Artificial multi-microbial system for degrading perishable garbage, application and degradation method

Abstract

The present application relates to the technical field of perishable garbage treatment, and particularly relates to an artificial multi-microbial system for degrading perishable garbage, application and degradation method, the artificial multi-microbial system comprises Bacillus amyloliquefaciens ZJB18046, Bacillus licheniformis ZJB19163 and Bacillus tequilensis ZJB19167, wherein the preservation number of the Bacillus amyloliquefaciens ZJB18046 is CCTCC NO:M2019423, the preservation number of the Bacillus licheniformis ZJB19163 is CCTCC NO:M2020014, and the preservation number of the Bacillus tequilensis ZJB19167 is CCTCC NO:M2020177. The three strains in the present application are obtained by artificial screening, each strain has a synergistic effect in the process of organic matter degradation, has high degradation activity and degradation efficiency for the main components of perishable garbage, is suitable for the degradation requirements of main components of perishable garbage such as starch, protein and oil, and through a continuous feeding strategy, the amount of microbial inoculum is effectively reduced, and cost-effectiveness is higher.

Description

Technical Field

[0001] The present invention relates to the technical field of perishable garbage treatment, and in particular to an artificial multi-bacteria system for degrading perishable garbage, and its application and degradation method. Background Art

[0002] Perishable waste, also known as wet waste, generally refers to kitchen waste generated during the production processes of restaurants, workplace canteens, and other organizations, as well as perishable waste generated in households. This includes leftovers, vegetable stems and leaves, meat and offal, fruit shells, and melon peels. Chemically, perishable waste contains starch, protein, cellulose, lipids, and inorganic salts, making it highly susceptible to decay and odor, spreading bacteria and viruses. Large accumulations not only affect urban appearance but also pollute the environment and harm public health. With the growth of the global population and improvements in living standards, the amount of perishable waste generated is increasing. This type of waste easily decays and deteriorates in the natural environment, becoming a source of pollution that hinders normal production and daily life, creating environmental hazards. Food waste accounts for over 60% of this waste. The World Food and Agriculture Organization (WFAO) indicates that 1.6 billion tons of perishable waste are generated globally each year, accounting for one-third of global food production. If improperly handled, food waste quickly decays and odors, producing leachate and breeding various bacteria, leading to serious environmental problems and potential health hazards. Therefore, effective strategies are needed to manage the rapidly growing amount of perishable waste.

[0003] Because perishable waste is rich in organic matter such as carbohydrates, proteins, and fats, it has the potential to be recycled. Traditional centralized treatment methods such as composting and anaerobic digestion can effectively recycle and reuse perishable waste, but due to the lengthy processing times, they struggle to effectively manage the rapidly growing volume of perishable waste. For example, a research team evaluated the use of composting systems to treat perishable waste in densely populated residential areas and found that labor, waste transportation, and sorting costs were $23.02 per ton of food waste, accounting for 83.95% of total expenses. Therefore, there is a need to develop efficient and controllable technologies for the reduction and harmless treatment of perishable waste.

[0004] As a kind of organic waste with strong biodegradability, perishable garbage can be treated in situ using microbial degradation technology to reduce the volume of garbage at the source, greatly reduce the cost of collection and transportation, and reduce secondary pollution. The residue after treatment can also be made into organic fertilizer. For example, in patent CN109182179A, a bacterial agent was prepared based on a strain of Bacillus subtilis. 1 kg of degradation bacterial agent was added to a perishable garbage degradation machine, and 0.4 kg of leftover food was added for degradation. The degradation rate was calculated based on the actual weight reduction. Within 48 hours, the weight reduction rate of perishable garbage reached 93.2%. In general, this patent adopts a single bacterial system in terms of substrate utilization. The types of substrates that can be used are relatively single, and a single microbial strain is difficult to achieve all-round degradation of perishable garbage. When targeting complex renewable biomass, the substrate utilization efficiency of the single bacterial system is low and the stability is poor. In terms of degradation effect, it focuses more on reducing the total weight, and the treatment cycle is longer. Considering that food waste contains a water content of up to 70-90%, a single weight loss rate cannot intuitively illustrate the degradation of organic matter in food waste. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide an artificial multi-bacteria system, application and degradation method for degrading perishable garbage. The artificial multi-bacteria system is adapted to the degradation needs of the main components of perishable garbage, has high degradation efficiency for different perishable garbage components, has good stability in the in-situ reduction process, and through a continuous feeding strategy, effectively reduces the amount of bacterial agent added, improves the reduction degree of perishable garbage, and has a short fermentation treatment cycle.

