Bacterial strain for aerobic composting of kitchen waste and preparation method of grease degrading bacterial agent of bacterial strain

Abstract

The invention discloses a preparation method of a bacterial strain for aerobic composting of kitchen waste and a grease degrading bacterial agent thereof, which is characterized by comprising the following steps of: enriching and domesticating a microbiota by using a hydrolysis ring method by adopting an enrichment domestication and separation purification method, separating and screening the bacterial strain for degrading animal grease, the method specifically comprises the following steps: collection of microbial communities, enrichment domestication, separation and purification of strains, and primary screening and secondary screening of the strains; the liquid grease degrading bacterial agent is prepared by compounding bacterial strain pseudomonas alcaligenes liquid, brevibacillus parabrevis liquid, candida lipolytica liquid and bacillus amyloliquefaciens liquid according to the volume ratio of 1: 1: 1: 1. The solid grease degrading microbial inoculum is prepared by mixing the liquid microbial inoculum and the conditioner according to the volume-weight ratio of the dry weight of the liquid microbial inoculum to the conditioner of 1: 1. Compared with the prior art, the method has the advantages that animal fat is efficiently degraded, the composting period is short, stink emission is avoided, the composting treatment effect is improved, efficient resourceful treatment of kitchen waste is promoted, the influence on the environment is reduced, and the method has good application prospects, economic value and social benefits.

Description

Technical Field

[0001] This invention relates to the field of aerobic composting technology for kitchen waste, specifically a method for preparing aerobic composting strains for kitchen waste and their oil-degrading agents. Background Technology

[0002] Since the implementation of waste sorting, the proper handling and resource utilization of kitchen waste has attracted widespread attention. Among various treatment methods, aerobic composting technology has become a promising and ideal choice due to its significant advantages. However, kitchen waste has a complex composition and a high concentration of oil. Furthermore, some small-scale biochemical treatments often directly compost kitchen waste without prior oil removal. The high oil content often causes compost material to clump together, affecting the composting effect. Therefore, direct composting of kitchen waste is difficult to achieve more efficient resource utilization. Introducing highly efficient oil-degrading bacteria can hydrolyze oil into intermediate products such as glycerol and fatty acids by secreting lipases. These intermediate products are then further decomposed and oxidized into metabolites such as water and carbon dioxide, alleviating the inhibitory effect of high oil content on composting and improving the aerobic composting efficiency of kitchen waste. In addition, numerous studies have been reported on oil-degrading bacteria in recent years, mainly focusing on screening and isolating microbial strains with strong lipase production and high oil degradation rates from the environment, optimizing their enzyme production conditions, and exploring the degradation capabilities of composite strains. However, these studies mainly focus on screening for oil-degrading bacteria with strong ability to degrade vegetable oils such as soybean oil, olive oil, and peanut oil, while there is relatively little research on highly efficient degrading bacteria for lard, tallow, and other oils containing high levels of saturated fatty acid glycerides. Furthermore, most studies were conducted under conditions of low oil concentration, with very few studies on strains capable of degrading animal fats under high oil concentration conditions.

[0003] Patent CN201110365608.0 describes the preparation of dormant microbial dry powder by high-density cultivation of Trichoderma viride, Bacillus subtilis, Rhizopus nigricans, photosynthetic bacteria, Candida lipolyticis, Bacillus licheniformis, Trichobacterium nitrite, and Nitrosporium vesiculosum. This powder is then mixed in a specific ratio to obtain a composite microbial agent for degrading oil in kitchen waste, used to treat kitchen waste and degrade the oil components. Patent application CN201310403853.5 describes the fermentation of the oil-degrading bacterium Acinetobacter UC13 in a culture medium with peanut oil as the sole carbon source. The fermentation product is then centrifuged, extracted, dried, filtered, and distilled to obtain a biosurfactant, which can be used to degrade oil in kitchen waste.

[0004] In summary, while existing technologies for oil-degrading bacteria can degrade some oils, their degradation capacity remains limited. For high-oil food waste with an oil content exceeding 8%, their activity decreases, preventing timely and complete oil degradation. Oil encapsulation of the bacteria leads to anaerobic conditions, affecting composting efficiency. Furthermore, aerobic composting methods for food waste are ineffective in degrading animal fats, resulting in long composting cycles and foul odors. Therefore, it is necessary to screen for highly efficient animal fat-degrading strains, construct animal fat-degrading bacterial agents, study the oil degradation characteristics of these agents, and investigate the effects of adding these agents on aerobic composting of food waste to promote efficient resource recovery from food waste. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for preparing bacterial strains and oil-degrading agents for aerobic composting of kitchen waste. The method employs a process of enrichment, domestication, isolation, and purification of bacterial strains. Microbial communities capable of oil metabolism are enriched, domesticated, isolated, and purified using a hydrolysis circle method. The resulting initial screening strains are then further screened to select highly efficient degrading strains for the preparation of animal fat-degrading agents. These agents offer significant advantages for aerobic composting of kitchen waste, particularly in the efficient degradation of animal fats such as lard and tallow, greatly improving the efficiency and quality of aerobic composting of kitchen waste. This invention demonstrates promising application prospects and development value.

[0006] The specific technical solution to achieve the purpose of this invention is: a method for preparing aerobic composting strains for kitchen waste, characterized by using enrichment, domestication, and separation purification methods, enriching and domesticating the microbial community using hydrolysis circle method, and separating and screening strains for animal fat degradation. The specific preparation includes the following steps:

[0007] Step 1: Collection of microbial communities

[0008] Oily soil near swill buckets or oily wastewater from kitchen waste were used as samples for screening target bacterial strains.

[0009] Step 2: Enrichment, domestication, isolation, and purification of strains

[0010] 1) Shake 10 g of the collected sample with 100 mL of physiological saline, and then dilute the sample with the resulting enrichment and acclimatization culture solution.

[0011] The bacterial culture was mixed at a volume ratio of 10:90 and cultured with shaking at 30°C for 6 acclimatization cycles, each lasting 5 days. After each acclimatization cycle, 10 mL of the bacterial culture was transferred to 90 mL of fresh culture medium. This process was repeated for 6 cycles to obtain an enriched acclimatized bacterial culture. The culture medium was porcine / cattle fat, with an initial fat concentration of 2.5 g / L. The fat concentration of the fresh culture medium increased by 0.5 g / L each cycle. The shaking speed was 160 r / min.

[0012] 2) Mix 10 mL of the enriched and acclimatized bacterial solution with 90 mL of physiological saline to prepare a 10⁻¹ dilution, and then dilute it tenfold to 10⁻¹. -7 Take 100 μL of each of the following 10 μL samples: -4 ~10 -7 Gradient bacterial suspensions were evenly spread on a primary screening medium containing hog / cattle fat and cultured upside down at 30°C for 2-3 days. After culture, single colonies with obvious blue hydrolysis zones were selected and purified to a single strain by streaking multiple times. Colonies with good growth were transferred to nutrient agar (NA) slants, stored at 4°C and transferred periodically to obtain the isolated and purified strain.

[0013] Step 3: Initial screening of strains

[0014] The isolated and purified strains were initially screened using the hydrolysis zone method. They were inoculated into screening medium containing hog / cattle fat and cultured upside down at 30°C for 3 days. The ratio of the colony diameter to the diameter of the surrounding blue hydrolysis zone was used as the enzyme activity evaluation index. The isolated and purified strains were then screened to obtain the initial screening strains.

[0015] Step 4: Secondary screening of strains

[0016] The initially screened strains were transferred to a liquid culture medium of bacteria-NB / yeast-PDB and cultured with shaking at 30°C for 24 hours. Then, they were transferred to a secondary screening medium containing pig / bovine fat at a 10% inoculation rate and cultured with shaking at 30°C for 72 hours. The degradation rate of the strains on pig / bovine fats and the activity of lipase were used as evaluation indicators to re-screen the initially screened strains. The obtained secondary screening strains were the strains for preparing animal fat degradation agents. The shaking speed was 160 r / min.

