Glycine bacillus, bacterial agent and application thereof

Abstract

The present application relates to a kind of glycine bacillus, is glycine bacillus YZQ-5 (Bacillus glycinifermentans YZQ-5), preserved in China typical culture preservation center (CCTCC), and the preservation number is CCTCCM2023121.The present application further provides a kind of microbial inoculant comprising above-mentioned glycine bacillus and its preparation method, and provides the application of above-mentioned microbial inoculant in degrading oil and kitchen waste compost.The glycine bacillus YZQ-5 of the present application can degrade oil up to 69.89% within 72h, and can tolerate high temperature, in the aerobic composting of kitchen waste, the degradation rate and weight loss rate of oil are increased by 33.15% and 26.30% respectively, and the composting degree is improved, with good application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Bacillus glycinus, its inoculum, and its applications. Background Technology

[0002] In recent years, food waste pollution from restaurants, canteens, and households has become an increasingly serious social problem. Food waste, also known as swill or swill, is household waste generated during daily consumption. China, the world's most populous country, saw its urban food waste production rise to 128 million tons in 2020 due to population growth. Sorted food waste has an extremely high moisture content (usually above 90%) and a very low calorific value. The continuous increase in food waste production easily leads to a series of environmental, economic, and social problems, such as groundwater pollution, greenhouse gas emissions, eutrophication of water bodies, and soil pollution. Common food waste treatment technologies in my country can be categorized into direct treatment technologies, feed production technologies, anaerobic digestion technologies, aerobic composting technologies, biological drying technologies, and biological reduction technologies. Aerobic composting technology, due to its low cost, high degree of harmlessness, large processing capacity, and the ability to directly apply the processed products to the soil, has become an important means of treating food waste.

[0003] Compared to livestock and poultry manure, garden waste, and agricultural straw, food waste has unique properties, such as a high oil content (1%–5 wt%, wet basis). However, a problem remains in the in-situ biodegradation of food waste (FW). Due to traditional Chinese cooking methods and customs, the oil content in food waste is typically very high (greater than 20% on a dry basis).

[0004] Waste cooking oil (WCO) refers to oil remaining after frying or steaming. Its structure is altered by oxidation reactions through a typical free radical mechanism, gradually oxidizing into toxic products such as 4-hydroxy-2-olefins and acrylamide. On the other hand, during cooking, unsaturated fatty acids are converted into trans fatty acids through thermal oxidative degradation, becoming more difficult to degrade. Previous studies indicate that nearly 16.5 million tons of WCO are produced globally each year, with China alone producing over 5 million tons annually. Recent reports suggest that microbial biodegradation of WCO is a low-cost and environmentally friendly treatment method. Sarac isolated the hemolytic Acinetobacter lipase LipAH02-3 from soil, which exhibited optimal enzymatic activity at 40°C and effectively hydrolyzed high concentrations of oil in kitchen wastewater. In previous reports, *Pseudomonas putida* and *Bacillus amyloliquefaciens* demonstrated their synergistic effect in waste oil degradation. These microorganisms can be used to degrade grease in wastewater but cannot be applied to composting environments. During aerobic composting, the temperature at the compost center typically exceeds 60°C when a large amount of heat is generated during the decomposition of organic matter. Therefore, while requiring microorganisms to have good lipid degradation effects, they must also have good temperature tolerance. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a high-temperature resistant oil-degrading bacterium that can be applied to the composting of kitchen waste.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] In a first aspect, the present invention provides a glyciniferous Bacillus, specifically Bacillus glycinifermentans YZQ-5, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2023121.

[0008] Secondly, the present invention provides the application of the above-mentioned Bacillus glycinus in the degradation of oils and fats.

[0009] Thirdly, the present invention provides the application of the above-mentioned Bacillus glycinus in the composting of kitchen waste.

[0010] Fourthly, the present invention provides a microbial agent comprising the above-mentioned Bacillus glycinus.

[0011] Fifthly, the present invention provides a method for preparing the above-mentioned microbial agent, wherein the glycine Bacillus is inoculated into a culture medium to obtain the microbial agent.

