CN116218729A - Artificial multi-bacterial system for degrading perishable garbage, application and degradation method - Google Patents

Abstract

The invention relates to the technical field of perishable garbage treatment, in particular to an artificial multi-fungus system for degrading perishable garbage, application and a degradation method, wherein the artificial multi-fungus system comprises bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZJB18046, bacillus licheniformis (Bacillus licheniformis) ZJB19163 and bacillus tertiaryalis (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 tertiaryother ZJB19167 is CCTCC NO: M2020177. The three strains in the invention are obtained through manual screening, each strain has a synergistic effect in the organic matter degradation process, has high degradation activity and degradation efficiency for main components of the perishable garbage, is suitable for degradation requirements of the main components of the perishable garbage such as starch, protein, grease and the like, effectively reduces the dosage of the microbial inoculum through a continuous feed supplement strategy, and has more cost effectiveness.

Description

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

Technical Field

The invention relates to the technical field of perishable garbage treatment, in particular to an artificial multi-bacterial system for degrading perishable garbage, application and a degradation method.

Background

The perishable garbage can also be called wet garbage, generally refers to kitchen waste generated in the production process of catering operators, unit canteens and the like, and perishable garbage generated in family life, and mainly comprises: leftovers, stems and leaves, viscera, shell and melon rinds, etc. The perishable garbage contains starch, protein, cellulose, lipid, inorganic salt and the like, is very perishable and odorous, transmits bacteria and viruses, and is accumulated in a large amount to influence the appearance of the city and pollute the environment and endanger the health of people. With the improvement of the world population growth living standard, the quantity of produced perishable garbage is larger and larger, the garbage is perishable and deteriorated in natural environment, and becomes a pollution source for preventing normal production and living, and environmental pollution is caused, wherein the food garbage accounts for more than 60 percent. The world's agricultural grain organization indicates that 16 million tons of perishable waste are produced annually worldwide, accounting for one third of the global grain yield. If improperly treated, food waste can quickly decay and become stink, creating leachate and breeding various bacteria, thereby causing serious environmental problems and potential health hazards. Therefore, some effective strategies need to be taken to handle the rapidly growing perishable garbage.

Because the perishable garbage is rich in carbohydrate, protein, fat and other organic matters, the perishable garbage has the potential of resource recovery. Traditional centralized treatment methods such as composting and anaerobic digestion can effectively recover and reuse the perishable waste, but due to the excessive treatment time, it is difficult to effectively treat the rapidly growing perishable waste. For example, there are research teams evaluating how perishable waste is treated using composting systems in densely populated areas, and the cost of labor, waste transportation and sorting per ton of food waste is found to be $23.02, accounting for 83.95% of the total expenditure. Therefore, a high-efficiency and controllable technology for reducing and harmless treatment of the perishable garbage is needed to be established.

The perishable garbage is used as organic garbage with strong biodegradability, the volume of the garbage can be reduced from the source by utilizing the microbial degradation technology to treat the perishable garbage in situ, the collection and transportation cost is greatly reduced, the secondary pollution is reduced, and the treated residues can be prepared into organic fertilizer. For example: the patent CN109182179A is based on a bacillus subtilis to prepare a microbial inoculum, 1 kg of the microbial inoculum is added into a perishable garbage degradation machine, 0.4 kg of the residual meal is added for degradation treatment, the degradation rate is calculated by actually reducing the weight, and the weight reduction rate of the perishable garbage within 48 hours reaches 93.2 percent. In general, in the aspect of substrate utilization, a single-fungus system is adopted, the types of usable substrates are single, and the single-fungus strain is difficult to realize the omnibearing degradation treatment of perishable garbage, and when aiming at complex renewable biomass, the single-fungus system has low substrate utilization efficiency and poor stability; the degradation effect is more focused on the reduction of the total weight, and the treatment period is longer. Considering that the kitchen waste contains water content of 70-90%, the degradation condition of organic matters in the kitchen waste cannot be intuitively illustrated by the single weight reduction rate.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide an artificial multi-fungus system for degrading perishable garbage, application and a degradation method thereof, wherein the artificial multi-fungus system is suitable for the degradation requirement of main components of the perishable garbage, has higher degradation efficiency for different perishable garbage components, has better stability in an in-situ reduction process, effectively reduces the dosage of a microbial inoculum through a continuous feeding strategy, improves the reduction degree of the perishable garbage, and has shorter fermentation treatment period.

