Salt-tolerant thermophilic strain and application thereof in degrading oil in kitchen waste

Abstract

The application aims to provide a kitchen waste or permeate grease degradation strain: Bacillus licheniformis SX-1, preferably the preservation number of which is CGMCC No.22532 in the China General Microbiological Culture Collection Center. The strain has the growth characteristics of salt tolerance and wide pH tolerance, and has a high grease degradation rate under the application of small test in a shake flask and under high-concentration grease content. The application has important significance for the development and application of high-efficiency microorganism treatment of high-salt kitchen waste or permeate, waste water grease.

Description

Technical Field

[0001] This invention belongs to the field of food waste grease degradation technology, specifically involving a salt-tolerant, thermophilic, and highly efficient bacterial strain that degrades food waste grease and its application. Background Technology

[0002] Food waste grease refers to the mixture of inedible animal and vegetable oils and various oils and waters generated by residents in daily life and production, as well as by factories, schools, and other activities such as food processing, catering services, and institutional catering. With my country's economic development and population growth, the amount of food waste has increased significantly in recent years. At the same time, the proportion of food waste in total urban waste is also constantly rising, often reaching around 70% in some large cities, and the grease content of food waste has increased accordingly.

[0003] The massive accumulation of food waste poses a significant challenge and pressure on urban environmental safety and waste management. However, while food waste is an environmental pollutant, it also contains enormous resource potential. Therefore, the rationality of food waste management directly determines its future use.

[0004] Among food waste, the treatment of grease is an extremely important aspect of food waste management. This is because food waste grease directly affects the operation of urban sewage systems. On the one hand, it hinders the reoxygenation and natural purification processes of water bodies, harming aquatic ecosystems and severely polluting the surrounding environment; on the other hand, it easily clogs sewage channels, causing other negative effects. Secondly, from a hygiene and epidemic prevention perspective, food waste grease, after being refined in open-air workshops in underground facilities to produce "gutter oil," cannot remove harmful components such as bacteria from the grease. Once it enters the market, it will seriously endanger people's health.

[0005] Analysis results show that the salt concentration of food waste in my country is between 2-6 g / L. However, with the continuous addition of food waste during the treatment process, grease and salt accumulate, leading to high concentrations of sodium. + Increased osmotic pressure in the degradation environment can affect the activity of microorganisms and interfere with their metabolism. The oil concentration in Chinese food waste is approximately 4%. High oil content hinders the intake of oxygen needed for microbial metabolism, thus affecting microbial growth and metabolism, and negatively impacting the rate of organic matter decomposition and the quality of degradation products. Furthermore, oil-containing organic fertilizers can cause soil degradation, directly affecting crop growth. Therefore, there is an urgent need to screen for thermophilic bacterial strains that are both salt-tolerant and capable of degrading oils for high-temperature treatment of food waste. Summary of the Invention

[0006] The purpose of this invention is to provide a salt-tolerant, wide pH-tolerant thermophilic strain for treating grease in kitchen waste, which can efficiently degrade grease in kitchen waste, thereby overcoming the shortcomings of the prior art.

[0007] The present invention first provides a Bacillus licheniformis SX-1, which was deposited on May 17, 2021, at the China General Microbiological Culture Collection Center, at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 22532.

[0008] The present invention also provides the application of this strain in the processing of oils.

[0009] The grease mentioned refers to the grease found in kitchen waste;

[0010] The present invention also provides a method for treating kitchen waste, which uses the screened Bacillus licheniformis strain SX-1 to treat kitchen waste;

[0011] One method of the method is to spray the fermentation broth of Bacillus licheniformis strain SX-1 into the kitchen waste to be treated;

[0012] In another aspect, the present invention provides an article for treating kitchen waste, the article containing live Bacillus licheniformis strain SX-1. Attached Figure Description

[0013] Figure 1 : Culture morphology diagram of the strain;

[0014] Figure 2 : Growth status of this strain under different salinity gradients;

[0015] Figure 3 : Growth status of this strain at different pH levels;

[0016] Figure 4 Standard curve of oil at absorbance of 225nm;

[0017] Figure 5 Comparison of lipid degradation effects between the strain and the blank control group after 48 hours;

[0018] Figure 6 A comparison of the changes in lipid degradation rate of this strain under different lipid concentrations;

[0019] Figure 7 Changes in the degradation rate of kitchen waste between the microbial agent group and the blank control group. Detailed Implementation

[0020] The following describes in detail, with reference to the embodiments, the strain screening, the effect of the present invention on environmental pressure tolerance, and its application effect in the small-scale degradation of grease in kitchen waste.

