during the cultivation process until 50% of pre-pupae appeared or the substrate was significantly changed into granular sand. The changes in body length and weight of black soldier fly were as follows: Figure 2 (wherein, "6d or 7d" means that the monitoring results of T-25 and TF-25 groups are on the 7th day, and the monitoring results of other groups are on the 6th day), from Figure 2 It can be seen that:

[0081] (1) In the early stage of cultivation (0-4d), the growth of black soldier fly in Examples 1-4 with the addition of algal sludge was significantly inhibited, and the growth of black soldier fly in the groups with the addition of fermenting agent was improved.

[0082] (2) The body length of the black soldier flies finally obtained in Examples 1-4 and Comparative Example 1 is relatively close, and there is no significant difference, but the body weight of the black soldier flies in Examples 1-2 is obviously decreased, and the body weight of the black soldier flies in Example 1-4 is not significantly different from that in the comparative example as the breeding time is prolonged, which may be due to the fact that the black soldier flies in Example 1-2 fail to adapt to the mixed feed environment in the early stage of breeding, and the black soldier flies are not fully grown in a short time in the later stage.

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

[0084] The structure and composition of the bacterial community in the intestinal tract of the larvae were analyzed by high-throughput sequencing of the bacterial 16S rRNA gene:

[0085] (1) First, the collected larvae were starved for 12 h to empty their digestive contents, and 10 larvae were randomly picked after being rinsed with pure water, the surface was wiped with 70% alcohol for 30 s, 0.25% sodium hypochlorite was soaked for 1 min, and then the external contaminants were removed by rinsing with sterile water for 3 times;

[0086] (2) Finally, in a sterile environment, the larvae were cut open at the abdomen using sterilized fine forceps, the entire intestinal tract was taken out, and immediately rinsed twice with 0.9% sterile NaCl solution, then the intestinal tract was slowly separated and placed in a microcentrifuge tube. The collected sample was quickly frozen in liquid nitrogen and stored at -80°C.

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

[0088] The combined graph of PCoA (A) and NMDS (B) of the bacterial community corresponding to the beta diversity is as shown in Figure 3 , and the heat map result of the abundance of the top 50 microbial genera in each treatment group is shown in Figure 4 .

[0089] From Figure 3 and Figure 4 , it can be seen that:

[0090] (1) Based on the dimensionality reduction analysis of beta diversity, the results show that the intestinal microorganisms in Example 1-2 are relatively similar to the control group, and the difference with Example 3-4 group is relatively obvious; the intestinal microorganisms change significantly after adding the fermenting agent compared with the group without adding the fermenting agent.

[0091] (2) With the increase of the proportion of algal sludge, the diversity and abundance of the intestinal microorganisms of black soldier fly larvae increase. At the phylum level, with the increase of the proportion of algal sludge, the relative abundance of Firmicutes decreases, and the relative abundance of Proteobacteria increases. The increase of the content of crude polysaccharides in the substrate will limit the utilization of protein by Firmicutes, which may be the reason for the decrease of the relative abundance of Firmicutes. At the genus level, the dominant bacteria Enterococcus in Examples 1-4 decrease, and the condition pathogen Peptostreptococcus increases, especially in Examples 3-4, from 0.034% (Comparative Example 1) to 8.61%-12.48%. At the same time in Examples 3-4, the abundance of Morganella and Providencia, which can protect the larvae from pathogens and promote the growth of black soldier fly, increases. This shows that the black soldier fly larvae will respond to the adverse environment to maintain their growth.

[0092] (3) The relative abundance of Bacillus in the intestines of black soldier flies in Example 2 and Example 4 increases, which can play a role in regulating intestinal health and protecting the intestines of larvae. In combination with the above conversion effect and nutritional components of black soldier fly, if blue-green algae and kitchen waste are used as substrates, the conversion effect decreases with the increase of the proportion of blue-green algae, and the effect is best when the fresh weight of algal sludge accounts for 25%. A high proportion of blue-green algae has a certain limiting effect on the conversion of black soldier fly and its nutritional components.

[0093] In summary, the growth of black soldier fly, nutritional components, and intestinal microbial community structure of black soldier fly are determined and analyzed, respectively, and it is found that the kitchen waste with added algal sludge has a good conversion effect on black soldier fly larvae. Using mature black soldier fly breeding factory technology, each breeding box can process 25 Kg of blue-green algal sludge, which can be used as one of the effective resource utilization methods for large-scale treatment of blue-green algal sludge, and can be popularized and applied in actual life.

[0094] Although the present application has been disclosed with reference to the preferred embodiments above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for resource utilization of cyanobacteria sludge and kitchen waste using black soldier fly larvae, the specific steps of which 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) Mix the kitchen waste treated in step (1) with blue-green algae mud with a water content of 65-95% to form a black soldier fly breeding substrate, and control its water content to be 65-85%; the blue-green algae mud is obtained by flocculation; Adding 0.1%-1% of Bacillus spp. by dry weight to the black soldier fly breeding substrate helps the black soldier fly larvae to simultaneously biotransform cyanobacteria and kitchen waste; (3) Place the black soldier fly larvae in the black soldier fly breeding substrate and raise them for 7 days. After the breeding is completed, separate the insect body from the insect sand. The insect sand is used as raw material for organic fertilizer, and the black soldier fly larvae are used as insect protein products. The conditions for the completion of the breeding are: half of the black soldier fly larvae enter the prepupal stage or the black soldier fly breeding substrate is basically transformed into granular insect sand. The fresh weight ratio of the blue-green algae sludge to kitchen waste is 1:3; The ratio of the weight of the black soldier fly larvae to the fresh weight of the black soldier fly culture substrate is 1:50; Based on the weight of Bacillus spp. inoculants, the effective viable count is ≥20 billion / g, of which the relative abundance of Bacillus spp. is 99.4%. Bacillus spp. inoculants include Bacillus spp. and its various extracellular enzymes, actinomycetes and their various extracellular enzymes, yeasts and their various extracellular enzymes, and filamentous fungi and their various extracellular enzymes.

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