[0017] A microbial agent for degrading animal fats, prepared using aerobic composting strains from food waste, is characterized by using aerobic composting strains from food waste to prepare liquid or solid animal fat degrading agents. The liquid animal fat degrading agent is prepared by inoculating a liquid culture medium with *Pseudomonas alba*, *Bacillus parabregas*, *Candida lipolyticis*, and *Bacillus amyloliquefaciens* strains, and then shaking and culturing at pH 7.0, 30℃, and 160 r / min for 24 h to obtain the liquid animal fat degrading agent. The *Pseudomonas alba*, *Bacillus parabregas*, *Candida lipolyticis*, and *Bacillus amyloliquefaciens* strains are mixed in a 1:1:1:1 volume ratio, with the volume percentage concentrations of the four strains being 25%, 25%, 25%, and 25%, respectively. The liquid culture medium is either bacteria-NB (Nutrient Broth) or yeast-PDB (Potato Bacteria). Dextrose Broth liquid culture medium; the contents of peptone, beef extract, and sodium chloride in the bacterial-NB culture medium are 5.0 g / L, 3.0 g / L, and 0.5 g / L, respectively; the contents of peptone, glucose, and yeast extract in the yeast-PDB liquid culture medium are 20.0 g / L, 20.0 g / L, and 10.0 g / L, respectively.

[0018] The solid animal fat degrading microbial agent is prepared by mixing the liquid animal fat degrading microbial agent with the dry weight of the conditioner at a volume-to-weight ratio of 1:1. The conditioner is composed of coconut coir and rice husk, with a dry weight ratio of coconut coir to rice husk of 5:3.

[0019] Compared with the prior art, the present invention has the following beneficial technical effects and significant technical progress:

[0020] 1) Adding animal fat degrading bacteria can promote the temperature rise of the compost pile, alleviate compost acidification, reduce odor during composting, and improve the maturity of the compost. The temperature in the middle and later stages of composting is above 48℃, and the pH is maintained at 6.0-6.5, higher than the control group. The seed germination index (GI) of the compost product is greater than 70%, meeting the requirement of NY / T 525-2021 for organic fertilizers with a GI ≥ 70%.

[0021] 2) Adding animal fat degrading microbial agents can promote fat degradation and reduce food waste volume. Compared with no added microbial agents and those with commercially available composting microbial agents, the fat degradation rate increased by 12.45% and 2.84%, respectively; the total dry matter reduction rate increased by 19.29% and 7.86%, respectively; the total organic matter reduction rate increased by 13.05% and 4.86%, respectively; the food waste dry matter reduction rate increased by 28.29% and 11.53%, respectively; and the total reduction rate increased by 12.26% and 3.89%, respectively.

[0022] 3) The addition of animal fat-degrading bacteria increased the relative abundance of Actinobacteria, and significantly increased the relative abundance of genes related to cellulose and hemicellulose degradation, including 1,4-β-cellobiosidase (exoglucanase), β-glucosidase, endo-1,4-β-xylanase, and 1,4-β-xylosidase. In secondary metabolic pathways, the relative abundance of functional genes related to carbohydrate and lipid metabolism increased, and in tertiary metabolic pathways, the relative abundance of functional genes related to multi-environmental microbial metabolism, cofactor synthesis, and carbon metabolism also increased. Prediction results of key enzymes for glycerol and fatty acid degradation showed that the addition of animal fat-degrading bacteria effectively increased the gene abundance of triglyceride lipase, monoglyceride lipase, alcohol dehydrogenase, acetaldehyde dehydrogenase, acyl-CoA dehydrogenase, long-chain acyl-CoA synthase, acetyl-CoA C-acetyltransferase, and acetyl-CoA acyltransferase.

[0023] 4) The group co-added with animal fat degrading bacteria and commercially available composting bacteria showed the best overall performance: pH remained stable above 6.0, odor during composting was lowest, and the GI of compost products was highest; the degradation rates of fat (53.46%), starch (82.32%), and protein (65.27%) were all the highest, with total dry matter, total organic matter, food waste dry matter reduction rates and total reduction rates reaching 52.34%, 55.35%, 76.77%, and 87.63%, respectively, effectively promoting food waste reduction; compared with the group that only added animal fat degrading bacteria, the number of microbial species was significantly increased, with the relative abundance of Pseudomonas (related to organic matter degradation) increasing from 0.5% to 32.23%, and the relative abundance of Bacillus (species) increasing from 0.17% to 10.52%.

[0024] 5) It effectively promotes the efficient resource utilization of kitchen waste and has good application prospects and commercial development value. Attached Figure Description

[0025] Figure 1 The degradation rates of lard and tallow for the initial screening strains;

[0026] Figure 2 The lipase activity of the primary screening strains in lard and butter secondary screening media was measured.

[0027] Figure 3 The degradation rate of lard for different strain combinations;

[0028] Figure 4 The degradation rate of butter for different strain combinations;

[0029] Figure 5 This shows the changes in CODCr concentration and CODCr removal rate over time in the treatment of kitchen wastewater by animal fat degrading bacteria in Example 1.

[0030] Figure 6 The following is an example of how the oil degradation rate and enzyme activity of the animal fat degrading bacteria in the treatment of kitchen wastewater changed over time.

[0031] Figure 7 This is a graph showing the pH change during the treatment of kitchen wastewater by the animal fat degrading bacteria agent in Example 1.

[0032] Figure 8 This shows the temperature changes in each group during the aerobic composting process in Example 2.

[0033] Figure 9 This shows the pH changes in each group during the aerobic composting process in Example 2.

[0034] Figure 10 This shows the changes in EC in each group during the aerobic composting process in Example 2;

[0035] Figure 11 This shows the changes in oil degradation rate in each group during the aerobic composting process in Example 2.

[0036] Figure 12 This shows the changes in starch degradation rate in each group during the aerobic composting process in Example 2.

[0037] Figure 13 This shows the changes in protein degradation rate in each group during the aerobic composting process in Example 2.

[0038] Figure 14 This shows the changes in the content of easily degradable organic matter in each group during the aerobic composting process in Example 2.

[0039] Figure 15 This shows the total reduction rate of each group after the aerobic composting process in Example 2;

[0040] Figure 16 This shows the changes in GI in each group during the aerobic composting process in Example 2;

[0041] Figure 17 This shows the changes in E4 / E6 in each group during the aerobic composting process in Example 2;

[0042] Figure 18 Example 2: Microbial community changes (domain level);

[0043] Figure 19 Example 2: Changes in the microbial community (phylum level);

[0044] Figure 20 Example 2: Microbial community changes (genus level);

[0045] Figure 21 This is a graph showing the abundance of genes related to glycerol ester degradation in the four groups of compost products in Example 2.

[0046] Figure 22 This is a graph showing the abundance of fatty acid degradation-related genes in the four compost products of Example 2. Specific implementation methods

[0047] This invention employs enrichment, domestication, and separation purification methods to prepare a bacterial strain for aerobic composting of kitchen waste, specifically including the following steps:

[0048] 1) Sample source

[0049] In oil-rich environments, numerous microbial communities possess the ability to metabolize oils, greatly increasing the likelihood of screening for highly efficient oil-degrading bacteria. Therefore, this study established two sample collection sites to screen for target strains with highly efficient oil-degrading capabilities. The two sampling sites were: oily soil next to the garbage bin at the restaurant entrance and oily wastewater from the kitchen.

[0050] 2) Enrichment, domestication, isolation, and purification of strains

[0051] a. Weigh 10 g of the collected sample and add it to 100 mL of physiological saline, shaking thoroughly. Take 10 mL of the sample dilution and add it to 90 mL of enrichment and acclimatization medium (initial lard / beef tallow concentration 2.5 g / L). Incubate at 30℃ and 160 r / min with shaking for 5 days as one acclimatization cycle. After each cycle, transfer 10 mL of bacterial culture to 90 mL of fresh medium, increasing the fat concentration by 0.5 g / L each cycle. Repeat this process for 6 cycles (30 days in total) to achieve the enrichment and acclimatization of the strain.