In order to achieve the aim of the invention, the invention is realized by the following technical scheme:

the first object of the invention is to provide an artificial multi-bacterial system for degrading perishable garbage, which comprises bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZJB18046, bacillus licheniformis (Bacillus licheniformis) ZJB19163 and bacillus tervelarius (Bacillus tequilensis) ZJB19167, wherein the preservation number of the bacillus amyloliquefaciens ZJB18046 is CCTCCNO: M2019423, the preservation number of the bacillus licheniformis ZJB19163 is CCTCCNO: M2020014, and the preservation number of the bacillus tervelarius ZJB19167 is CCTCCNO: M2020177.

According to the composition characteristics of the perishable garbage in China, the strain with high degradation activity for the main components of the perishable garbage is obtained through screening, and the strain comprises bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZJB18046, and is preserved in China center for type culture collection, which is positioned in the university of Wuhan in Hubei province, wherein the preservation date is 2019, 6, 4 and the preservation number is CCTCC NO: M2019423; bacillus licheniformis ZJB19163 is preserved in China center for type culture Collection, with preservation date of 2020, 1 month and 6 days, and preservation number of CCTCC NO: M2020014; bacillus tertequilensis (Bacillus tequilensis) ZJB19167 is preserved in China center for type culture Collection, with a preservation date of 2020, 6 months and 3 days, and a preservation number of CCTCC NO: M2020177.

Preferably, the mass ratio of the bacillus amyloliquefaciens ZJB18046 to the bacillus licheniformis ZJB19163 to the bacillus tertiaryalis ZJB19167 is 1-1.5:1:1 based on the weight of wet bacteria.

Preferably, the artificial multi-bacterial system further comprises straw powder, grass carbon powder and diatomite for fixing bacterial agents.

When the artificial multi-fungus system is prepared, the three bacteria are uniformly mixed with the straw powder, the grass carbon powder and the diatomite according to the proportion, wherein the weight ratio of the straw powder to the grass carbon powder to the diatomite is 50:30:20.

Preferably, the addition amount of three bacterial wet thalli in the artificial multi-fungus system accounts for 5% -15% of the total mass, and the artificial multi-fungus system is dried at 40 ℃ -50 ℃ for later use.

Activating bacillus amyloliquefaciens ZJB18046, bacillus licheniformis ZJB19163 and bacillus tervalicarpus ZJB19167 strains, collecting thalli by high-density fermentation, mixing the three wet thalli according to a proportion, adding straw powder, grass carbon powder and diatomite, uniformly mixing, and drying at 40-50 ℃ to prepare an artificial multi-fungus system dry preparation for later use. Wherein the addition amount of the three bacterial wet thalli accounts for 5% -15% of the total mass.

A second object of the present invention is to provide the use of said artificial multi-bacterial system for degrading perishable waste.

The third object of the invention is to provide a method for degrading perishable garbage, which uses the artificial multi-bacterial system to ferment and degrade the perishable garbage.

Preferably, the mass ratio of the addition amount of the artificial multi-fungus system to the perishable garbage is 0.1-10:100.

Preferably, the moisture content of the perishable garbage is 50% -60%.

Before fermentation degradation treatment, the perishable garbage is subjected to preliminary filtration and water removal, so that the water content is 50% -60%, and the treatment capacity and the treatment effect of the perishable garbage can be improved.

Preferably, the fermentation degradation is carried out in a fermentation bin, the temperature is controlled, ventilation is carried out, and the mixture is intermittently stirred for 24 hours, so that primary degradation is completed; after the primary degradation is finished, the same amount of perishable garbage as the primary degradation is continuously put into the fermentation tank for secondary degradation, and the process is repeated in batches until the fermentation tank is full.

Preferably, the fermentation temperature is controlled to 45.+ -. 2 ℃.