[0012] Sixthly, the present invention provides the application of the above-mentioned microbial agents in the degradation of oils and fats.

[0013] In a seventh aspect, the present invention provides the application of the above-mentioned microbial agents in the composting of kitchen waste.

[0014] The beneficial effects of this invention are as follows: The glycine-containing Bacillus YZQ-5 of this invention has the highest degradation rate of oils and fats (69.89%) within 72 hours, and it can withstand high temperatures. At 50°C, the degradation rate of oils and fats is 55.99%, and the lipase activity is as high as 1550 U / L. It also has high tolerance to pH and salt concentration. In aerobic composting of kitchen waste, the degradation rate of oils and fats and the weight loss rate are increased by 33.15% and 26.30%, respectively, and the degree of decomposition is improved. This indicates that YZQ-5 not only promotes the decomposition of organic matter, but also improves the humification of compost. Attached Figure Description

[0015] Figure 1 This is an electron microscope image of Bacillus glycinus YZQ-5 of the present invention;

[0016] Figure 2 This is the phylogenetic tree of Bacillus glycinus YZQ-5 of the present invention;

[0017] Figure 3 The growth curve of Bacillus glycinus YZQ-5 of this invention;

[0018] Figure 4 The effect of pH on the degradation ability of Bacillus glycinus YZQ-5;

[0019] Figure 5 The effect of temperature on the degradation ability of Bacillus glycinus YZQ-5;

[0020] Figure 6 The effect of salt concentration on the degradation ability of Bacillus glycinus YZQ-5;

[0021] Figure 7 This is an enzyme activity assay plate for Bacillus glycinoides YZQ-5 degrading organic matter, in which... Figure 7 A, 7B, 7C, and 7D are the enzyme activity indicator zones for amylase, protease, lipase, and cellulase, respectively.

[0022] Figure 8 This is a graph showing the changes in the number of viable bacteria during aerobic composting of kitchen waste. Detailed Implementation

[0023] The principles and features of the present invention are described below with reference to the accompanying drawings and specific embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0024] Experimental materials

[0025] The source of the grease-degrading bacteria was a sample of kitchen waste from the Jixian canteen at the East Lake Campus of Zhejiang Agriculture and Forestry University, collected during the high-temperature aerobic composting process. 500g of sample was taken, placed in ultraviolet-sterilized centrifuge tubes, and stored at -20℃ for later use.

[0026] culture medium

[0027] LB medium: 10.0 g / L peptone -1 5.0g·L yeast extract -1 NaCl 5.0 g·L -1 pH = 7.0, natural pH.

[0028] Enrichment medium: LB medium + 20.0 g / L soybean oil -1 , natural pH.

[0029] Selective culture medium: KH2PO4 0.3 g·L -1 MgSO4·7H2O 0.1g·L - 1, K₂HPO₄ 1.5 g·L⁻¹ -1 (NH4)2SO4 1.0 g·L -1 NaCl 5.0 g·L -1 3.0g·L soybean oil -1 Neutral red indicator, agar 18 g·L -1 , natural pH.

[0030] Degradation medium: KH2PO4 0.3 g·L -1 MgSO4·7H2O 0.1g·L -1 K2HPO4 1.5 g·L -1 (NH4)2SO4 1.0 g·L -1 NaCl 5.0 g·L -1 3.0g·L soybean oil -1 5.0g·L yeast extract -1 , natural pH.

[0031] Lipase fermentation medium: CaSO4·2H2O 0.1 g·L⁻¹ -1 MgSO4·7H2O 0.1g·L -1 20.0 g / L glucose -1 ,KH2PO4 0.5g·L -1 Yeast extract 20.0g·L -1 Natural pH.

[0032] All culture media were sterilized at 121°C for 20 min.

[0033] Methods for determining the degradation rate of oils and fats

[0034] Construction of the soybean oil standard curve: Accurately weigh 1.0 g of soybean oil and dissolve it in a 100 mL volumetric flask. Dilute to the mark with petroleum ether to prepare a stock solution containing 10 mg / mL soybean oil. Subsequently, prepare soybean oil standard solutions with concentrations of (0.25, 0.5, 0.75, 1, 1.5, 2) mg / mL. Using ultraviolet spectrophotometry at a wavelength of 225 nm, with petroleum ether as a blank solution, measure the absorbance of the above series of soybean oil standard solutions to construct the standard curve. Extract the oil from the fermentation broth with petroleum ether, shake thoroughly, let stand for 30 min, take the supernatant, dilute appropriately, and measure the absorbance. Calculate the concentration of the remaining oil based on the standard curve.