[0006] To achieve the above-mentioned purpose, the present invention is implemented through the following technical solutions:

[0007] The first object of the present invention is to provide an artificial multi-bacteria system for degrading perishable garbage, comprising Bacillus amyloliquefaciens ZJB18046, Bacillus licheniformis ZJB19163 and Bacillus tequilensis ZJB19167, wherein the preservation number of Bacillus amyloliquefaciens ZJB18046 is CCTCCNO: M2019423, the preservation number of Bacillus licheniformis ZJB19163 is CCTCC NO: M2020014, and the preservation number of Bacillus tequilensis ZJB19167 is CCTCCNO: M 2020177.

[0008] According to the composition characteristics of perishable garbage in my country, the present invention screened and obtained bacterial strains with high degradation activity for the main components of perishable garbage, including Bacillus amyloliquefaciens ZJB18046, which was deposited in the China Center for Type Culture Collection at Wuhan University in Hubei, China, with a deposit date of June 4, 2019, and a deposit number of CCTCC NO: M2019423; Bacillus licheniformis ZJB19163, which was deposited in the China Center for Type Culture Collection at Wuhan University in Hubei, China, with a deposit date of January 6, 2020, and a deposit number of CCTCC NO: M2020014; and Bacillus tequilensis ZJB19167, which was deposited in the China Center for Type Culture Collection at Wuhan University in Hubei, China, with a deposit date of June 3, 2020, and a deposit number of CCTCC NO: M2020014. 2020177.

[0009] Preferably, based on the weight of wet cells, the mass ratio of the Bacillus amyloliquefaciens ZJB18046, the Bacillus licheniformis ZJB19163, and the Bacillus tequila ZJB19167 is 1-1.5:1:1.

[0010] Preferably, the artificial multi-bacteria system further comprises straw powder, peat powder and diatomaceous earth for fixing the bacterial agent.

[0011] The weight ratio of straw powder, peat powder and diatomaceous earth in the artificial multi-bacteria system is 50:30:20. When preparing the artificial multi-bacteria system, the above three bacteria are evenly mixed with straw powder, peat powder and diatomaceous earth according to the proportion.

[0012] Preferably, the amount of wet cells of the three bacteria in the artificial multi-bacteria system is 5% to 15% of the total mass, and the artificial multi-bacteria system is dried at 40° C. to 50° C. and is ready for use.

[0013] Bacillus amyloliquefaciens ZJB18046, Bacillus licheniformis ZJB19163, and Bacillus tekila ZJB19167 were activated and fermented at high density. The wet cells of the three bacteria were mixed in proportion and then added with straw powder, peat powder, and diatomaceous earth. The mixture was then dried at 40°C to 50°C to prepare a dry preparation of the artificial multi-bacteria system. The wet cells of the three bacteria were added in an amount of 5% to 15% of the total weight.

[0014] The second object of the present invention is to provide the application of the artificial multi-bacteria system in degrading perishable garbage.

[0015] The third object of the present invention is to provide a method for degrading perishable garbage, using the artificial multi-bacteria system to ferment and degrade the perishable garbage.

[0016] Preferably, the mass ratio of the amount of the artificial multi-bacteria system added to the perishable waste is 0.1~10:100.

[0017] Preferably, the water content of the perishable garbage is 50% to 60%.

[0018] Before fermentation and degradation treatment, the perishable garbage needs to be initially filtered and dehydrated to reduce the water content to 50%~60%, which can increase the processing capacity and treatment effect of the perishable garbage.