[0021] Example 1: Screening of thermophilic lipid-degrading strains

[0022] Main culture medium:

[0023] Selection medium for lipid-degrading bacteria (g / L): NaCl 5.0, potassium dihydrogen phosphate 0.3, magnesium sulfate heptahydrate 0.1, dipotassium hydrogen phosphate 1.5, ammonium sulfate 1.0, soybean oil 5ml, agar 20.0, pH=7.2~7.4.

[0024] Luria-Bertani (LB) medium (g / L): sodium chloride 10.0, tryptone 10.0, yeast extract 5.0, distilled water added to 1L, and pH adjusted to 7.2.

[0025] In this invention, unless otherwise specified, the culture medium is sterilized by high-pressure steam at 120°C for 20 minutes.

[0026] The specific experimental method is as follows:

[0027] Take 10g of soil sample from the kitchen waste dumping area of ​​the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, into a 250ml Erlenmeyer flask, add 90ml of sterile water, and shake thoroughly for 30min at 30℃ and 180r / min to obtain a suspension. Then, serially dilute to 10⁻⁶. -4 Choose 10 -2 -10 -4 Diluted samples were spread onto oil-based screening medium plates at different dilutions, with three plates for each dilution. Each plate contained 200 μL of diluted sample suspension. The plates were sealed and labeled, and then incubated at 50°C for 24–72 h. Single colonies of different types were picked from the different plates and isolated and purified using the three-zone streak method on the corresponding screening plates. Single colonies were selected and inoculated into LB liquid medium and cultured at 50°C for 24 h on a shaker at 180 rpm to obtain single bacterial suspensions. These suspensions were then stored at -80°C in 25% glycerol for later use.

[0028] Each bacterial culture was spotted onto its corresponding screening plate, with 3 drops of 10 μL culture evenly spotted onto each plate (3 replicates). After the culture was dried, the plates were inverted and incubated at 50°C for 72 hours. The degradation ability of the strains for various substances was preliminarily estimated by comparing the ratio of the diameter of the clear zone (D) to the diameter of the colony (d) (D / d). Strains with stronger degradation ability (higher ratio) were selected as the screening results. DNA was extracted from each strain obtained from the initial screening, and PCR amplification of 16S rRNA was performed using universal primers 27F and 1492R. Sequencing was then used to identify the genus.

[0029] The experimental results are as follows:

[0030] 1. Screening results of grease-degrading bacteria in kitchen waste

[0031] Seven single bacteria were initially isolated from soil samples using screening culture medium plates for lipid-degrading bacteria. The degradation ability of these seven strains was then tested by measuring the diameter of the hydrolysis zone and the colony diameter. Five strains with stronger degradation ability were identified by comparing the ratios. The degradation results are shown in Table 1.

[0032] Table 1: Initial Screening Results of Oil-Degrading Bacteria

[0033]

[0034] 2. Results of strain identification

[0035] Based on sequencing results comparison and comprehensive analysis of the morphological characteristics of the strains, the genus of the screened strains was determined. The strain Y1 with the best degradation effect was identified as *Bacillus licheniformis*, and named *Bacillus licheniformis SX-1*. Its colony culture morphology is as follows: Figure 1 As shown.

[0036] Example 2: Effects of salinity and pH on thermophilic lipid-degrading bacterium Bacillus licheniformis SX-1 strain

[0037] Main culture medium:

[0038] Luria-Bertani (LB) medium (g / L): sodium chloride 10.0, tryptone 10.0, yeast extract 5.0, distilled water added to 1L, and pH adjusted to 7.2.

[0039] In this invention, unless otherwise specified, the culture medium is sterilized by high-pressure steam at 120°C for 20 minutes.

[0040] The specific experimental method is as follows:

[0041] Salinity tolerance test: The target strain was inoculated at a 10% inoculum into LB liquid medium with NaCl concentrations of 10, 50, 100, 150, and 200 g / L, respectively. Three replicates were set up for each strain. The cultures were incubated at 50°C and 180 rpm for 24 h on a shaker, and the OD value of the bacterial culture was measured. 600 The value is used to determine the growth status of the target strain in culture media with different salt concentration gradients.