[0052] b. Mix 10 mL of the enriched and acclimatized bacterial culture with 90 mL of physiological saline to prepare a 10⁻¹ dilution, and then dilute it tenfold to 10⁻¹. -7 Take 100 μL of each of the 10... -4 -10 -7 Gradient bacterial suspensions were evenly spread on primary screening medium containing lard / butter and incubated upside down at 30°C for 2-3 days. After incubation, single colonies with a clear blue hydrolysis zone were selected and purified to a single strain through repeated streak plating. Colonies in good growth condition were then transferred to NA slant agar, stored at 4°C, and subcultured periodically.

[0053] 3) Preliminary screening of bacterial strains

[0054] Newly isolated and laboratory-preserved strains were initially screened using the hydrolysis zone method. They were then inoculated onto screening medium containing lard / butter and cultured upside down at 30°C for 3 days. The ratio of the colony diameter to the diameter of the surrounding blue hydrolysis zone was measured as an indicator of enzyme activity.

[0055] 4) Secondary screening of strains

[0056] The initially screened strains were transferred to the corresponding liquid culture medium (bacteria-NB / yeast-PDB) and cultured at 30℃ and 160 r / min for 24 h. Then, they were transferred to lard / butter rescreening medium at a 10% inoculum and cultured under the same conditions for 72 h. The degradation rate and lipase activity of the strains on the two oils were measured to screen for highly efficient degradation strains.

[0057] The culture medium and its components are shown in Table 1 below.

[0058] Table 1 Culture medium and components

[0059]

[0060] See Figure 1 After 72 h of culture, strain DY-7 showed the highest oil degradation rate, with 71.48% for lard and 69.67% for tallow. Strain XY-4 followed, with 67.62% for lard and 62.10% for tallow. Strains XY-3, Y96, Y98, and W1 also showed good oil degradation, with lard degradation rates of 60.89%, 62.11%, 60.20%, and 65.02% for lard and 61.68%, 60.29%, 58.65%, and 63.32% for tallow after 72 h of culture. All strains XY-3, XY-4, DY-7, Y96, Y98, and W1 showed lard degradation rates greater than 60% after 72 h of culture. Compared to the strains screened by Wang Sen et al. using the gradient acclimation method, which showed a lard degradation rate of less than 50% after 72 hours; the strain D-4 screened by Xiao Han et al. using the gradient acclimation method showed a lard degradation rate of 57.21% after 96 hours; and the Staphylococcus saprophyticus DX2-6 screened by Yan Hongmei et al. using the gradient acclimation method also showed a degradation rate of less than 60% for lard and tallow, the strains isolated and screened in this invention have advantages in degrading animal fats such as lard and tallow.

[0061] See Figure 2The results showed the lipase activities of each strain after 72 h of culture. Strain DY-7 exhibited the highest lipase activity, with lipase activities of 9.33 U / mL (in lard screening medium) and 8.97 U / mL (in butter screening medium). Strains XY-3, XY-4, Y96, Y98, and W1 followed, with lipase activities of 8.01 U / mL, 8.29 U / mL, 8.02 U / mL, and 8.48 U / mL in lard screening medium, and 8.24 U / mL, 8.01 U / mL, 7.89 U / mL, and 8.36 U / mL in butter screening medium, respectively. After 72 h of culture, the lipase activities of strains XY-3, XY-4, DY-7, Y96, Y98, and W1 were all greater than 7.50 U / mL, which was higher than that of strain SLB-1 (5.67 U / mL) screened by Xu Weifang et al. and two lipid-degrading bacteria screened by Qin Zhentian et al. (both of which did not exceed 6.00 U / mL). This proves that the strains screened in this study have a strong ability to produce lipase.

[0062] This invention employs initial screening using hydrolysis zones, followed by secondary screening based on lipase activity and oil degradation rate. Ultimately, seven strains with high degradation rates of lard and tallow, as well as high lipase activity, were selected: strains XY-2, XY-3, XY-4, Y96, Y98, DY-7, and W1. Among these, strains XY-2, XY-3, XY-4, and DY-7 are newly isolated lipase-producing strains, derived from oily soil and oily wastewater. Y96 and Y98 are laboratory-preserved *Candida* species, and W1 is a laboratory-preserved *Bacillus amyloliquefaciens* species.

[0063] The amplification products of the newly isolated strain were verified by agarose gel electrophoresis to meet the requirements for subsequent sequencing. The obtained gene sequences were assembled using BioEdit software and then uploaded to the NCBI website for BLAST alignment. The alignment results are shown in Table 2.

[0064] Table 2. Alignment results of 16S rRNA gene sequences of strains XY-2, XY-3, XY-4, and DY-7

[0065]

[0066] Reference strain sequences with high similarity to the newly isolated strains were selected from the NCBI alignment results. The target and reference sequences were imported into MEGA, and multiple alignments were performed, with redundant regions pruned to construct a phylogenetic tree. Based on the BLAST alignment results, XY-2 was preliminarily identified as *Klebsiella oxytoca*, XY-3 as *Klebsiella pneumoniae*, XY-4 as *Pseudomonas salcaligenes*, and DY-7 as *Brevibacillus parabrevis*.

[0067] 5) The degradation effects of different combinations of animal fat degrading microbial agents on lard and tallow.

[0068] The five selected strains, XY-4, DY-7, Y96, Y98, and W1, were named a, b, c, d, and e respectively, and 31 combinations were created using permutation and combination methods. The combinations are set as a-1, b-2, c-3, d-4, e-5, ab-6, ac-7, ad-8, ae-9, bc-10, bd-11, be-12, cd-13, ce-14, de-15, abc-16, abd-17, abe-18, acd-19, ace-20, ade-21, bcd-22, bce-23, bde-24, cde-25, abcd-26, abce-27, abde-28, acde-29, bcde-30, and abcde-31. Subsequently, the OD600 of each strain culture was adjusted to 1.0 with sterile water. After mixing equal volumes of each bacterial culture, 10% of the culture was inoculated into 50 mL of simulated oily wastewater containing 10 g / L lard and tallow, respectively, to verify the oil degradation ability of each combination after 72 h at 30℃ and 160 r / min.

[0069] See Figures 3-4 Group 27 exhibited the highest degradation rates in simulated lard and tallow wastewater, reaching 80.19% and 77.04%, respectively, significantly outperforming single strains and combinations of other strains. Therefore, the composite bacterial agent composed of *Bacillus parabrittlesis* DY-7, *Pseudomonas alkaligenes* XY-4, *Bacillus amyloliquefaciens* W1, and *Candida lipolyticis* Y96 showed the most ideal degradation effect on lard and tallow, and can be used to construct a highly efficient animal fat degradation bacterial agent for further research.

[0070] 6) Preparation of liquid animal fat degradation bacteria

[0071] Selected strains of *Pseudomonas alcaligenes*, *Bacillus parabrachii*, *Candida lipolyticis*, and *Bacillus amyloliquefaciens* were inoculated into their respective liquid culture media (bacteria-NB / yeast-PDB) and cultured with shaking at pH 7.0, 30℃, and 160 r / min for 24 h. The four bacterial solutions were then mixed in an equal volume ratio (1:1:1:1) to finally prepare a liquid animal fat degradation bacterial agent.

[0072] The contents of peptone, beef extract, and sodium chloride in the bacterial-NB (Nutrient Broth) liquid culture medium are 5.0 g / L, 3.0 g / L, and 0.5 g / L, respectively.

[0073] The contents of peptone, glucose, and yeast extract in the yeast-PDB (Potato Dextrose Broth) liquid culture medium are 20.0 g / L, 20.0 g / L, and 10.0 g / L, respectively.