[0019] Preferably, the fermentation degradation is carried out in a fermentation bin with controlled temperature, ventilation and intermittent stirring for 24 hours to complete one degradation; after one degradation is completed, the same amount of perishable garbage as that of the first degradation is continuously added for further degradation, and the process is repeated in batches until the fermentation bin is full and the fermentation degradation is completed.

[0020] Preferably, the fermentation temperature is controlled at 45±2°C.

[0021] Pick out the non-degradable solid waste such as bones and plastics from the perishable waste; then preliminarily filter and remove water from the perishable waste to make the water content account for 50%~60%; add the artificial multi-bacteria system and mix evenly, and add appropriate amount of sawdust to adjust the C / N ratio to 30~40, control the temperature at 45±2℃ and ventilate, and stir intermittently for 24 hours to complete the first degradation; after 24 hours, continue to add the same amount of perishable waste as the first time, control the same reaction conditions, and repeat the process of adding perishable waste until the fermentation bin is close to overflowing (about 30 days), and take out the degraded perishable waste from the fermentation bin; the degraded perishable waste taken out can be used as organic fertilizer.

[0022] Batch continuous fed-batch fermentation matches the growth of the degradation strain with the coupling of the enzyme production process, which can effectively realize the continuous degradation process of a single-batch artificial multi-bacteria system (one-time feeding and continuous operation for one month), reducing the feeding cost of the strain.

[0023] Preferably, the intermittent stirring is stirring for 15 to 30 minutes and resting for 5 to 10 minutes.

[0024] The present invention has the following beneficial effects:

[0025] (1) The three strains of Bacillus amyloliquefaciens ZJB18046, Bacillus licheniformis ZJB19163, and Bacillus tequila ZJB19167 in this artificial multi-bacteria system were obtained through artificial screening and have high degradation activity against the main components of perishable waste, and are suitable for the degradation needs of the main components of perishable waste, such as starch, protein, and oil;

[0026] (2) The various bacterial species in this artificial multi-bacteria system have a synergistic effect in the organic matter degradation process, significantly improving the degradation effect and the stability of the in-situ reduction process, and have a high degradation efficiency for different perishable waste components;

[0027] (3) Without modifying the treatment equipment, the continuous feeding strategy effectively reduced the amount of microbial agents added, improved the degree of reduction of perishable waste, and was more stable and cost-effective in reducing perishable waste in situ. The degraded perishable waste could be used as organic fertilizer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the degradation rate of starch, protein, cellulose and oil by the strain in the liquid culture medium in Example 1. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only a portion of the embodiments of the present invention, rather than all of the embodiments. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0030] Example 1

[0031] Screening and identification of bacterial strains that degrade the main components of perishable garbage

[0032] Organic matter in perishable waste is mainly composed of protein, starch, oil and cellulose. 2 grams of fresh soil sample was taken from the Yuxiu Restaurant of Zhejiang University of Technology and suspended in sterile water. The suspension was shaken at 30°C and 150 rpm for 30 minutes. The suspension was diluted with sterile water in a gradient of 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 200 μL of the bacterial suspension at each concentration was inoculated onto an agar plate and incubated in a 37°C incubator for 24 hours. Representative colonies were selected and repeatedly streaked until distinct single colonies were isolated. The agar plates used were Luria-Bertani (LB) solid medium (1% peptone, 0.5% yeast extract, 1% NaCl, and 2% agar) and potato dextrose agar (PDA) medium (2% potato, 2% glucose, and 2% agar).

[0033] Because oils are difficult to degrade, separate screening for oil-degrading strains was necessary. Soil from an oil-contaminated area was collected near the exhaust vent of the Yuxiu Canteen at Zhejiang University of Technology. Oil-degrading strains were enriched using a mineral salt medium containing 2.0% vegetable oil and then isolated on agar using the same gradient dilution method.

[0034] The genomic DNA of the strain was extracted using the FastDNATM Spin Kit for Soil and used as a template for PCR amplification. The universal primers for fungi were:

[0035] ITS1: 5'-TCCGTAGGTGAACCTGCGG-3',

[0036] ITS4:5'-TCCTCCGCTTATTGATATGC-3',

[0037] Bacterial universal primers:

[0038] 27F: 5'-AGAGTTTTGATCCTGGCTCA-3',

[0039] 1492R: 5'-AAGGAGGTGATCCAGCCGCA-3'.