[0035] Soybean oil standard curve: After soybean oil is dissolved in petroleum ether (optically pure) with a boiling point of 60℃~90℃, its concentration satisfies the following regression equation: y=0.6414x+0.1968, R 2 =0.9986.

[0036] The oil degradation rate is calculated based on the following:

[0037] Oil degradation rate / % = (initial oil concentration - remaining oil concentration) / initial oil concentration × 100% Example 1: Isolation, screening and identification of oil-degrading bacteria

[0038] 1) Domestication, isolation, and purification of lipid-degrading bacteria

[0039] Acclimation: Take 5 mL of sample and add it to 100 mL of enrichment medium. Incubate at 50 °C and 150 rpm. -1 The culture was incubated in a constant temperature shaking incubator, with each cycle lasting 7 days. 1 mL of the culture medium was transferred to a new enrichment medium, and the degradation of the lipids was observed during this period. If the lipid degradation in the medium was good, a small amount of soybean oil was added. This process was repeated for 4 cycles.

[0040] Initial screening: The acclimatized bacterial culture was serially diluted and spread onto selective medium. It was then incubated at 50°C for 24 hours. During this period, the growth of the bacterial strain was observed. Colonies that turned red in the medium were picked and then streaked to obtain pure culture.

[0041] Secondary screening: Pick up the strain with an inoculation loop and inoculate it into 100 mL of degradation medium. Incubate at 50 °C and 160 rpm. -1 The culture was incubated in a constant temperature shaking incubator for 72 hours, with an uninoculated culture medium as a control. The oil content in the culture medium was determined by ultraviolet spectrophotometry, and the oil-degrading bacterium YZQ-5 was screened out.

[0042] 2) Identification of lipid-degrading strain YZQ-5

[0043] a. Morphological characteristics of lipid-degrading bacteria YZQ-5

[0044] The selected strain YZQ-5 was streaked onto LB plates and incubated in a biochemical incubator at 50℃ for 24 hours. The colony morphology was observed. The colony growth morphology was yellowish-white, with wrinkles in the middle, irregular small particles, and serrated edges.

[0045] The bacterial strain cultured for 24 hours was subjected to an induction temperature of 12000 rpm. -1 Centrifuge for 5 minutes under the specified conditions, then add 2.5% glutaraldehyde electron microscopy protection solution to the centrifuge tube. Send the sample to the Electron Microscopy Center of Zhejiang University for transmission electron microscopy (TEM) to observe the bacterial morphology. The morphological characteristics under TEM are as follows: Figure 1 As shown.

[0046] b. Molecular identification of lipid-degrading bacteria YZQ-5

[0047] The selected single bacteria were sent to Zhejiang Youkang Biotechnology Co., Ltd. for 16S rDNA gene sequencing analysis, obtaining a sequence of the target strain. The obtained sequence was searched and compared in the NCBI database using BLAST. The similarity between strain YZQ-5 and Bacillus glycinifermentans was over 98%. A phylogenetic tree was constructed using MEGA 7.0, as follows... Figure 2 As shown.

[0048] Based on the above morphological characteristics and molecular identification, strain YZQ-5 was determined to belong to Bacillus glycinifermentans and named Bacillus glycinifermentans YZQ-5.

[0049] Bacillus glycinifermentans YZQ-5 was deposited on February 14, 2023, at the China Center for Type Culture Collection (CCTCC), located at Luojia Mountain, Wuchang, Wuhan, Hubei Province, with accession number CCTCC M 2023121.