Picking out non-degradable solid wastes such as bones, plastics and the like in the perishable wastes; then, preliminarily filtering and dewatering the perishable garbage to ensure that the water content is 50% -60%; adding an artificial multi-bacterial system, uniformly mixing, adding a proper amount of sawdust, adjusting the C/N ratio to 30-40, controlling the temperature to 45+/-2 ℃ and ventilating, and intermittently stirring for 24 hours to finish primary degradation; continuously adding the same amount of the perishable garbage as the first time after 24 hours, controlling the same reaction conditions, and circularly adding the perishable garbage until the fermentation bin is close to overflowed (about 30 days), and taking out the degraded perishable garbage from the fermentation bin; the taken out degraded perishable garbage can be used as an organic fertilizer.

The batch continuous feed-supplementing fermentation is matched with the coupling of the growth of the degradation strain and the enzyme production process, so that the continuous degradation process of single-batch artificial multi-bacterial system feeding (one-time feeding is operated for one month) can be effectively realized, and the feeding cost of strains is reduced.

Preferably, the intermittent stirring is carried out for 15-30 min and the intermittent stirring is carried out for 5-10 min.

The invention has the following beneficial effects:

(1) Three strains of bacillus amyloliquefaciens ZJB18046, bacillus licheniformis ZJB19163 and bacillus tervalicarpus ZJB19167 in the artificial multi-strain system are obtained through artificial screening, have high degradation activity on main components of perishable garbage, and are suitable for the degradation requirements of the main components of the perishable garbage such as starch, protein, grease and the like;

(2) Each strain in the artificial multi-strain system has a synergistic effect in the degradation process of organic matters, so that the degradation effect and the stability of the in-situ reduction process are obviously improved, and the artificial multi-strain system has higher degradation efficiency for different perishable garbage components;

(3) On the premise of not modifying treatment equipment, the method effectively reduces the input amount of the microbial inoculum through a continuous feeding strategy, improves the reduction degree of the perishable garbage, is more stable and cost-effective in reducing the perishable garbage in situ, and can be used as an organic fertilizer after degradation.

Drawings

FIG. 1 shows the degradation rate of starch, protein, cellulose and oil by the strain in the liquid medium in example 1.

Detailed Description

The invention is further described below with reference to the drawings and specific examples. Those of ordinary skill in the art will be able to implement the invention based on these descriptions. In addition, the embodiments of the present invention referred to in the following description are typically only some, but not all, embodiments of the present invention. Therefore, all other embodiments, which can be made by one of ordinary skill in the art without undue burden, are intended to be within the scope of the present invention, based on the embodiments of the present invention.

Example 1

Screening and identification of strain degrading main component of perishable garbage

The organic matter in the perishable garbage is mainly composed of protein, starch, grease and cellulose. 2g of fresh sample from a soil sample from a commercial university of Zhejiang near Xiu restaurant was suspended in sterile water and shaken at 30℃for 30 minutes at 150 rpm. Diluting the suspension with sterile water at a concentration gradient of 10 ^-4 、10 ^-5 、10 ^-6 、10 ^-7 、10 ^-8 、10 ^-9 200 mu L of bacterial suspension with each concentration is sucked and inoculated on an agar plate, and the agar plate is placed in a constant temperature incubator at 37 ℃ for culturing for 24 hours. Representative colonies were picked and streaked out for isolation until distinct single colonies were isolated. Agar plates used were Luria-Bertani (LB) solid medium (peptone 1%, yeast powder 0.5%, naCl 1% and agar 2%) and Potato Dextrose Agar (PDA) medium (potato 2%, dextrose 2% and agar 2%).

Since grease is difficult to degrade, separate screening of grease-degrading strains is required. Soil in the greasy dirt region was collected near the exhaust outlet of the Zhejiang university industrial council. After enrichment of the lipid-degrading strains with mineral salts medium containing 2.0% vegetable oil, the separation was also performed on agar medium using a gradient dilution method.