[0040] The products were sequenced by Qingke Biotechnology Co., Ltd. The obtained DNA sequences were entered into GenBank and compared with all sequences in the database using the Blast program. Appropriate DNA sequences were selected to establish a phylogenetic tree to determine the species of the strains obtained by screening.

[0041] A total of 87 strains were screened and obtained. The clear zone method was used to screen the strains. All screened strains were inoculated into agar medium containing starch, fat, protein and cellulose. The clear zone around the colony was used as an indicator of degradation activity. The ten strains with the largest clear zone diameter in each culture medium were selected for rescreening and inoculated into starch degradation medium, oil degradation medium, protein degradation medium and cellulose degradation medium respectively. After three days of culture, the strains with the highest starch, oil, protein and cellulose degradation activity were selected to prepare new microbial agents. The degradation rates of starch, protein, cellulose and oil by each strain in liquid culture medium are as follows: Figure 1As shown. The starch degradation medium components are: peptone 10.0g / L, soluble starch 10.0g / L, beef extract 5.0g / L, NaCl 5.0g / L, pH 7.0-7.2. The protein degradation medium components are: skim milk powder 10g / L, peptone 10.0g / L, beef extract 5.0g / L, MgSO4·7H2O0.2g / L, pH 7.0-7.2. The oil degradation medium components are: (NH4)2SO42g / L, K2HPO41g / L, KCl 0.5g / L, MgSO4·7H2O 0.5g / L, FeSO40.01g / L, pH natural, vegetable oil 20 g / L. The cellulose degradation medium consists of 10.0 g / L peptone, 5.0 g / L beef extract, 0.5 g / L K₂HPO₄, 0.25 g / L MgSO₄, and 1 g / L wheat straw. All culture media, except the protein selection medium, were sterilized at 121°C for 20 minutes. Protein medium was sterilized by sterilizing skim milk powder at 115°C for 20 minutes, while all other culture media were sterilized at 121°C for 20 minutes.

[0042] Finally, one strain, Bacillus amyloliquefaciens, one strain, and one strain, Bacillus tequila, were screened from the 87 strains and named and deposited. Bacillus amyloliquefaciens ZJB18046, deposited with CCTCCM2019423, was deposited at the China Center for Type Culture Collection, Wuhan University, Hubei, China, on June 4, 2019; Bacillus licheniformis ZJB19163, deposited with CCTCC M2020014, was deposited at the China Center for Type Culture Collection, Wuhan University, Hubei, China, on January 6, 2020; and Bacillus tequila ZJB19167, deposited with CCTCC NO: M2020177, was deposited at the China Center for Type Culture Collection, Wuhan University, Hubei, China, on June 3, 2020.

[0043] Example 2

[0044] Preparation of an artificial multi-bacteria system for efficient degradation of perishable waste

[0045] (1) Streak the frozen glycerol tubes of the three strains on a plate using activated LB medium and incubate at 37°C for 12 hours. Pick the well-grown colonies into a 2L shake flask containing 700mL LB for fermentation, incubate at 37°C for 48 hours at 200r / min, and centrifuge at 8000r / min for 10 minutes to obtain the wet cells of the three strains. Mix the three strains in a wet cell mass ratio of 1:1:1 and mix them evenly with the bacterial agent fixative (straw powder, peat powder and diatomaceous earth in a mass ratio of 50:30:20) in proportion. The amount of the three bacterial wet cells added accounts for 5% of the total mass of the mixture. After natural drying at 40°C, the new artificial multi-bacteria system dry preparation is obtained and stored at 4°C.