[0074] 7) Preparation of solid animal fat degrading bacteria

[0075] The prepared liquid animal fat degradation bacteria agent is mixed with the conditioner in a 1:1 ratio of total liquid bacteria agent volume to dry weight to obtain a solid bacteria agent. The conditioner is composed of coconut coir and rice husk in a 5:3 dry weight ratio.

[0076] 8) Optimal conditions for liquid animal fat degradation bacteria

[0077] The animal fat degrading bacteria showed strong degradation ability for various types of oils, with higher degradation rates for vegetable oils than animal oils. The degradation rates for lard, soybean oil, and olive oil were all greater than 80% after 72 hours. Good oil removal effects were observed within the ranges of salt concentration (5-40 g / L), oil concentration (10-50 g / L), pH (5.5-10), rotation speed (80-200 r / min), and temperature (30-50℃). Both excessively high and low temperatures affected bacterial activity; 30℃ was the optimal degradation temperature, with degradation rates of 87.52% for soybean oil and 80.39% for lard. The optimal conditions for bacterial growth and degradation were a slightly alkaline pH of 7.0-8.5; strongly acidic or alkaline environments led to decreased enzyme activity. The highest degradation rate was observed at pH 8.5, with soybean oil reaching 89.11% and lard reaching 84.13%. The optimal rotation speed was 160 r / min, with degradation rates of 87.56% for soybean oil and 80.31% for lard. The highest degradation rate was observed at a salt concentration of 10 g / L, with soybean oil reaching 90.01% and lard reaching 83.61%. The degradation rate decreased significantly when the salt concentration exceeded 20 g / L.

[0078] The following detailed examples of treating actual kitchen wastewater and aerobic composting of kitchen waste with animal fat degrading bacteria further illustrate the technical solution and effects of the present invention.

[0079] Example 1

[0080] This embodiment describes the treatment of kitchen wastewater using animal fat-degrading bacteria. The wastewater treatment process is as follows:

[0081] The prepared liquid animal fat degrading bacterial agent was added to actual kitchen wastewater at an inoculum of 10% (denoted as SY). The control group was set with 10% inactivated animal fat degrading bacterial agent (denoted as CK). Degradation experiments were carried out in a shaking incubator (set at a speed of 160 r / min and a temperature of 30℃). The fat degradation rate, pH, lipase activity, and COD removal rate were measured every 12 h. Each group was set up in duplicate.

[0082] Kitchen wastewater is taken from the grease trap of the canteen and is filtered by sedimentation to remove impurities such as leftover vegetable scraps and food. The basic parameters of kitchen wastewater are shown in Table 3 below.

[0083] Table 3 Basic Indicators of Kitchen Wastewater

[0084]

[0085] See Figure 5Overall, the CODcr removal rates of both the liquid animal fat degrading microbial agent group (SY) and the control group (CK) showed a trend of first increasing and then leveling off with degradation time. During the period of 12 h to 24 h, the growth rate of CODcr removal rate was highest in both groups, reaching 39.28% and 34.59% at 24 h, respectively. This is because kitchen wastewater contains abundant nutrients such as oils, proteins, and starches, and its complex composition, with most of it existing in a colloidal or suspended state, allows microorganisms to preferentially degrade easily degradable substances, resulting in a rapid increase in CODcr removal rate in both groups during the initial degradation phase. Subsequently, during the period of 24–48 h, the growth rate of CODcr removal rate fluctuated in both groups, but the overall trend remained upward, with the CODcr removal rate of the liquid animal fat degrading microbial agent group consistently higher than that of the control group. This may be because the bacterial agent contains highly efficient grease-degrading bacteria that specifically degrade grease. After the easily degradable substances in the kitchen wastewater are decomposed, the grease-degrading bacteria begin to degrade more difficult-to-degrade substances such as grease, with a grease degradation rate superior to the control group, thus significantly reducing the CODcr content in the wastewater. After 60 hours, the CODcr removal rate of both groups slowly increased. After 72 hours of treatment, the CODcr removal rate of the group with added liquid animal fat-degrading bacterial agent was 57.46%, higher than the control group by 12.61%. At 120 hours, the CODcr removal rates of both groups reached their highest values, at which point the CODcr removal rates were 59.81% and 47.38%, respectively. The CODcr removal rate of the group with added highly efficient liquid grease-degrading bacteria is close to the CODcr removal rate (62.4%) of the actual kitchen wastewater treated with the animal fat-degrading bacterial agent developed by Ke et al. after 72 hours. The bacterial agent prepared in this invention also has a good CODcr removal rate. It can be seen that in the practical application of kitchen wastewater treatment, after inoculating highly efficient liquid oil-degrading bacteria degrades large molecules such as oils into smaller molecules, a small number of other strains can further utilize these small molecule degradation products, thereby improving the degradation efficiency.

[0086] See Figure 6As can be seen, the lipid degradation rate of the group with added liquid animal lipid-degrading bacteria was higher than that of the control group at all time points, indicating that it exhibited superior performance in lipid degradation. With the change in degradation time, the lipid degradation rate of both groups showed an increasing trend, but the rate of increase in the group with added liquid animal lipid-degrading bacteria was higher than that of the control group, especially between 24 h and 72 h, where the lipid degradation rate increased rapidly. Although it slowed down afterward, it still maintained a high degradation rate. The lipase activity of the group with added liquid animal lipid-degrading bacteria was higher than that of the control group at all time points, which is consistent with the results of the lipid degradation rate. Its lipase activity increased rapidly in the initial stage (0-24 h), reached a high level, and then remained relatively stable; while the lipase activity of the control group showed a relatively gradual upward trend, with an overall activity level lower than that of the group with added bacteria. After 72 h, although the lipase activity of the group with added liquid animal lipid-degrading bacteria decreased somewhat, it still remained at a relatively high level and was higher than that of the control group. This further indicates that the liquid animal lipid-degrading bacteria also has an advantage in maintaining lipase activity. At 72 h, the differences between the two groups in terms of lipid degradation rate and lipase activity were significant. This may be because the lipase activity in the group with added liquid animal fat-degrading bacteria reached its peak at this time. The fat-degrading bacteria secreted a large amount of lipase, which decomposed the fat into small molecules such as fatty acids and glycerol, thus making the fat-degrading bacteria significantly higher than the control group. At this time, the fat degradation rate of the group with added liquid animal fat-degrading bacteria was 97.36%, which was higher than the fat degradation rate (47.49%) of strain JY34 selected by Yipuhong et al. after 3 days of fermentation in oily wastewater from kitchen waste. After 72 hours, the fat degradation rate of both groups tended to level off. By 120 hours, the fat degradation rate of the group with added animal fat-degrading bacteria reached 98.68%, which was higher than the control group's 47.41%. Adding animal fat-degrading bacteria can effectively improve the fat degradation rate.

[0087] See Figure 7This chart shows the pH changes during the degradation of kitchen wastewater. From 0 h to 24 h, the pH values ​​of both the group with added liquid animal fat-degrading bacteria and the control group dropped sharply, from an initial pH of 7.0 to around 5.2. At this time, a large amount of easily degradable organic matter in the wastewater was degraded, leading to the pH decrease. Subsequently, by 84 h, the pH value continued to decrease to a minimum of 4.7. This pH decrease had two reasons: firstly, the fat-degrading bacteria functioned, degrading the fats in the kitchen wastewater into glycerol and fatty acids, and the accumulation of fatty acids caused the pH to drop. Secondly, the accumulation of lactic acid, the final product of carbohydrate fermentation, caused the pH to continue to decrease. This indicates that while the liquid animal fat-degrading bacteria promoted fat degradation, it may also have accelerated the production or release of certain acidic substances in the wastewater. The pH value subsequently gradually rose to 5.5, possibly due to the gradual degradation of small molecule acids. The pH of the control group gradually increased from 5.2 to 6.8 after 48 h. This may be because some strains in the wastewater produced alkaline substances during the degradation process, which led to a continuous increase in pH of the control group from 48 h to 120 h. This is consistent with the study by Zhao et al.