Extracting genome DNA of the strain by using a FastDNATM Spin Kit for Soil kit, and carrying out PCR amplification by using the genome DNA as a template, wherein the general primers are as follows:

ITS1：5’-TCCGTAGGTGAACCTGCGG-3’，

ITS4：5’-TCCTCCGCTTATTGATATGC-3’，

bacterial universal primers:

27F：5'-AGAGTTTGATCCTGGCTCA-3'，

1492R：5'-AAGGAGGTGATCCAGCCGCA-3'。

the products were sequenced by PCR products from the Optimus Praeparata of the family Praeparata, the obtained DNA sequence was input into GenBank, and (3) comparing the Blast program with all sequences in a database, selecting a proper DNA sequence to establish a phylogenetic tree, and determining and screening the species of the obtained strain.

And co-screening to obtain 87 strains. The strain is subjected to primary screening by adopting a transparent ring method. All sievesThe selected strains were inoculated into agar medium containing starch, fat, protein and cellulose. Transparent circles around colonies were used as an indicator of degradation activity. Ten strains with the largest transparent circle diameter in each culture medium are selected for re-screening, and are respectively inoculated into a starch degradation culture medium, a grease degradation culture medium, a protein degradation culture medium and a cellulose degradation culture medium. After three days of culture, strains with the highest starch, grease, protein and cellulose degradation activity were selected to prepare new microbial agents. The degradation rates of starch, protein, cellulose and oil by the respective strains in the liquid medium are shown in FIG. 1. The starch degradation culture medium comprises the following components: 10.0g/L of peptone, 10.0g/L of soluble starch, 5.0g/L of beef extract, 5.0g/L of NaCl and pH 7.0-7.2. Wherein the protein degradation culture medium comprises the following components: 10g/L of skimmed milk powder, 10.0g/L of peptone, 5.0g/L of beef extract and MgSO ₄ ·7H ₂ O0.2 g/L, pH 7.0-7.2. Wherein the grease degradation culture medium comprises the following components: (NH) ₄ ) ₂ SO ₄ 2g/L，K ₂ HPO ₄ 1g/L，KCl 0.5g/L，MgSO ₄ ·7H ₂ O 0.5g/L，FeSO ₄ 0.01g/L, natural pH, 20. 20 g/L vegetable oil. Wherein the cellulose degradation culture medium comprises the following components: peptone 10.0g/L, beef extract 5.0g/L, K ₂ HPO ₄ 0.5g/L，MgSO ₄ 0.25g/L, and 1g/L of wheat straw. The conditions for sterilization of the media except protein selection media were 121℃for 20 minutes. Protein medium sterilization conditions: the skimmed milk powder is independently sterilized at 115deg.C for 20min, and the rest at 121deg.C for 20min.

And finally screening 87 strains to obtain a bacillus amyloliquefaciens strain, a bacillus licheniformis strain and a bacillus tequila strain, and naming and preserving the bacillus tequila strain and the bacillus tequila strain respectively. Bacillus amyloliquefaciens ZJB18046 with a preservation number of CCTCC M2019423 is preserved in China center for type culture collection (China center) at university of Wuhan in Hubei province, and the preservation time is as follows: 2019, 6 and 4; bacillus licheniformis ZJB19163 with the preservation number of CCTCC M2020014 is preserved in China center for type culture collection (China center for type culture collection) at university of Wuhan in Hubei province, china, with the preservation time: 2020, 1 month and 6 days; bacillus tertequila ZJB19167 with a preservation number of CCTCC NO: M2020177, and is preserved in China center for type culture collection (China university of Wuhan in Hubei province) for a preservation time of: and 2020, 6 months and 3 days.

Example 2

Preparation of artificial multi-bacterial system for efficiently degrading perishable garbage

(1) The three frozen strain glycerol tubes were streaked on each plate using plate-activated LB medium and incubated at 37℃for 12 hours. The well-grown colony is picked into a 2L shaking flask containing 700mL LB for fermentation medium, cultured for 48 hours at 37 ℃ at 200r/min, centrifuged at 8000r/min for 10min, and then the wet thalli of the three strains are obtained. Mixing the three strains according to the mass ratio of the wet thalli of 1:1:1, and uniformly mixing with a microbial inoculum fixing agent (the mass ratio of straw powder, grass carbon powder and diatomite of 50:30:20) according to the proportion, wherein the addition amount of the three bacterial wet thalli accounts for 5% of the total mass of the mixture, naturally drying at 40 ℃ to obtain the novel artificial multi-fungus system dry preparation, and storing at 4 ℃.