[0050] Example 2: Degradation characteristics of Bacillus glycinus YZQ-5

[0051] 1) Growth curve determination

[0052] Inoculate 1% (v / v) seed culture into 100 mL of liquid LB medium and incubate at 50 °C and 160 rpm. -1 The samples were cultured in a constant temperature shaking incubator, and samples were taken every 4 hours. After appropriate dilution, the OD values ​​were measured using a UV spectrophotometer. 600The growth curve of the strain was plotted based on the measured absorbance values, and the results are as follows: Figure 3 As shown, *Bacillus glycinoides* YZQ-5 quickly adapted to the new environment from 0 to 27 hours, with a rapid increase in cell count. From 27 to 30 hours, strain YZQ-5 entered a stable growth phase, with growth gradually slowing down. This indicates that the accumulation of metabolic products and an imbalance in nutrient ratios affected microbial growth. The cell count reached its peak at 30 hours, with OD... 600 The value was 4.44. After 30 hours, the curve showed a gradual downward trend, indicating that the strain entered the death phase from this point.

[0053] 2) Effects of pH, temperature and salt concentration on the degradation ability of Bacillus glycinus YZQ-5

[0054] 5% (v / v) seed culture was inoculated into 100 mL of degradation medium with pH values ​​of 5.0, 6.0, 7.0, 8.0, and 9.0, respectively, and incubated at 50 °C and 160 rpm. -1 The strains were cultured in a constant temperature shaking incubator for 72 hours, and the degradation rate of soybean oil by different pH values ​​was measured. The results are as follows: Figure 4 As shown, the oil removal rates of strain YZQ-5 were 7.69%, 25.81%, 57.79%, 73.17%, and 55.30% at pH values ​​of 5, 6, 7, 8, and 9, respectively. The oil degradation rate of strain YZQ-5 reached its maximum at pH 7.

[0055] Transfer 5% (v / v) seed culture to a 250 mL Erlenmeyer flask containing 100 mL of degradation medium. Incubate the medium at different temperatures: 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃, at 160 rpm. -1 The strain was cultured in a constant temperature shaking incubator for 72 hours, and the degradation rate of soybean oil by the strain at different temperatures was measured. The results are as follows: Figure 5 As shown, strain YZQ-5 exhibits varying levels of growth within the temperature range of 30–50℃, demonstrating a certain degree of degradation effect on oils. The highest oil degradation rate (69.86%) was observed at 40℃. Even at 50℃, YZQ-5 maintained an oil degradation rate of 55.99%. This indicates that this strain can adapt to the high temperatures of aerobic composting of kitchen waste, while simultaneously degrading animal and vegetable oils in the compost.

[0056] The 5% (v / v) seed culture was transferred to a 250 mL Erlenmeyer flask containing 100 mL of degradation medium with an initial NaCl concentration of 1 g·L⁻¹. -1 5.0 g·L -1 10.0 g·L -1 15.0 g·L -1 20.0 g·L-1 At 50℃, 160 r·min -1 The strain was cultured in a constant temperature shaking incubator for 72 hours, and the degradation rate of soybean oil by the strain was measured at different NaCl concentrations. The results are as follows: Figure 6 As shown, when the salinity was 1, 5, 10, 15, and 20 g / L, the oil removal rates of strain YZQ-5 were 16.11%, 21.39%, 34.46%, 50.74%, and 34.27%, respectively. The oil degradation rate of strain YZQ-5 reached its maximum at a salinity of 15 g / L. At a salt concentration of 0 g / L, the strain showed almost no degradation of soybean oil.

[0057] 3) Lipase activity of Bacillus glycinoidis YZQ-5

[0058] YZQ-5 was cultured in LB medium for 24 hours, then inoculated into 100 mL of degradation medium containing 2.0 g soybean oil and cultured at 50°C for 72 hours. The unit lipase activity of the fermentation broth was determined using the P-np method. YZQ-5 exhibited high lipase activity (2600 U / L) at 37°C. At 50°C, YZQ-5 maintained strong lipase activity (1550 U / L), demonstrating its adaptability to high-temperature composting systems.