[0088] Example 2

[0089] This embodiment describes an experiment using a solid animal fat degrading microbial agent in the aerobic composting of kitchen waste. The process is as follows:

[0090] The prepared liquid animal fat degrading bacteria were mixed with the conditioner at a ratio of 1:1 (total volume of liquid bacteria to dry weight) to form the solid bacteria. The conditioner was composed of coconut coir and rice husks in a 5:3 dry weight ratio. Commercially available kitchen waste composting bacteria were produced by a certain company, and their main components were yeast, lactic acid bacteria, photosynthetic bacteria, fermenting filamentous bacteria, and actinomycetes. A simulated kitchen waste composting formula consisted of 600 g of vegetables, 108 g of rice, 60 g of fruit peels, 23.75 g of cooked pork, 8 g of cooking oil, and 0.25 g of salt. The purchased frozen pork was cooked thoroughly and then cut into small pieces approximately 0.5 cm in diameter. The rice was cooked in a rice cooker. The vegetables were sourced from a school cafeteria, mainly leafy greens, while the fruit peels included apple, banana, and citrus peels, which were processed into small pieces approximately 1 cm in diameter using an electric meat grinder. Four treatment groups were set up: Group 1 (700 g conditioner + 700 mL inactivated bacteria agent), Group 2 (700 g conditioner + 700 mL animal fat degradation bacteria agent), Group 3 (700 g conditioner + 700 mL commercially available composting bacteria agent), and Group 4 (700 g conditioner + 700 mL animal fat degradation bacteria agent and commercially available composting bacteria agent in a 1:1 mixture). All bacteria agents were prepared as solid agents and added. After the equipment was started and activated for one day, 800 g of simulated kitchen waste was added daily for 16 consecutive days of aerobic composting to evaluate the impact of different bacteria agent combinations on fat degradation and composting efficiency.

[0091] The food waste biological treatment machine used in this experiment has a rectangular structure with gears at the bottom for easy relocation. Its dimensions (length × width × height) are 535 × 235 × 580 mm, with an effective processing volume of 6 L and a total weight of approximately 16 kg. The machine integrates an axial flow fan, mixing components, and a temperature regulation and insulation mechanism. When the internal chamber temperature is below 40℃, the auxiliary electric heating system automatically activates; conversely, if the temperature exceeds 45℃, the system automatically shuts off. Furthermore, the processor is equipped with a deodorization function. Through a built-in photocatalytic purification system, ultraviolet radiation combined with a catalyst effectively treats the gas molecules generated during the treatment process, ultimately releasing clean air. Sample collection employed a multi-point sampling and thorough mixing strategy, dividing the samples into three portions each time. One sample, taken immediately and fresh, was used for rapid testing of multiple indicators, including moisture content, pH, EC, E4 to E6 ratio, and seed germination rate. Another sample, after being air-dried, was ground through a 40-mesh sieve to obtain a powder of suitable particle size, facilitating subsequent analysis of key parameters such as easily decomposable organic matter, starch, protein, and fat content. The third sample was stored at -80°C for metagenomic sequencing analysis.

[0092] The experimental results and discussion of this embodiment are as follows:

[0093] 1) Temperature

[0094] The aerobic composting process of food waste generally involves four stages: the heating stage, the high-temperature stage, the cooling stage, and the maturation stage. Therefore, the temperature during composting reflects different stages of the process, indicating the activity of microorganisms and the degradation of organic matter. The duration of the high-temperature stage is also a key indicator for evaluating whether the fermentation has achieved harmlessness. The decomposition of organic matter releases heat, causing the temperature of the composting system to rise.

[0095] See Figure 8 It can be seen that the temperature of treatment group #4 was consistently higher than that of the other groups, indicating its strong ability to degrade organic matter. This is because commercially available composting agents and animal fat degrading agents were added to treatment group #4. Compared to treatment group #2, it contained various strains of commercially available composting agents capable of degrading starch, protein, and cellulose. These microorganisms have a stronger ability to degrade various organic matter. The overall temperature of treatment group #3 was lower than that of treatment group #4 at different stages, indicating a good synergistic effect between the animal fat degrading agents and the commercially available composting agents. The animal fat degrading agents preferentially decomposed the fat covering the surface, allowing the remaining organic matter to be better degraded by the various effective strains in the commercially available composting agents, releasing heat and causing the pile temperature to rise. Both control group #1 and treatment group #2 showed temperature peaks on day 9. However, thereafter, because control group #1 had difficulty degrading continuously fed kitchen waste, the pile temperature decreased faster than that of treatment group #2, and the temperature continued to decrease until the end of the composting process, while the temperature of treatment group #2 remained relatively stable until the end of the composting process. This indicates that the microorganisms in the group inoculated with animal fat-degrading bacteria have strong activity and can continue to utilize organic matter to generate heat, thus ensuring that the generated heat is well maintained.

[0096] 2) pH

[0097] During the composting process of food waste, the degradation of organic matter produces small-molecule organic acids, which affect the pH. Therefore, observing pH changes during composting can reflect the degradation of organic matter during aerobic composting.

[0098] See Figure 9The pH of all groups dropped significantly in the early stages of composting, ranging from 6.0 to 6.5. This was due to the rapid degradation of food waste, which released a large amount of organic acids, consistent with Wu Hailong's findings. Subsequently, the pH of treatment groups #2, #3, and #4, inoculated with different microbial agents, fluctuated within a small range, while the pH of the control group #1 dropped sharply after 10 days. This may be because this group had a poorer ability to degrade food waste, leading to a large accumulation of undegradable food waste in the later stages, creating an anaerobic environment within the pile. The anaerobic bacteria inside produced a large amount of organic acids through anaerobic respiration. At the end of composting, the pH of treatment groups #2 and #4 was greater than 6.0, while the pH of treatment group #3, inoculated with a commercially available composting microbial agent, was lower than 6.0, but still higher than the control group #1. This indicates that inoculation with animal fat degrading bacteria has a significant effect on improving acidification during composting. Furthermore, a large amount of undegraded grease was observed on the surface of the compost at the end of treatment group #3, while the grease on the surface of the compost at the end of treatment group #4 was significantly reduced. This demonstrates that adding the solid animal fat degrading bacteria of this invention to commercially available composting agents can achieve better results. By decomposing a large amount of difficult-to-degrade grease covering the surface with the solid animal fat degrading bacteria, and combining it with various protein, starch, and cellulose degrading bacteria in commercially available composting agents, the food waste can be better fermented, enhancing its buffering capacity against compost acidification.

[0099] 2) Electrical Conductivity

[0100] Electrical conductivity is an important parameter for assessing the agricultural applicability of compost products, reflecting the soluble salt content during aerobic fermentation. The optimal electrical conductivity range for safe plant growth is less than 4 mS / cm, while higher conductivity inhibits plant growth.

[0101] See Figure 10 The EC values ​​of all experimental groups showed a slow upward trend, with slight fluctuations throughout, but the overall trend was upward. Treatment group #4 showed the fastest growth in the early stages, indicating that this group had the strongest microbial activity. The degradation of large amounts of organic matter promoted the generation of a large number of soluble ions, leading to a rapid increase in EC. At the end of the composting process, the EC values ​​of control group #1, treatment group #2, treatment group #3, and treatment group #4 were 3.065 mS / cm, 3.345 mS / cm, 3.500 mS / cm, and 3.700 mS / cm, respectively, all less than 4.0 mS / cm, which preliminarily indicates that the compost products of each group have no toxic effect on plants.

[0102] 4) Oil degradation rate

[0103] Fats and oils, a collective term for oils and lipids, are important carbon and energy sources available to many microorganisms. Different types of fats and oils differ in the composition of their fatty acids and the length of their carbon chains, and this structural characteristic often makes their degradation difficult.