(2) Three single bacteria and artificial multi-bacteria systems are respectively inoculated into a simulated perishable garbage culture medium, the mass ratio of the inoculated mass to the perishable garbage culture medium is 2:100, the culture is carried out for 72 hours at 45 ℃, the degradation rate of starch and protein is measured after 24 hours of culture, the degradation rate of grease and cellulose is measured after 72 hours of culture, and the results are shown in table 1. Wherein, perishable garbage simulation medium: 5g of starch, 5g of casein, 6.5g of soybean oil, 1.0g of carboxymethyl cellulose, 80g of water and K ₂ HPO ₄ 0.2g，KCl 0.1g，MgSO ₄ 0.05g，FeSO ₄ ·7H ₂ O 0.01g。

TABLE 1 degradation of organic substances by single strains and artificial multi-bacterial systems

。

The highest starch and protein degradation rate in the single-strain degradation is bacillus amyloliquefaciens ZJB18046, the highest cellulose degradation rate is bacillus licheniformis ZJB19163, and the highest lipid degradation rate is bacillus tertagatose ZJB19167. Compared with single strain degradation, the degradation rate of starch, protein, grease and cellulose is greatly improved by the artificial multi-strain system, which shows that the synergistic degradation of three bacillus strains can effectively promote the degradation of main organic components in perishable garbage.

Example 3

Preparation of artificial multi-bacterial system for efficiently degrading perishable garbage

(1) The three frozen strain glycerol tubes were streaked on each plate using plate-activated LB medium and incubated at 37℃for 12 hours. The well-grown colony is picked into a 2L shaking flask containing 700mL LB for fermentation medium, cultured for 48 hours at 37 ℃ at 200r/min, centrifuged at 8000r/min for 10min, and then the wet thalli of the three strains are obtained. Mixing the three strains according to the mass ratio of the wet thalli of 1.3:1:1, uniformly mixing the three strains with a microbial inoculum fixing agent (the mass ratio of straw powder to grass carbon powder to diatomite of 50:30:20), wherein the addition amount of the three bacterial wet thalli accounts for 10% of the total mass of the mixture, naturally drying at 45 ℃ to obtain a novel artificial multi-fungus system dry preparation, and storing at 4 ℃.

(2) Three single bacteria and artificial multi-bacteria systems are respectively inoculated into a simulated perishable garbage culture medium, the mass ratio of the inoculated mass to the perishable garbage culture medium is 2:100, the culture is carried out for 72 hours at 45 ℃, wherein the degradation rate of starch and protein is measured after 24 hours of culture, the degradation rate of grease and cellulose is measured after 72 hours of culture, and the results are shown in table 2. Wherein, perishable garbage simulation medium: 5g of starch, 5g of casein, 6.5g of soybean oil, 1.0g of carboxymethyl cellulose, 80g of water and K ₂ HPO ₄ 0.2g，KCl 0.1g，MgSO ₄ 0.05g，FeSO ₄ ·7H ₂ O 0.01g。

TABLE 2 degradation of organic substances by single strains and artificial multi-bacterial systems

。

The highest starch and protein degradation rate in the single-strain degradation is bacillus amyloliquefaciens ZJB18046, the highest cellulose degradation rate is bacillus licheniformis ZJB19163, and the highest oil degradation rate is bacillus teryaensis ZJB19167, compared with the embodiment 2, the wet thallus proportion in the microbial inoculum is increased from 5% to 10%, so that the degradation rate of starch and protein is greatly improved, and the degradation rate of oil and cellulose is less.