[0042] Acid-base tolerance test: The target strain was inoculated at a 10% inoculum in LB liquid medium at pH 3, 5, 9, and 11, with three replicates. The cultures were incubated at 50°C and 180 rpm for 24 h on a shaker, and the OD values ​​of the bacterial solutions were measured. 600 The pH value is used to determine the tolerance of the target strain to different pH levels.

[0043] Statistical analysis was performed using SPSS 20.0 statistical software. Quantitative data are expressed as mean ± standard deviation (x ± s).

[0044] The experimental results are as follows:

[0045] 1. Growth status of Bacillus licheniformis SX-1 under different salinity gradients

[0046] During the processing of food waste, inorganic salts accumulate as organic matter is consumed, leading to a gradual increase in salinity. Therefore, strains that degrade food waste have certain salt tolerance requirements. The salinity tolerance of the selected grease-degrading bacterium, *Bacillus licheniformis* SX-1, was tested using OD values. 600 As an indicator of bacterial growth, the tolerance of the strain to high salinity environments is tested. For example... Figure 2 As shown, strain SX-1 is tolerant of high salinity environments. At a NaCl concentration of 100 g / L, the strain exhibits significant growth, with a 24-hour OD value... 600 The value is 0.624. Furthermore, it is capable of surviving in environments with salt concentrations as high as 150 g / L, and its 24-hour OD value is [missing value]. 600 The value is 0.283.

[0047] 2. Growth status of Bacillus licheniformis SX-1 at different pH levels

[0048] During the fermentation and degradation of food waste, various reaction products and microbial metabolites accumulate, affecting the pH of the system. For example, substances such as fatty acids and lactic acid produced during fermentation lower the pH. As products accumulate, the pH gradually decreases. Although the pH of the system is monitored regularly during composting to maintain it within a certain range, abnormal situations can still occur where the pH exceeds the range. Therefore, bacteria capable of surviving within a wide pH range are needed to ensure that even if abnormal situations occur, there is still a chance for recovery.

[0049] The lipid-degrading bacterium Bacillus licheniformis SX-1, screened in this invention, was cultured in LB liquid medium with different pH values ​​for 24 h, and the OD was calculated. 600 Determine the strain's tolerance to pH levels.

[0050] like Figure 3 As shown, since the pH changes during the food waste treatment process are basically between 5 and 8, this bacterium has a certain tolerance to an environment with a pH of 9. The 24-hour OD... 600 The value was 0.323. The strain also showed signs of growth at pH 3 and 5, with a 24-hour OD value of [missing value]. 600 The mean values ​​are 0.182 and 0.129, respectively.

[0051] Example 3: Small-scale application effect of thermophilic lipid-degrading bacterium Bacillus licheniformis SX-1 under different lipid concentration gradients

[0052] Main culture medium:

[0053] Oil-degrading bacteria rescreening medium (g / L): peptone 1.0, ammonium nitrate 0.2, dipotassium hydrogen phosphate 0.5, potassium dihydrogen phosphate 0.5, magnesium sulfate heptahydrate 0.1, soybean oil 3.0 ml, pH = 7.2-7.4.

[0054] In this invention, unless otherwise specified, the culture medium is sterilized by high-pressure steam at 120°C for 20 minutes.

[0055] Specific experimental methods:

[0056] The selected oil-degrading bacterial strain Bacillus licheniformis SX-1 was inoculated into a small-scale oil-degrading shake flask culture medium as the bacterial agent treatment group, and a CK group was set up as a blank control. The initial oil concentration was 3 g / L. Both treatment groups were placed in the same environment and cultured at 180 r / min and 50 ℃ for 48 h. The change in oil content was measured.

[0057] Oil degradation rate = (CK soybean oil concentration - treated soybean oil concentration) / CK soybean oil concentration

[0058] Preparation of the standard curve for kitchen waste oil: Research found that the maximum absorption peak of kitchen waste oil is at 225 nm. Therefore, seven 50 ml volumetric flasks were used to prepare solutions of soybean oil at concentrations of 0.1, 0.2, 0.4, 0.5, 0.6, 0.8, and 1.0 mg / ml using petroleum ether as the solvent. The OD values ​​were then measured. 225 A standard curve was plotted with soybean oil concentration on the x-axis and absorbance values ​​on the y-axis. Three replicate samples were used for each concentration. Based on this method, a standard curve was obtained with standard oil concentration on the x-axis and absorbance OD values ​​on the y-axis. 225 Using the vertical axis as the ordinate, a standard curve for the grease in kitchen waste is plotted.