[0104] See Figure 11 It can be seen that the oil degradation rate of each group showed an increasing trend over time. Furthermore, at different time points, the oil degradation rate was highest in treatment group #4, followed by #2, #3, and #1. This indicates that adding solid animal fat degrading bacteria can effectively promote oil degradation, which is consistent with Ling Ling's research, namely, inoculation with oil-removing bacteria can increase the oil degradation rate by 11.15% compared to the control group. The dominant period of oil degradation was concentrated in the high-temperature stage of composting. The mechanism may be that high temperature causes solid oil in kitchen waste to melt and liquefy, significantly improving the decomposition efficiency of lipids through a physical mechanism that increases the contact area between microorganisms and oil. At the end of composting, treatment group #4 had the highest oil degradation rate at 53.46%, followed by treatment group #2 at 48.67%, treatment group #3 at 45.83%, and control group #1 at 36.22%. Compared to the 38.62% fat degradation rate observed by the animal fat degrading microbial agent developed by Zhan Yabin at the end of aerobic composting (i.e., day 21), the solid animal fat degrading microbial agent of this invention, and the combined addition of the solid animal fat degrading microbial agent of this invention with commercially available composting microbial agents, exhibits better fat degradation effects. This indicates that in treatment #4, the solid animal fat degrading microbial agent of this invention and the commercially available composting microbial agent have a good synergistic effect, jointly promoting fat degradation.

[0105] 5) Starch degradation rate

[0106] Starch is a large carbohydrate molecule formed by the polymerization of glucose units. In the simulated food waste designed in this embodiment, rice and other foods rich in starch were added. Due to the characteristics of the starch molecule structure, it is easily metabolized and utilized by microorganisms as a carbon source.

[0107] See Figure 12On day 4 of the composting process, treatment groups #2, #3, and #4 all exhibited high starch degradation efficiencies, all exceeding 70%, which was more than 27% higher than the starch degradation rate of the control group #1. During the 10-day operation, the starch degradation rates of treatment groups #3 and #4 were higher than that of treatment group #2. This is because treatment groups #3 and #4 were inoculated with commercially available composting microbial agents containing strains specifically and efficiently degrading starch, thus resulting in higher starch degradation rates than #2. After 10 days, the starch degradation rate of treatment group #2 was higher than that of treatment group #3, while treatment group #4 maintained the highest starch degradation rate. This is likely because treatment groups #2 and #4 incorporated the solid animal fat degrading microbial agent of this invention, which, in the later stages, decomposed the fat covering the surface of the food waste due to the highly efficient fat-degrading bacteria, thereby improving starch degradation. This finding is consistent with the conclusions of Nakasaki's research, which indicates that lower concentrations of fat can mitigate the inhibitory effect on the organic matter degradation process. In the later stages of treatment group #3, due to the group's poor efficiency in degrading grease, a large amount of undecomposed grease accumulated and covered the surface of the kitchen waste, forming an oxygen-isolated oil film that made it difficult for microorganisms to decompose and utilize the starch inside the pile, resulting in a decrease in the starch degradation rate.

[0108] 6) Protein degradation rate

[0109] Proteins are a class of high-molecular-weight organic compounds. Microorganisms first need to secrete proteases to hydrolyze proteins into dipeptides or polypeptides, and then further hydrolyze them into amino acids that can be directly utilized by the microorganisms. In this experiment, many protein-containing substances were added to the simulated kitchen waste; therefore, analyzing the protein reduction rate can verify its composting effect.

[0110] See Figure 13 The trends in protein degradation rate during composting were similar to those in starch degradation rate. The protein degradation rates of treatment groups #2, #3, and #4, which had different microbial agents added, were significantly higher than those of the control group #1, which had no microbial agents added, indicating that inoculation with microbial agents can improve the protein degradation rate. In the early and middle stages of composting, the protein degradation rates of treatment groups #3 and #4 were higher than those of treatment group #2. This is because both groups contained strains specifically capable of degrading proteins, resulting in higher protein degradation rates. In the later stages of composting, the protein degradation rates of treatment groups #2 and #4 continued to increase, while the protein degradation rate of treatment group #3 decreased. This may be because treatments #2 and #4 were inoculated with the solid animal fat degrading microbial agent of this invention, which enhanced the microbial decomposition of proteins by decomposing the fat coating the material surface.

[0111] 7) Content of easily degradable organic matter

[0112] Easily degradable organic matter is characterized by its ease of decomposition and utilization by microorganisms, and its degradation process is usually relatively rapid. It contains polysaccharides, proteins, fats, and fatty acids. Therefore, the determination of the content of easily degradable organic matter can reflect the residual amounts of proteins, polysaccharides, fats, and fatty acids in compost materials.

[0113] See Figure 14 Because the machine operates by adding kitchen waste daily, the content of readily biodegradable organic matter in the compost pile continuously increases. In the early stages of composting, the readily biodegradable organic matter content in each group increases rapidly due to the presence of lignocellulose, a conditioner primarily composed of solid microbial agents, which is difficult to degrade. Subsequently, as microorganisms become more active, a large amount of organic matter begins to degrade. In treatment groups #2, #3, and #4, which were inoculated with microbial agents, the readily biodegradable organic matter content initially increases slowly before showing a slight decrease, while in the control group #1, the readily biodegradable organic matter content continues to rise. This may be because the control group #1, lacking microbial agents, suffers from a large amount of undegradable oils, proteins, and starches in the compost pile. With the continuous accumulation of organic acids, the system lacks an effective buffering mechanism to neutralize these acidic substances, thus inhibiting microbial activity. Under this influence, the readily biodegradable organic matter content in the control group #1 is higher than that in treatment group #2, reaching 25.39% at the end of composting. The group without microbial agents showed weaker organic matter degradation capabilities. This also demonstrates that the solid animal fat degrading microbial agent of the present invention can reduce the inhibitory effect of continuous accumulation of stockpile on the degradation of organic matter by degrading fats.

[0114] 8) Total reduction rate

[0115] The reduction rate can reflect the reduction effect of aerobic composting of kitchen waste. In this embodiment, the reduction rate is comprehensively evaluated by the total dry matter reduction rate, the total organic matter reduction rate, the kitchen waste dry matter reduction rate, and the total wet weight reduction rate, in order to achieve a more comprehensive evaluation.

[0116] See Figure 15The total dry matter reduction rate, total organic matter reduction rate, kitchen waste dry matter reduction rate, and total wet weight reduction rate of treatment groups 2#, 3#, and 4#, which were inoculated with different microbial agents, were all significantly higher than those of the control group 1#, indicating that the inoculation of microbial agents achieved good results in all aspects of weight reduction. At the end of composting, the total dry matter reduction rate, total organic matter reduction rate, kitchen waste dry matter reduction rate, and total wet weight reduction rate of treatment group 4# were 52.34%, 55.35%, 76.77%, and 87.63%, respectively, all of which were higher than those of treatment groups 2# (50.02%, 51.61%, 73.36%, and 84.23%) and 3# (42.15%, 46.75%, 61.83%, and 80.34%). The wet weight degradation rate of food waste is similar to that of Lü Xianzhe (who achieved 88% wet weight degradation rate of food waste at the end of composting) and is superior to that of commercially available food waste microbial agents (77.94%) and food waste degradation microbial agents (81.23%) developed by Lutong. This indicates that the co-addition of animal fat degradation microbial agents and commercially available composting microbial agents effectively promotes the reduction of food waste.

[0117] 9) Seed germination index

[0118] The Germination Index (GI), a key biological evaluation indicator, is widely used to assess the maturity and potential phytotoxicity of compost products. A GI of 50% or higher indicates that the organic material has eliminated phytotoxic effects and can be considered basically mature. Referring to the industry standard for organic fertilizers (NY / T 525-2021), the Ministry of Agriculture and Rural Affairs has set requirements for the GI value of organic fertilizers. Compost products must meet the stringent requirement of a GI value of no less than 70% to be recognized as qualified organic fertilizers.