Example 4

Preparation of artificial multi-bacterial system for efficiently degrading perishable garbage

(1) The three frozen strain glycerol tubes were streaked on each plate using plate-activated LB medium and incubated at 37℃for 12 hours. The well-grown colony is picked into a 2L shaking flask containing 700mL LB for fermentation medium, cultured for 48 hours at 37 ℃ at 200r/min, centrifuged at 8000r/min for 10min, and then the wet thalli of the three strains are obtained. Mixing the three strains according to the mass ratio of the wet thalli of 1.5:1:1, uniformly mixing the three strains with a microbial inoculum fixing agent (the mass ratio of straw powder to grass carbon powder to diatomite of 50:30:20), wherein the addition amount of the three bacterial wet thalli accounts for 15% of the total mass of the mixture, naturally drying at 50 ℃ to obtain a novel artificial multi-fungus system dry preparation, and storing at 4 ℃.

(2) Three single bacteria and artificial multi-bacteria systems are respectively inoculated into a simulated perishable garbage culture medium, the mass ratio of the inoculated mass to the perishable garbage culture medium is 2:100, the culture is carried out for 72 hours at 45 ℃, the degradation rate of starch and protein is measured after 24 hours of culture, the degradation rate of grease and cellulose is measured after 72 hours of culture, and the results are shown in table 3. Wherein, perishable garbage simulation medium: 5g of starch, 5g of casein, 6.5g of soybean oil, 1.0g of carboxymethyl cellulose, 80g of water and K ₂ HPO ₄ 0.2g，KCl 0.1g，MgSO ₄ 0.05g，FeSO ₄ ·7H ₂ O 0.01g。

TABLE 3 degradation of organic substances by single strains and artificial multi-bacterial systems

。

The highest starch and protein degradation rate in the single-strain degradation is bacillus amyloliquefaciens ZJB18046, the highest cellulose degradation rate is bacillus licheniformis ZJB19163, and the highest oil degradation rate is bacillus teryaensis ZJB19167, compared with the embodiment 3, the wet thallus proportion in the microbial inoculum is increased from 10% to 15%, and the degradation promotion of main organic components such as starch, protein, oil, cellulose and the like is smaller.

Example 5

Selection of inoculum size for artificial multi-bacterial systems

The artificial multi-bacterial system obtained in example 2 was put into a perishable garbage simulation medium in different inoculation ratios for effect detection, the mass ratio of inoculation mass to the perishable garbage medium was 0.1:100, 0.5:100, 1:100, 2:100, 5:100, 10:100, respectively, and the results are shown in table 4.

TABLE 4 influence of the input of the Artificial Multibacteria System on the degradation of organic substances

。

When the mass ratio of the inoculation amount of the artificial multi-fungus system to the perishable garbage culture medium is 2:100-5:100, good degradation effect can be obtained under the condition of less input of the artificial multi-fungus system, the inoculation amount is increased, and the degradation rate of main organic matters is limited.

Example 6

Reduction of perishable garbage by artificial multi-bacterial system

Perishable waste was collected from the fine macro canteen at the university of Zhejiang industry, manually drained gravity water and removed bones and plastic products. Wood chips are used as fillers to adjust the moisture content and C/N ratio of the substrate. All experiments were performed in a home-made bioreactor. 1000g of perishable waste (moisture content 55%), 500g of wood chips and 50g of artificial multi-bacterial system (artificial multi-bacterial system in example 2 was used) were added to the bioreactor. Setting the temperature to 45 ℃ and reacting for 24 hours. Two control groups were simultaneously set, and two commercial perishable garbage degrading microbial agents (commercial microbial agent 1 and commercial microbial agent 2) were added respectively, and the reduction results of the different agents on the perishable garbage are shown in table 5.

TABLE 5 reduction of perishable waste by different formulations

。

As can be seen from Table 5, the degradation effect of the artificial multi-bacterial system is better than that of two commercial bacterial agents. In order to increase the load of the perishable garbage degradation equipment, gravity water is removed from the perishable garbage, so the total weight reduction rate is low, but the artificial multi-bacterial system has good effect on dry weight reduction. The number of viable bacteria in the remaining sample is low, probably due to too short degradation time.