[0046] (2) The three single-strain bacteria and the artificial multi-strain system were inoculated into simulated perishable waste culture medium, respectively. The mass ratio of inoculum mass to perishable waste culture medium was 2:100. The culture was held at 45°C for 72 h. The degradation rates of starch and protein were measured after 24 h of culture, and the degradation rates of oil and cellulose were measured after 72 h of culture. The results are shown in Table 1. The perishable waste simulated culture medium consisted of: starch 5 g, casein 5 g, soybean oil 6.5 g, carboxymethyl cellulose 1.0 g, water 80 g, K2HPO4 0.2 g, KCl 0.1 g, MgSO4 0.05 g, and FeSO4·7H2O 0.01 g.

[0047] Table 1 Degradation of organic matter by single bacterial strain and artificial multi-bacteria system

[0048] .

[0049] Among the single-bacteria degradation systems, Bacillus amyloliquefaciens ZJB18046 showed the highest starch and protein degradation rates, Bacillus licheniformis ZJB19163 the highest cellulose degradation rate, and Bacillus tekila ZJB19167 the highest oil degradation rate. Compared to single-strain degradation, the artificial multi-bacteria system significantly improved the degradation rates of starch, protein, oil, and cellulose, demonstrating that the synergistic degradation of the three Bacillus strains can effectively promote the degradation of major organic components in perishable waste.

[0050] Example 3

[0051] Preparation of an artificial multi-bacteria system for efficient degradation of perishable waste

[0052] (1) Streak the frozen glycerol tubes of the three strains on a plate using activated LB medium and incubate at 37°C for 12 hours. Pick the well-grown colonies into a 2L shake flask containing 700mL LB for fermentation, incubate at 37°C for 48 hours at 200r / min, and centrifuge at 8000r / min for 10 minutes to obtain the wet cells of the three strains. Mix the three strains in a wet cell mass ratio of 1.3:1:1 and mix them evenly with the bacterial agent fixative (straw powder, peat powder and diatomaceous earth in a mass ratio of 50:30:20) in proportion. The amount of the three bacterial wet cells added accounts for 10% of the total mass of the mixture. After natural drying at 45°C, the new artificial multi-bacteria system dry preparation is obtained and stored at 4°C.

[0053] (2) The three single bacteria strains and the artificial multi-bacteria system were inoculated into simulated perishable waste culture medium, with the mass ratio of inoculum mass to perishable waste culture medium being 2:100. The culture was held at 45°C for 72 h. The degradation rates of starch and protein were measured after 24 h of culture, and the degradation rates of oil and cellulose were measured after 72 h of culture. The results are shown in Table 2. The perishable waste simulated culture medium consisted of: starch 5 g, casein 5 g, soybean oil 6.5 g, carboxymethyl cellulose 1.0 g, water 80 g, K2HPO4 0.2 g, KCl 0.1 g, MgSO4 0.05 g, and FeSO4·7H2O 0.01 g.

[0054] Table 2 Degradation of organic matter by single bacterial strain and artificial multi-bacteria system

[0055] .

[0056] Among them, the starch and protein degradation rates in the single bacterial degradation were highest in Bacillus amyloliquefaciens ZJB18046, the cellulose degradation rate was highest in Bacillus licheniformis ZJB19163, and the oil degradation rate was highest in Bacillus tekila ZJB19167. Compared with Example 2, increasing the proportion of wet bacteria in the bacterial agent from 5% to 10% significantly improved the degradation rates of starch and protein, but had a smaller improvement on the degradation of oil and cellulose.

[0057] Example 4

[0058] Preparation of an artificial multi-bacteria system for efficient degradation of perishable waste

[0059] (1) Streak the frozen glycerol tubes of the three strains on a plate using activated LB medium and incubate at 37°C for 12 hours. Pick the well-grown colonies into a 2L shake flask containing 700mL LB for fermentation, incubate at 37°C for 48 hours at 200r / min, and centrifuge at 8000r / min for 10 minutes to obtain the wet cells of the three strains. Mix the three strains in a wet cell mass ratio of 1.5:1:1 and mix them evenly with the bacterial agent fixative (straw powder, peat powder and diatomaceous earth in a mass ratio of 50:30:20) in proportion. The amount of the three bacterial wet cells added accounts for 15% of the total mass of the mixture. After natural drying at 50°C, the new artificial multi-bacteria system dry preparation is obtained and stored at 4°C.