[0119] See Figure 16 In the initial stage of the composting process, the GI values ​​of all groups showed a significant decreasing trend. This phenomenon is attributed to the extremely vigorous microbial activity during this period, which generated a large amount of organic acids and phenolic compounds during the decomposition of organic matter. These metabolites had a significant inhibitory effect on the seed germination process. Subsequently, the GI values ​​of treatment group 1 (without added microbial agent) continued to decrease, while the GI values ​​of treatment groups 2, 3, and 4 gradually increased. At the end of the process, the GI values ​​of treatment groups 2 and 4 were both greater than 70%, meeting the requirement of ≥70% seed germination rate in the "Organic Fertilizer" standard (NY / T 525-2021). The results indicate that adding the solid animal fat degrading microbial agent of this invention can improve the compost maturity.

[0120] 10) E4 / E6

[0121] E4 / E6 refers to the ratio of absorbance of compost product leachate at wavelengths of 465 nm and 665 nm. It is one of the standards for measuring the maturity of compost products. The lower the ratio, the higher the degree of aromatization of humic substances, the larger the molecular weight, the higher the degree of humification, and the stronger the stability of organic matter.

[0122] See Figure 17 The E4 / E6 values ​​of all groups showed a decreasing trend over time. From 1 to 4 days, the E4 / E6 values ​​of all groups showed a rapid decreasing trend. This change may be attributed to the mineralization of organic matter within the composting system, or the initial condensation reaction of aromatic compounds, leading to a decrease in the E4 / E6 ratio. Subsequently, with the increased activity of microorganisms, the continuous degradation of organic matter resulted in the continuous generation of small-molecule organic acids, thus leading to a slow decrease. At the end of the composting process, the E4 / E6 value of group #4 was the lowest, significantly lower than that of treatment groups #1, #2, and #3. The results indicate that the co-addition of the solid animal fat degrading microbial agent of this invention and commercially available composting microbial agents can accelerate the humification process of food waste composting.

[0123] 11) Composition and properties of compost products

[0124] The composition and properties of the compost products from each experimental group are detailed in Table 4 below:

[0125] Table 4. Composition and properties of compost products from each experimental group

[0126]

[0127] The organic fertilizer standard is: organic matter ≥30%, pH 5.5-8.5, GI ≥70%. As can be seen from Table 4 above, treatment groups 2# and 4# both meet the organic fertilizer standard. Among them, the GI of control group 1# and treatment group 3# is less than 70%, and the group with animal fat degrading bacteria agent + commercially available degrading bacteria agent added together has the highest GI.

[0128] 12) Microbial community structure analysis

[0129] See Figure 18 Among the four groups of compost products, bacteria had the highest relative abundance at the domain level, holding an absolute advantage. The relative abundance of bacteria in samples M1-16d, M2-16d, M3-16d, and M4-16d were 97.89%, 99.57%, 92.50%, and 96.31%, respectively. In contrast, the relative abundance of eukaryotic microorganisms in samples M1-16d, M2-16d, M3-16d, and M4-16d were 1.85%, 0.27%, 2.41%, and 3.33%, respectively. Therefore, it can be concluded that bacteria are the primary means of degrading food waste during the composting process in all four groups.

[0130] See Figure 19The abundance of Firmicutes was relatively high in samples M1-16d, M2-16d, M3-16d, and M4-16d, at 92.01%, 94.67%, 85.23%, and 61.53%, respectively. This is because Firmicutes have good heat resistance and can form heat-resistant spores to adapt to the high-temperature environment during composting, thus resulting in a relatively high abundance of Firmicutes in the final compost products. The relative abundance of Actinobacteria in samples M2-16d and M4-16d at the end of composting was 4.36% and 2.05%, respectively, higher than that in samples M1-16d and M3-16d. Actinobacteria can degrade lignocellulose and other recalcitrant organic matter by producing enzymes that degrade lignocellulose. Therefore, the addition of animal fat-degrading bacteria to promote fat decomposition allows actinobacteria to begin degrading the accumulated lignocellulose in the compost, leading to a higher relative abundance of Actinobacteria. The relative abundance of Pseudomonas was only 0.5% in sample M2-16d, while it was 32.23% in sample M4-16d. This indicates that adding commercially available composting agents to the animal fat degrading microbial agent increased the relative abundance of Pseudomonas, thereby promoting the degradation of food waste. This is because Pseudomonas can degrade proteins, fats, carbohydrates, and some cellulose in food waste by secreting various extracellular enzymes (such as proteases, lipases, amylases, and cellulases), thus promoting the degradation of food waste. Therefore, the total reduction rate of the group with both animal fat degrading microbial agent #4 and commercially available composting agents added was better than that of the group with only animal fat degrading microbial agent #2.

[0131] See Figure 20After adding the microbial agent, the main bacterial species are more evenly distributed. In the compost products containing only the solid animal fat degradation microbial agent of this invention, the main bacterial genera are Pediococcus, Lactiplantibacillus, Limosilactobacillus, Ligilactobacillus, and Microbacterium. In the compost products containing only commercially available composting microbial agents, the main bacterial genera are Pediococcus, Lactiplantibacillus, Limosilactobacillus, Ligilactobacillus, Acinetobacter, and Pseudomonas. The main bacterial genera in the final compost product of animal fat degradation bacteria and commercially available composting bacteria are Pseudomonas, Bacillus, Ligilactobacillus, Pediococcus, Pseudochrobactrum, Paenibacillus, and Rhizophagus.

[0132] In all three groups with added microbial agents, the abundance of *Pediococcus* was high. *Pediococcus* can convert easily degradable organic matter such as sugars and starches in kitchen waste into lactic acid through glycolysis, and inhibit the production of acetic acid, thereby lowering the pH of the compost pile, reducing ammonia volatilization, and indirectly retaining nitrogen nutrition. Adding the solid animal fat degrading microbial agent of this invention increased the relative abundance of *Pediococcus*, by 56.23% and 48.87% compared to the control group and the group with commercially available composting microbial agent, respectively. In groups #2 (containing only the solid animal fat degrading microbial agent of this invention) and #3 (containing only commercially available composting microbial agent), the relative abundance of *Lactobacillus plantarum* was high, at 8.09% and 26.71%, respectively. The metabolic process of *Lactobacillus plantarum* produces vitamins, plant hormone analogs (such as indoleacetic acid), and antimicrobial peptides. These substances can be retained in the finished compost, enhancing the compost's ability to promote plant growth and disease resistance.

[0133] In group #4, which added both the solid animal fat degrading microbial agent of this invention and a commercially available composting microbial agent, the relative abundance of Bacillus was 10.52%, higher than that of group #2 (0.17%) which only added the solid animal fat degrading microbial agent of this invention and group #3 (0.87%) which only added the commercially available composting microbial agent. This indicates that the co-addition of the high-efficiency solid fat degrading microbial agent of this invention and the commercially available composting microbial agent significantly increased the relative abundance of Bacillus. Most Bacillus species possess characteristics such as high-temperature resistance, strong enzyme activity, and metabolic diversity, and are widely used for the decomposition of recalcitrant organic matter such as fats, cellulose, and hemicellulose. He et al. studied the effects of composite microbial agents on the decomposition of organic matter and nitrogen retention in a composting system. The selected agents consisted of Bacillus, Streptomyces, and Saccharomonospora. Analysis of the microbial community structure revealed that the intervention of exogenous agents significantly activated the activity of indigenous microorganisms, especially promoting the increase in the abundance of Bacillus-like organisms. Simultaneously, this treatment enhanced polyphenol oxidase activity, driving the efficient operation of the tricarboxylic acid cycle and amino acid metabolic pathways. Final data showed that the organic matter decomposition rate of the composting system increased by 9.9%, and nitrogen content increased by 20.6%, confirming that the microbial agent enhancement strategy has a positive regulatory effect on the humification process. This is similar to the situation in treatment group #4 in this embodiment, where the co-addition of the solid high-efficiency oil-based microbial agent of this invention and a commercially available composting microbial agent activated the activity of indigenous microorganisms, thereby increasing the relative abundance of Bacillus.