Example 7

Reduction of perishable garbage by artificial multi-bacterial system

Perishable waste was collected from the fine macro canteen at the university of Zhejiang industry, manually drained gravity water and removed bones and plastic products. Wood chips are used as fillers to adjust the moisture content and C/N ratio of the substrate. All experiments were performed in a home-made bioreactor. 1000g of perishable waste (water content 50%), 500g of wood chips and 50g of artificial multi-bacterial system (artificial multi-bacterial system in example 2 was used) were added to the bioreactor. Setting the temperature at 43 ℃ and reacting for 24 hours. Two control groups were simultaneously set, and two commercial perishable garbage degrading microbial agents (commercial microbial agent 1 and commercial microbial agent 2) were added respectively, and the reduction results of the different agents on the perishable garbage are shown in table 6.

TABLE 6 reduction of perishable garbage by different formulations

。

The result shows that the total weight reduction rate and the dry weight reduction rate of the artificial multi-bacterial system on the perishable garbage are better than those of two commercial bacterial agents. The number of viable bacteria in the residual sample after 24 hours of reaction reaches 8.7X10 ⁶ cfu/g, which is far higher than the number of viable bacteria in the residual samples of two commercial bacteria agents.

Example 8

Reduction of perishable garbage by artificial multi-bacterial system

Perishable waste was collected from the fine macro canteen at the university of Zhejiang industry, manually drained gravity water and removed bones and plastic products. Wood chips are used as fillers to adjust the moisture content and C/N ratio of the substrate. All experiments were performed in a home-made bioreactor. 1000g of perishable waste (water content 60%), 500g of wood chips and 50g of artificial multi-bacterial system (artificial multi-bacterial system in example 2 was used) were added to the bioreactor. The temperature was set at 47℃and the reaction was carried out for 24 hours. Two control groups were simultaneously set, and two commercial perishable garbage degrading microbial agents (commercial microbial agent 1 and commercial microbial agent 2) were added respectively, and the reduction results of the different agents on the perishable garbage are shown in table 7.

TABLE 7 reduction of perishable garbage by different formulations

。

The results show that the total weight reduction rate and the dry weight reduction rate of the artificial multi-bacterial system on the perishable garbage are better than those of two commercial bacterial agents, and compared with the examples 6 and 7, the dry weight reduction rate of the reaction for 24 hours is reduced, probably because the number of the residual living bacteria of the sample is lower.

Example 9

Stability of artificial multi-bacterial system in quick degradation of perishable garbage

Perishable waste was collected from the fine macro canteen at the university of Zhejiang industry, manually drained gravity water and removed bones and plastic products. 1 kg perishable garbage, 500g wood chips and 50g artificial multi-bacterial system (using the artificial multi-bacterial system of example 2) were added to the bioreactor. The reaction was carried out at 45℃for 7 days. During this period, 1 kg fresh perishable garbage was placed into the bioreactor every 24 hours. After 7 days, no perishable garbage is put into the container, and the residual product is degraded to remove the residual moisture. Samples were taken after day 1,4,7, bacterial genomic DNA was extracted from the samples using a Mag-Bind soil DNA kit (Omega Biotek inc., usa) and the purity and concentration of DNA was detected using a NanoDrop ND-1000 spectrophotometer.

The bacterial 16S rRNA gene V4 region was PCR amplified using forward primer 515F (5 '-GTGCCAGCMGCCGCGGTAA-3') and reverse primer 806R (5 '-GGACTACHVGGGTWTCTAAT-3'). 50ul of PCR reaction system: 25. mu.l of High-fidelity enzyme Phusion High-Fidelity PCR Master Mix with HF Buffer; 3. Mu.l (10 uM) of each of the front and rear F/R primers; 10. mu.l of DNA template; 6 μl dd H ₂ O. The prepared PCR system is subjected to PCR amplification according to the following reaction conditions: pre-denaturation at 98 ℃ for 30s, followed by 25 cycles: denaturation at 98℃for 15s, annealing at 58℃for 15s, extension at 72℃for 15s. Finally, the mixture is extended at 72 ℃ for 1min. The PCR product was purified using AMPure XP Beads and allowed to passQuantification was performed using PicoGreen dsDNA, assay Kit. After quantification, the bacterial community structure of the artificial multi-bacterial system in the quick degradation of perishable garbage is obtained according to the sequencing result by using an IlluminaHiSeq 4000 platform for sequencing, and is shown in Table 8.