[0060] (2) The three single-strain bacteria and the artificial multi-strain system were inoculated into simulated perishable waste culture medium, respectively. The mass ratio of inoculum mass to perishable waste culture medium was 2:100. The culture was held at 45°C for 72 h. The degradation rates of starch and protein were measured after 24 h of culture, and the degradation rates of oil and cellulose were measured after 72 h of culture. The results are shown in Table 3. The perishable waste simulated culture medium consisted of: starch 5 g, casein 5 g, soybean oil 6.5 g, carboxymethyl cellulose 1.0 g, water 80 g, K2HPO4 0.2 g, KCl 0.1 g, MgSO4 0.05 g, and FeSO4·7H2O 0.01 g.

[0061] Table 3 Degradation of organic matter by single bacterial strain and artificial multi-bacteria system

[0062] .

[0063] Among them, the starch and protein degradation rates of single bacteria were the highest in Bacillus amyloliquefaciens ZJB18046, the cellulose degradation rate was the highest in Bacillus licheniformis ZJB19163, and the oil degradation rate was the highest in Bacillus tekila ZJB19167. Compared with Example 3, the increase in the proportion of wet bacteria in the bacterial agent from 10% to 15% had little effect on the degradation of major organic components such as starch, protein, oil and cellulose.

[0064] Example 5

[0065] Selection of inoculation amount for artificial multi-bacteria system

[0066] The artificial multi-bacteria system obtained in Example 2 was put into the perishable garbage simulation culture medium at different inoculation ratios for effect testing. The mass ratios of inoculation mass to perishable garbage culture medium were 0.1:100, 0.5:100, 1:100, 2:100, 5:100, and 10:100, respectively. The results are shown in Table 4.

[0067] Table 4 Effect of input amount of artificial multi-bacteria system on organic matter degradation

[0068] .

[0069] When the mass ratio of the inoculation amount of the artificial multi-bacteria system to the perishable garbage culture medium is 2:100~5:100, a good degradation effect can be obtained with a relatively small investment in the artificial multi-bacteria system. However, increasing the inoculation amount will only have a limited effect on the improvement of the degradation rate of major organic matter.

[0070] Example 6

[0071] Effect of artificial multi-bacteria system on reducing perishable waste

[0072] Perishable waste was collected from the Jinghong Canteen at Zhejiang University of Technology. Water was manually drained by gravity, and bones and plastics were removed. Sawdust was used as a filler to adjust the moisture content and C / N ratio of the substrate. All experiments were conducted in a homemade bioreactor. 1000g of perishable waste (55% moisture content), 500g of sawdust, and 50g of an artificial multi-bacteria system (using the artificial multi-bacteria system described in Example 2) were added to the bioreactor. The temperature was set at 45°C and the reaction was allowed to proceed for 24 hours. Two control groups were also set up, each containing two commercially available perishable waste-degrading microbial agents (Commercial Agent 1 and Commercial Agent 2). The results of the reduction of perishable waste with the different agents are shown in Table 5.

[0073] Table 5 Effect of different preparations on reducing the amount of perishable waste

[0074] .

[0075] Table 5 shows that the artificial multi-bacteria system performed better than the two commercially available agents. To increase the load on the perishable waste degradation equipment, gravity water was removed from the perishable waste, resulting in a lower overall weight loss. However, the artificial multi-bacteria system demonstrated a good dry weight reduction. The low viable bacterial count in the remaining samples may be due to the short degradation time.

[0076] Example 7

[0077] Effect of artificial multi-bacteria system on reducing perishable waste

[0078] Perishable waste was collected from the Jinghong Canteen at Zhejiang University of Technology. Water was manually drained by gravity, and bones and plastics were removed. Sawdust was used as a filler to adjust the moisture content and C / N ratio of the substrate. All experiments were conducted in a homemade bioreactor. 1000g of perishable waste (50% moisture content), 500g of sawdust, and 50g of an artificial multi-bacteria system (using the artificial multi-bacteria system described in Example 2) were added to the bioreactor. The temperature was set at 43°C and the reaction was allowed to proceed for 24 hours. Two control groups were also set up, each receiving two commercially available perishable waste-degrading microbial agents (Commercial Agent 1 and Commercial Agent 2). The results of the reduction of perishable waste with the different agents are shown in Table 6.