[0134] 13) Abundance analysis of key enzyme genes in lipid metabolism

[0135] See Figure 21It can be seen that K01046 and K01054 are triacylglycerol lipase (EC: 3.1.1.3) and acylglycerol lipase (EC: 3.1.1.23), respectively, which are key enzymes in the glycerol lipase metabolism process. The gene abundance of triacylglycerol lipase and acylglycerol lipase in sample M4-16d was significantly higher than that in the other three groups, indicating that the co-addition of the highly efficient oil-degrading microbial agent of this invention and the commercially available composting microbial agent increased the abundance of triacylglycerol lipase and acylglycerol lipase-related genes. The gene abundance of triacylglycerol lipase was also high in sample M2-16d. Triacylglycerol lipase hydrolyzes the ester bonds of triacylglycerol, gradually breaking it down into diacylglycerol and free fatty acids, which is beneficial for further metabolism. K13979, K00128, K15918, and K00011 are alcohol dehydrogenase (NADP+) EC:1.1.1.2, aldehyde dehydrogenase (NAD+) EC:1.2.1.3, D-glycerate 3-kinase EC:2.7.1.31, and aldehyde reductase EC:1.1.1.21, respectively. The gene abundance of alcohol dehydrogenase and aldehyde dehydrogenase was higher in samples M2-16d and M4-16d than in the other two groups. In the absence of glycerol kinase, alcohol dehydrogenase can work with aldose reductase to convert glycerol into glyceraldehyde, which is then acted upon by aldehyde dehydrogenase to produce D-glycerides. D-glycerides can be catalyzed by D-glycerate 3-kinase to produce 3-phosphate-D-glycerides, thereby promoting the breakdown of glycerol into glycolysis. Therefore, the addition of the highly efficient oil-degrading microbial agent of the present invention, as well as the co-addition of the highly efficient oil-degrading microbial agent and the commercially available composting microbial agent, can increase the gene abundance of triglyceride lipase, monoglyceride lipase, alcohol dehydrogenase and acetaldehyde dehydrogenase in glycerol metabolism, thereby achieving more efficient glycerol metabolism.

[0136] See Figure 22In the metagenomic sequencing of the four groups of compost products, a large number of genes related to fatty acid degradation were annotated. It can be seen that K00249, K01897, K00626 and K00632 are acyl-CoA dehydrogenase (EC:1.3.8.7), long-chain acyl-CoA synthetase (EC:6.2.1.3), acetyl-CoA C-acetyltransferase (EC:2.3.1.9) and acetyl-CoA acyltransferase (EC:2.3.1.16), respectively. The abundance of genes related to acyl-CoA dehydrogenase, long-chain acyl-CoA synthase, acetyl-CoA C-acetyltransferase, and acetyl-CoA acyltransferase was higher in samples M4-16d and M2-16d than in M1-16d and M3-16d. Long-chain acyl-CoA synthase can catalyze the combination of long-chain fatty acids and coenzyme A to form acyl-CoA, thereby improving the metabolic efficiency of long-chain fatty acids. Acyl-CoA dehydrogenase plays a crucial role in fatty acid β-oxidation, catalyzing the dehydrogenation reaction of the fatty acid-CoA complex, enhancing the breakdown of long-chain fatty acids, and promoting lipid degradation. Acetyl-CoA C-acetyltransferase and acetyl-CoA acyltransferase can convert acetyl-acetyl-CoA to acetyl-CoA, which is one of the core reactions in lipid metabolism.

[0137] In summary, the addition of the highly efficient oil-degrading microbial agent of the present invention, as well as the co-addition of the highly efficient oil-degrading microbial agent of the present invention with commercially available composting microbial agents, can effectively increase the gene abundance of several key enzymes in the fatty acid β-oxidation process, such as acyl-CoA dehydrogenase, long-chain acyl-CoA synthase, acetyl-CoA C-acetyltransferase, and acetyl-CoA acyltransferase, thereby degrading oils more efficiently.

[0138] The above specific embodiments further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing bacterial strains for aerobic composting of kitchen waste, characterized in that, The microbial community was enriched and domesticated using the hydrolysis circle method, and strains suitable for animal fat degradation were isolated and screened. The specific preparation process included: Step 1: Collection of microbial communities Oily soil near swill buckets or oily wastewater from kitchen waste were used as samples for screening target bacterial strains. Step 2: Enrichment, Acclimation, and Purification of the Strains. 10 g of the collected sample was shaken with 100 mL of physiological saline. The resulting sample dilution was then used for enrichment and acclimation culture. The bacterial culture was mixed at a volume ratio of 10:90 and cultured with shaking at 30°C for 6 acclimatization cycles, each lasting 5 days. After each acclimatization cycle, 10 mL of the bacterial culture was transferred to 90 mL of fresh culture medium. This process was repeated for 6 cycles to obtain an enriched acclimatized bacterial culture. The culture medium was porcine / cattle fat, with an initial fat concentration of 2.5 g / L. The fat concentration of the fresh culture medium increased by 0.5 g / L each cycle. The shaking speed was 160 r / min. 2) Mix 10 mL of enriched and domesticated bacterial solution with 90 mL of physiological saline to prepare 10 mL of the solution. - ¹Diluent, diluted tenfold to 10. -7 Take 100 μL of each of the following 10 μL samples: -4 ~10 -7 Gradient bacterial suspensions were evenly spread on a primary screening medium containing hog / cattle fat and cultured upside down at 30°C for 2-3 days. After culture, single colonies with obvious blue hydrolysis zones were selected and purified to a single strain by streaking multiple times. Colonies with good growth were transferred to nutrient agar (NA) slants, stored at 4°C and transferred periodically to obtain the isolated and purified strain. Step 3: Initial screening of strains The isolated and purified strains were initially screened using the hydrolysis zone method. They were inoculated into screening medium containing hog / cattle fat and cultured upside down at 30°C for 3 days. The ratio of the colony diameter to the diameter of the surrounding blue hydrolysis zone was used as the enzyme activity evaluation index. The isolated and purified strains were then screened to obtain the initial screening strains. Step 4: Secondary screening of strains The initially screened strains were transferred to a liquid culture medium of bacteria-NB / yeast-PDB and cultured with shaking at 30°C for 24 h. Then, they were transferred to a secondary screening medium containing hog / bovine fat at a 10% inoculation rate and cultured with shaking at 30°C for 72 h. The degradation rate of the strains on hog / bovine fats and the activity of lipase were used as evaluation indicators to re-screen the initially screened strains and obtain the strains for preparing animal fat degradation agents. The shaking speed was 160 r / min.

2. A method for preparing the grease-degrading bacterial agent using the aerobic composting strains of claim 1 for food waste, characterized in that, Liquid or solid animal fat degrading agents were prepared using aerobic composting strains of food waste. The liquid animal fat degrading agent was prepared by inoculating a liquid culture medium with *Pseudomonas alkaligenes*, *Bacillus parabregas*, *Candida lipolyticis*, and *Bacillus amyloliquefaciens* strains. The medium was then shaken and cultured for 24 h at pH 7.0, 30℃, and 160 r / min. The *Pseudomonas alkaligenes*, *Bacillus parabregas*, *Candida lipolyticis*, and *Bacillus amyloliquefaciens* strains were mixed at a volume ratio of 1:1:1:1, with volume percentage concentrations of 25%, 25%, 25%, and 25%, respectively. The liquid culture medium was either bacteria-NB or yeast-PDB liquid culture medium. The contents of peptone, beef extract, and sodium chloride in the bacteria-NB culture medium were 5.0 g / L, 3 g / L, and 3 g / L, respectively. The contents of peptone, glucose, and yeast extract in the yeast-PDB liquid culture medium are 0 g / L and 0.5 g / L, respectively. The solid animal fat degrading agent is prepared by mixing the liquid animal fat degrading agent with the dry weight of the conditioner at a volume-to-weight ratio of 1:

1. The conditioner is composed of coconut coir and rice husk, and the dry weight ratio of coconut coir to rice husk is 5:3.

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