TABLE 8 bacterial community structure of artificial multi-bacterial system in quick degradation of perishable garbage

。

Based on the sequencing results, bacterial community structure succession of the artificial multi-bacterial system in the rapid degradation of perishable waste is listed in table 8. By using the artificial multi-bacterial system, bacillus becomes dominant bacteria from the first day, and the abundance ratio of bacillus exceeds 91% from the 4 th day, which shows that the artificial multi-bacterial system greatly improves the stability of the in-situ decrement process, and the three degradation strains can cooperate with each other to improve the degradation rate of each main component in the perishable garbage. These results indicate that the present artificial multi-bacterial system is more stable and cost effective in reducing perishable waste in situ.

Example 10

Application of artificial multi-bacterial system in quick degradation of perishable garbage

Perishable waste was collected from the fine macro canteen at the university of Zhejiang industry, manually drained gravity water and removed bones and plastic products. 1 kg perishable garbage, 500g wood chips and 100 g artificial multi-bacterial system (using the artificial multi-bacterial system of example 2) were added to the bioreactor. The reaction was carried out at 45℃for 7 days. During this period, 1 kg fresh perishable garbage was placed into the bioreactor every 24 hours. After 7 days, no perishable garbage is put into the container, and the residual product is degraded to remove the residual moisture. Two control groups were simultaneously set, and two purchased commercial perishable garbage degrading microbial agents were added, respectively, and the reduction results of the different agents on the perishable garbage are shown in table 9.

TABLE 9 Effect of different microbial agents in quick degradation of perishable waste

。

As can be seen from Table 9, the degradation effect of the artificial multi-bacterial system is better than that of two commercial bacterial agents. The total weight reduction rate and the dry weight reduction rate are obviously superior to those of two commercial bacterial agents, and the number of viable bacteria in the residual sample is also higher.

Claims (10)

1. The artificial multi-bacterial system for degrading perishable garbage is characterized by comprising bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZJB18046, bacillus licheniformis (Bacillus licheniformis) ZJB19163 and bacillus tertiaryalis (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 tertiaryother ZJB19167 is CCTCC NO: M2020177.

2. The artificial multi-bacterial system for degrading perishable garbage according to claim 1, wherein the mass ratio of bacillus amyloliquefaciens ZJB18046 to bacillus licheniformis ZJB19163 to bacillus tervalicalis ZJB19167 is 1-1.5:1:1 based on the weight of wet bacteria.

3. An artificial multi-bacterial system for degrading perishable waste according to claim 1 or 2, further comprising straw powder, grass carbon powder and diatomaceous earth for bacterial agent fixation.

4. The artificial multi-fungus system for degrading perishable garbage according to claim 3, wherein the addition amount of three wet bacterial cells in the artificial multi-fungus system is 5% -15% of the total mass, and the artificial multi-fungus system is dried at 40 ℃ -50 ℃ for later use.

5. The use of an artificial multi-bacterial system according to any one of claims 1 to 4 for degrading perishable waste.

6. A method for degrading perishable garbage, characterized in that the method comprises fermenting and degrading the perishable garbage by using the artificial multi-bacterial system according to any one of claims 1-4.

7. The method for degrading perishable garbage according to claim 6, wherein the mass ratio of the addition amount of the artificial multi-bacterial system to the perishable garbage is 0.1-10:100.

8. The method for degrading perishable garbage of claim 6, wherein the moisture content of the perishable garbage is 50% -60%.

9. The method for degrading perishable garbage as claimed in claim 6, wherein the fermentation degradation is performed in a fermentation bin, the temperature is controlled, ventilation is performed, and the intermittent stirring is performed for 24 hours, so as to complete the primary degradation; after the primary degradation is finished, the same amount of perishable garbage as the primary degradation is continuously put into the fermentation tank for secondary degradation, and the process is repeated in batches until the fermentation tank is full.

10. A method of degrading perishable waste as claimed in claim 9, wherein the fermentation temperature is controlled to 45±2 ℃.

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