[0059] The specific method of the P-np method is as follows: 2.1 mL of Tris-HCl (pH = 8.0) solution, 200 μL of p-nitrophenol palmitate (p-NPP) (7.5 mM, acetonitrile as solvent), and 100 μL of appropriately diluted fermentation supernatant are added to a 10 mL EP tube. After mixing and reacting at 37°C with shaking for 10 minutes, the EP tube is placed on an ice bath, and 100 μL of 0.1 M zinc sulfate solution is added to terminate the reaction. The reaction solution is filtered through a 0.45 μm water membrane, and the absorbance is measured at a wavelength of 405 nm. Lipase activity is evaluated according to the p-nitrophenol absorbance standard curve.

[0060] 4) Analysis of organic matter degradation and enzyme activity of Bacillus glycinoidis YZQ-5

[0061] Extracellular protease activity was analyzed using LB agar plates containing 2% skim milk powder; extracellular cellulase activity was analyzed using LB agar plates containing 0.5% soluble sodium carboxymethyl cellulose, stained with Congo red for 30 min, and then destained with 1% NaCl; extracellular amylase activity was analyzed using LB agar plates containing 1% amylose, stained with 10% KI, and then destained with 1% NaCl; lipase activity was analyzed using LB agar plates containing 1% peanut oil or animal fat. YZQ-5 colonies were picked with an inoculation loop and inoculated onto the above test plates, then incubated at 50℃ for 48 h. The size of the clear zone around the colonies was observed and recorded to determine the relative activity of YZQ-5 degrading enzymes.

[0062] Tablet photos such as Figure 7 As shown in the figure, the size of the indicator ring was measured, and the results are shown in Table 2 below.

[0063] Table 1. Activities of enzymes related to organic matter degradation in Bacillus glycinus YZQ-5

[0064] amylase protease Lipase Cellulase YZQ-3 ++ ++ +++ +++

[0065] +: The diameter of the indicator ring corresponding to enzyme activity is <1cm; ++: The diameter of the indicator ring corresponding to enzyme activity is 1-2cm; +++: The diameter of the indicator ring corresponding to enzyme activity is 2-3cm; ++++: The diameter of the indicator ring corresponding to enzyme activity is >4cm.

[0066] 5) Determination of the overall physiological and biochemical characteristics of Bacillus glycinoidis YZQ-5

[0067] The salt tolerance of *Bacillus glycinus* YZQ-5 was determined using the following method: NaCl (sodium chloride) was used as a representative salt compound. Liquid culture media were prepared by adding NaCl to LB medium to achieve final concentrations of 2%, 4%, 6%, 8%, 10%, and 12%. After sterilization and overnight standing, YZQ-5 was inoculated onto the NaCl-containing liquid culture media in a clean bench, and the strain growth was monitored in a 50°C constant-temperature shaker.

[0068] Gelatin liquefaction test: The target strain cultured for 24 hours was punctured and inoculated onto gelatin slant culture medium. Another uninoculated culture medium was used as a blank control. The culture was carried out in an incubator at 20℃ for 24-72 hours. The growth of each strain, whether the gelatin was liquefied and the degree of liquefaction were observed at 20℃.

[0069] The formula for the gelatin primary screening culture medium is as follows: 3g beef extract, 200g gelatin, 5g sodium chloride, 5g peptone, 1000ml distilled water, and pH adjusted to 7.2-7.4.

[0070] Based on the above analysis results of organic matter degradation and enzyme activity, as well as the effect of pH on Bacillus glycinus YZQ-5, the physiological and biochemical characteristics of Bacillus glycinus YZQ-5 are shown in Table 2 below.

[0071] Table 2 Physiological and biochemical characteristics of Bacillus glycinoidis YZQ-5

[0072] Properties Performance Properties Performance pH=4 + amylase production + pH = 10 + Protease production + 8% NaCl + Lipase production + 10% NaCl + Cellulase production + 12% NaCl + Liquefied gelatin +

[0073] 6) Application of Bacillus glycinoides YZQ-5 in aerobic composting of kitchen waste

[0074] a. Sampling: Collect kitchen waste (in a plastic bucket) from Jixian Canteen on the East Lake Campus of Zhejiang Agriculture and Forestry University. Stir the waste as thoroughly as possible during sampling.

[0075] b. Sample preparation (determination of the physicochemical properties of kitchen waste)

[0076] 1. Remove bones, plastic, and other debris. (Do not shred.)