[0079] Table 6 Effect of different preparations on reducing the amount of perishable waste

[0080] .

[0081] The results showed that the total weight reduction rate and dry weight reduction rate of the artificial multi-bacteria system for perishable waste were better than those of two commercially available microbial agents. The number of viable bacteria remaining in the sample after 24 hours of reaction reached 8.7×10 6 cfu / g, which is much higher than the number of viable bacteria in the remaining samples of two commercially available microbial agents.

[0082] Example 8

[0083] Effect of artificial multi-bacteria system on reducing perishable waste

[0084] Perishable waste was collected from the Jinghong Canteen at Zhejiang University of Technology. Water was manually drained by gravity, and bones and plastics were removed. Sawdust was used as a filler to adjust the moisture content and C / N ratio of the substrate. All experiments were conducted in a homemade bioreactor. 1000g of perishable waste (60% moisture content), 500g of sawdust, and 50g of an artificial multi-bacteria system (using the artificial multi-bacteria system described in Example 2) were added to the bioreactor. The temperature was set at 47°C and the reaction was allowed to proceed for 24 hours. Two control groups were also set up, each receiving two commercially available perishable waste-degrading microbial agents (Commercial Agent 1 and Commercial Agent 2). The results of the reduction of perishable waste with the different agents are shown in Table 7.

[0085] Table 7 Effect of different preparations on reducing the amount of perishable waste

[0086] .

[0087] The results showed that the total weight loss rate and dry weight loss rate of the artificial multi-bacteria system for perishable waste were better than those of the two commercially available microbial agents. Compared with Examples 6 and 7, the dry weight loss rate after 24 h of reaction was lower, which may be due to the lower number of viable bacteria in the remaining samples.

[0088] Example 9

[0089] Stability of artificial multi-bacteria system in rapid degradation of perishable waste

[0090] Perishable waste was collected from the Jinghong Canteen at Zhejiang University of Technology. Water was manually drained by gravity, and bones and plastics were removed. One kilogram of perishable waste, 500 grams of sawdust, and 50 grams of an artificial multi-bacteria system (using the artificial multi-bacteria system described in Example 2) were added to a bioreactor. The temperature was set at 45°C and the reaction was allowed to proceed for 7 days. During this period, 1 kilogram of fresh perishable waste was added to the bioreactor every 24 hours. After the 7-day period, no more perishable waste was added, and the remaining product was degraded to remove excess water. Samples were collected after 1, 4, and 7 days. Bacterial genomic DNA was extracted from the samples using the Mag-Bind Soil DNA Kit (Omega Biotek Inc., USA), and DNA purity and concentration were determined using a NanoDrop ND-1000 spectrophotometer.

[0091] The bacterial 16S rRNA gene V4 region was amplified using forward primer 515F (5'- GTGCCAGCMGCCGCGGTAA -3') and reverse primer 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The 50-μl PCR reaction system consisted of 25 μl of Phusion High-Fidelity PCR Master Mix with HF Buffer, 3 μl (10 uM) of each forward and backward F / R primer, 10 μl of DNA template, and 6 μl of ddH2O. PCR amplification was performed using the following reaction conditions: initial denaturation at 98°C for 30 seconds, followed by 25 cycles of denaturation at 98°C for 15 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 15 seconds. A final extension at 72°C for 1 minute was performed. PCR products were purified using AMPure XP Beads and quantified using the PicoGreen dsDNA Assay Kit. After quantification, sequencing was performed using the Illlumina HiSeq 4000 platform. Based on the sequencing results, the bacterial community structure of the artificial multi-bacteria system in the rapid degradation of perishable waste was obtained as shown in Table 8.

[0092] Table 8 Bacterial community structure of artificial multi-bacteria system in rapid degradation of perishable waste

[0093] .