[0059] Determination of oil content in culture medium: Transfer the culture medium from the shake flask to a 250ml separatory funnel, add 1ml of concentrated hydrochloric acid for acidification, then add approximately 2% (m / v) NaCl. Wash the shake flask with 5ml of petroleum ether, pour the washings into the separatory funnel, and repeat the washing once with 5ml of petroleum ether. Shake the separatory funnel thoroughly and allow it to separate into layers. After separation, transfer the aqueous layer to an Erlenmeyer flask, and transfer the petroleum ether layer to a 100ml ceramic crucible and cover it to reduce petroleum ether evaporation (or transfer it to a small beaker and cover it with a petri dish). Transfer the aqueous layer from the Erlenmeyer flask back to the separatory funnel, and repeat the extraction once with 10ml of petroleum ether. Add an appropriate amount of anhydrous sodium sulfate to the recovered petroleum ether, stir, cover, and let stand for at least half an hour to ensure complete dehydration. Then transfer the petroleum ether to a 25ml volumetric flask, add petroleum ether dropwise to the mark, mix thoroughly, and measure the OD. 225 The oil content of the culture medium is determined based on the standard curve.

[0060] Degradation efficiency at different initial oil contents: Oil degradation culture media with initial soybean oil concentrations of 3, 9, 15, 30, and 45 g / L were prepared respectively. The target strain was inoculated into the oil degradation culture media at an inoculum of 10% and cultured in a shaker at 180 r / min at 50℃ for 48 h. The soybean oil content in the culture medium was measured and the degradation rate was calculated.

[0061] Statistical analysis was performed using SPSS 20.0 statistical software. Quantitative data are expressed as mean ± standard deviation (x ± s).

[0062] Experimental results:

[0063] 1. Standard curves for oils at different concentration gradients

[0064] Based on the prepared petroleum ether solutions of soybean oil at different concentrations, the corresponding absorbance was obtained. A standard curve for soybean oil was plotted with soybean oil concentration as the x-axis and absorbance as the y-axis. To accurately reflect the relationship between the corresponding sample concentration and absorbance value, the least squares method was used to perform regression analysis on the experimental data, obtaining a working curve with the smallest error for each data point, thus making the measurement results closer to the true value. Based on the obtained data, the coefficients and constant terms of the regression equation were calculated, and the regression equation was established. The resulting working curve is shown below. Figure 4 .

[0065] pass Figure 4 The standard curve equation for the absorbance of kitchen waste grease at 225 nm can be obtained as Y = 0.58893X + 0.00548, R 2 =0.99375, the linear relationship is very good.

[0066] 2. Comparison of the morphological characteristics of lipid degradation

[0067] During the degradation of kitchen waste grease, observations (after a 30-minute settling period) revealed that after 48 hours of treatment, the liquid phase in the group with added microbial agents was more homogeneous, with no obvious stratification. However, the control group (CK) showed significant oil-water stratification. Comparison of different treatment groups suggests that the degradation results with added microbial agents are excellent. After a 30-minute settling period, the oil layer in the microbial agent-treated group was dispersed and had a more uniform composition, indicating a better treatment effect. Figure 5 ).

[0068] 3. The degradation rate of this oil-degrading bacteria in a shake-flask small-scale test at an oil concentration of 3 g / L.

[0069] Based on the standard curve of the petroleum ether solution of the oil, a shake-flask test was conducted on the strain Bacillus licheniformis SX-1 obtained from the preliminary screening. The degradation rate of the oil by this strain after 48 hours was determined to quantitatively assess its degradation ability. The preliminary calculation showed that its degradation rate of soybean oil after 48 hours was 85.3%.

[0070] 4. The degradation rate of this oil-degrading bacteria at different initial oil contents

[0071] The effectiveness of microbial degradation is influenced to some extent by factors such as the oil content in kitchen waste. The oil concentration in Chinese kitchen waste is approximately 1%-3%. High oil content hinders the intake of oxygen required for microbial metabolism, thus affecting microbial growth and metabolism, and negatively impacting the rate of organic matter decomposition and compost quality. Furthermore, organic fertilizer containing oil can cause soil degradation, directly affecting crop growth. Therefore, the screened highly efficient oil-degrading bacterium *Bacillus licheniformis* SX-1 was cultured in media with different soybean oil concentrations. After 48 hours, the soybean oil content was measured, and the soybean oil degradation rate was calculated. The degradation rates of the strain in media with different soybean oil concentrations are as follows: Figure 6 As shown in the figure. The results showed that the degradation rate of the oil-degrading bacterium Bacillus licheniformis SX-1 gradually decreased with the increase of the initial oil content. Its degradation rate was 49.9% when the initial oil content was 30 g / L, and it still maintained a degradation rate of 48.4% when the concentration was as high as 45 g / L.