[0077] II. Drain the water and collect the wastewater separately for subsequent wastewater tests.

[0078] III. Determination of basic properties of kitchen waste: moisture content, pH, oil content, and salt content.

[0079] c. Add 2500g of wet food waste, 500g of sawdust (adjusted for moisture content and C / N ratio with sawdust), and 13.5ml of microbial inoculum (13.5g of centrifuged bacteria dissolved in 50ml of water) to the bioreactor as A1. Add 2500g of food waste, 500g of sawdust, and 3.5ml of microbial inoculum (13.5g of centrifuged bacteria dissolved in 50ml of water) to the bioreactor at the early stage (day 1) and middle stage (day 4) of composting, respectively, as A2. Add 2500g of food waste, 500g of sawdust, and 13.5g of sterile water to the bioreactor as CK. The bioreactor reaction lasts for 8 days at 50℃. Samples of the product from the bioreactor are taken every 24 hours.

[0080] d. Parameters: Determine the oil degradation rate, weight loss rate, maturity, and colony count in the sample.

[0081] Weight reduction rate: (composted compost pile - auxiliary materials) / initial weight of kitchen waste

[0082] Humics (HS) were extracted and measured using a method improved by Wang et al. (2021b). Air-dried compost samples were mixed with a 1:20 weight-volume ratio of 0.1M NaOH and 0.1M Na₄P₂O₇ (1:1, v / v) and shaken at 180 rpm for 30 min. The mixture was then centrifuged at 4000 rpm for 20 min to obtain a supernatant for HS quantification. This process was repeated three times to ensure complete extraction of HS from the solid sample. The pH of the supernatant was adjusted to 1 with 6M HCl and then allowed to stand at room temperature for 12 h to precipitate humic acid (HA) from fulvic acid (FA). The HA precipitate was redissolved with 0.1M NaOH. Both HA and FA extracts were passed through a 0.45 μm membrane filter and then quantified using a TOC analyzer (TOC-l CPH, Shimadzu, Japan). Compost maturity = humic acid (HA) / fulvic acid (FA), typically reflecting the degree of compost completion.

[0083] After 8 days, the grease degradation rate, food waste weight reduction rate, and decomposition degree are shown in Table 3 below.

[0084] Table 3 Comparison of various indicators of food waste under different treatment methods.

[0085]

[0086] The results of changes in viable bacteria count during aerobic composting are as follows: Figure 8 As shown.

[0087] YZQ-5 bacteria performed well in the in-situ aerobic composting of kitchen waste. In aerobic composting without the addition of microbial agents, even when temperature and moisture content were controlled to maintain microbial activity in the control group (CK group containing unacclimated YZQ-5 bacteria), the number of microorganisms began to decline on the third day, with an oil degradation rate of only 35.92% and a weight loss rate of only 55%. However, after adding microbial agents (acclimated YZQ-5, at 0.45% of kitchen waste) to A1 and A2, compared to CK, the oil degradation rate increased to 59.84% and 69.07%, respectively; the weight loss rate reached 60.10% and 67.81%. After adding microorganisms, the compost maturity of A1 and A2 products was higher than that of CK, by 1.07 and 1.22, respectively. This indicates that inoculation with YZQ-5 bacteria not only promoted the decomposition of organic matter but also improved the humification process of composting.

[0088] The above description is only a preferred embodiment of the present invention and is 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 type of glycine-containing Bacillus, characterized in that, Bacillus glycinoides ( Bacillus glycinifermentans YZQ-5 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M2023121.

2. The application of Bacillus glycinus as described in claim 1 in the degradation of oils and fats.

3. The application of Bacillus glycinus as described in claim 1 in the composting of kitchen waste.

4. A microbial inoculant, characterized in that, It includes the glycine-containing Bacillus as described in claim 1.

5. The method for preparing the microbial inoculant as described in claim 4, characterized in that, The microbial agent was obtained by inoculating the Bacillus glycinus into a culture medium.

6. The application of the microbial agent as described in claim 4 in the degradation of oils and fats.

7. The application of the microbial agent as described in claim 4 in the composting of kitchen waste.

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