[0094] Based on the sequencing results, Table 8 lists the bacterial community structure succession during the rapid degradation of perishable waste using the artificial multi-bacteria system. Using this artificial multi-bacteria system, Bacillus became the dominant genus from day one, and from day four onward, its abundance exceeded 91%. This indicates that the artificial multi-bacteria system significantly improved the stability of the in situ waste reduction process and that the three degradation strains synergistically enhanced the degradation rate of the major components of perishable waste. These results demonstrate that this artificial multi-bacteria system is more stable and cost-effective for in situ reduction of perishable waste.

[0095] Example 10

[0096] Application of artificial multi-bacteria system in rapid degradation of perishable waste

[0097] Perishable waste was collected from the Jinghong Canteen at Zhejiang University of Technology. Water was manually drained by gravity, and bones and plastics were removed. One kilogram of perishable waste, 500 grams of sawdust, and 100 grams of an artificial multi-bacteria system (using the artificial multi-bacteria system described in Example 2) were added to a bioreactor. The temperature was set at 45°C and the reaction was allowed to proceed for seven days. During this period, 1 kilogram of fresh perishable waste was added to the bioreactor every 24 hours. After the seven-day period, no more perishable waste was added, and degradation of the remaining product continued to remove excess moisture. Two control groups were also set up, each containing two commercially available perishable waste-degrading microbial agents. The results of the reduction of perishable waste with the different agents are shown in Table 9.

[0098] Table 9 Effects of different microbial agents on the rapid degradation of perishable waste

[0099] .

[0100] As can be seen from Table 9, the degradation effect of the artificial multi-bacteria system was better than that of the two commercially available agents. In terms of total weight loss rate and dry weight loss rate, the artificial multi-bacteria system was significantly better than the two commercially available agents, and the number of viable bacteria in the remaining samples was also higher.

Claims

1. An artificial multi-bacteria agent for degrading perishable garbage, characterized in that: The active ingredients are composed of Bacillus amyloliquefaciens ZJB18046, Bacillus licheniformis ZJB19163 and Bacillus tequilensis ZJB19167, wherein the preservation number of Bacillus amyloliquefaciens ZJB18046 is CCTCC NO: M2019423, the preservation number of Bacillus licheniformis ZJB19163 is CCTCC NO: M2020014, and the preservation number of Bacillus tequilensis ZJB19167 is CCTCC NO: M 2020177; Calculated by wet cell weight, the mass ratio of the Bacillus amyloliquefaciens ZJB18046, the Bacillus licheniformis ZJB19163, and the Bacillus tequila ZJB19167 is 1~1.5:1:

1.

2. The artificial multi-bacteria agent for degrading perishable garbage according to claim 1, characterized in that: The artificial multi-bacteria inoculant further comprises straw powder, peat powder and diatomaceous earth for fixing the inoculant.

3. The artificial multi-bacteria agent for degrading perishable garbage according to claim 2, characterized in that: The addition amount of the three kinds of wet bacterial cells in the artificial multi-bacteria inoculant accounts for 5% to 15% of the total mass, and the artificial multi-bacteria inoculant is dried at 40° C. to 50° C. and then used.

4. Use of the artificial multi-bacteria agent according to any one of claims 1 to 3 in degrading perishable garbage.

5. A method for degrading perishable waste, characterized in that: The artificial multi-bacteria agent as claimed in any one of claims 1 to 3 is used to ferment and degrade perishable garbage.

6. A method for degrading perishable waste according to claim 5, characterized in that: The mass ratio of the added amount of the artificial multi-bacteria agent to the perishable garbage is 0.1~10:

100.

7. The method for degrading perishable waste according to claim 5, wherein: The water content of the perishable garbage is 50% to 60%.

8. The method for degrading perishable waste according to claim 5, wherein: The fermentation degradation is carried out in a fermentation bin, with temperature controlled, ventilation and intermittent stirring for 24 hours to complete one degradation; after one degradation is completed, the same amount of perishable garbage as the first degradation is continuously added for another degradation, and the process is repeated in batches until the fermentation bin is full and the fermentation degradation is completed.

9. A method for degrading perishable waste according to claim 8, characterized in that: The fermentation temperature was controlled at 45±2℃.

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