[0072] Example 4: Application of the thermophilic lipophilic bacterium Bacillus licheniformis SX-1 in a small-scale food waste treatment device

[0073] Specific experimental methods:

[0074] The highly efficient degrading bacterium Bacillus licheniformis SX-1, selected through screening, was cultured in LB medium at 50℃ and 180 rpm for 24 hours, and the bacterial solution was diluted to 10. 8 250 mL of CFU bacterial solution was thoroughly mixed with sawdust and bran in a 1:3 ratio in a device. Then, food waste (salt concentration: 5%) prepared according to international high-salt, high-fat dietary standards was added. The high-salt, high-fat food waste consisted of: 564 g rice, 156 g fatty meat, 388.5 g cabbage, 0.54 g spice powder, 125 g salt, 4.8 g sugar, 100 mL oil, and approximately 2500 g of distilled water. The mixture was weighed every eight hours and run for 72 hours. A control group (CK) was also set up, with the bacterial solution replaced by the same amount of sterile water, following the same procedure as above.

[0075] The actual degradation rate was calculated using the weight loss method:

[0076] Total weight loss rate (%) = [C - (BA)] / C × 100%

[0077] A: Initial total weight

[0078] B: Total weight at 0, 8, 16… and 72 hours respectively

[0079] C: Amount of high-salt and high-oil kitchen waste added

[0080] Experimental results:

[0081] The weight loss rate was calculated by measuring the residual weight of the food waste during the entire degradation process in a small-scale test every eight hours. The weight loss rate (…) Figure 7 It is evident that the total mass reduction rate of food waste is one of the most important indicators in the biodegradation process of high-salt and oily food waste. Therefore, the total weight loss rate was chosen as the indicator for detecting the degradation rate of the newly constructed microbial agent. Figure 6 The results showed that the total weight loss rate of the new agent-inoculated food waste biodegradation system was higher than that of the system without functional strains. In the initial 8 hours, the difference in total weight loss rate between the two systems was small, but this difference increased significantly from 8 to 72 hours. Ultimately, the total weight loss rate with the addition of the highly efficient degrading bacterium *Bacillus licheniformis* SX-1 was approximately 80.6%, while the control group had a total weight loss rate of approximately 60.5%. Most importantly, the biodegradation system with the added bacteria operated stably, while the control group continuously released an unpleasant acidic odor throughout the food waste biodegradation process. It is well known that distributed and on-site food waste biodegradation equipment is generally located near residential areas or workplaces, and odor is a primary concern in actual operation. The weight loss rate also demonstrates that the addition of the bacteria promotes the degradation of food waste.

[0082] In summary, this strain exhibits excellent oil degradation ability, salt tolerance, and stress resistance under wide pH conditions. Most importantly, it maintains a high oil degradation rate even at oil concentrations as high as 45 g / L. Furthermore, in the final on-site pilot application, the degradation effect shows that the oil-degrading bacterium *Bacillus licheniformis* SX-1 of this invention can significantly improve the degradation rate of high-salt, high-oil food waste, achieving a total weight loss rate of 80.6%, demonstrating significant application and promotion value, especially for the degradation of high-salt, high-oil food waste.

Claims

1. A Bacillus licheniformis, characterized in that, The Bacillus licheniformis ( Bacillus licheniformis The accession number for this work is CGMCC No. 22532.

2. The application of Bacillus licheniformis as described in claim 1 in the processing of oils.

3. The application as described in claim 2, characterized in that, The grease mentioned is grease from kitchen waste.

4. A method for treating kitchen waste, characterized in that, The method described herein uses Bacillus licheniformis as described in claim 1 to treat kitchen waste.

5. The method as described in claim 4, characterized in that, The method involves spraying the fermentation liquid of Bacillus licheniformis as described in claim 1 onto the kitchen waste to be treated.

6. An article for processing kitchen waste, characterized in that, The product contains live Bacillus licheniformis as described in claim 1.

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