CN113388552B - Thermophilic cellulose degradation strain and application thereof - Google Patents

Abstract

The present invention specifically relates to a salt-tolerant thermophilic high-efficiency bacterial strain Aneurinibacillus thermoaerophilus SX‑2 capable of degrading cellulose and oil in solid waste at the same time. The deposit number of the Microbiology Center is CGMCC No.22533. The novelty of this strain is that it has good growth characteristics under thermophilic (50°C) and acidic (pH5) conditions, and it also has the ability to degrade cellulose and oil. Most noteworthy is that in the shake flask test application, its ability to degrade oil increases with the increase of oil content. When the initial oil concentration was 30g/L, the degradation rate was as high as 69.6%. The microbial strain involved in the present invention can not only efficiently degrade cellulose, but also degrade oil, and has very important application value for influencing current carbon emission reduction policies and realizing harmless treatment of waste.

Description

A Thermophilic Cellulose Degrading Bacteria Strain and Its Application

technical field

The invention belongs to the technical field of solid waste degradation treatment, and specifically relates to a salt-tolerant thermophilic bacterial strain capable of simultaneously degrading cellulose and oil in solid waste and its application.

Background technique

With the continuous development of urbanization and the rapid growth of population in my country, the volume of municipal solid waste has also increased significantly. In addition, the proportion of urban green plants, kitchen waste and other wastes in the total urban solid waste content is also increasing. Therefore, the degradation of cellulose and oil in urban solid waste has become the key to waste treatment.

The accumulation of urban green plants and kitchen waste is a huge challenge and pressure for the development of the city, and has a great impact on human health. The strict management of this kind of solid waste also shows the urgency and necessity of the country to deal with the problem of solid waste. Therefore, as these problems become increasingly prominent, there is an urgent need for environmentally friendly technologies for efficiently degrading such solid wastes.

Contents of the invention

The purpose of the present invention is to provide a thermophilic cellulose-degrading bacterial strain and its application. The provided bacterial strain has the ability to efficiently degrade cellulose and kitchen waste oil, so as to make up for the deficiencies in the prior art.

The present invention firstly provides a thermophilic bacillus Aneurinibacillusthermoaerophilus SX-2, which was preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on May 17, 2021, and the preservation number is CGMCC No.22533;

The thermophilic aerophilic Bacillus thiamine SX-2 strain provided by the present invention is used to degrade oil;

The thermophilic Bacillus aerothiamine SX-2 strain provided by the present invention can also be used to degrade and treat cellulose;

The present invention also provides a fungus product for degrading oil and/or cellulose, which contains the thermophilic aerophilic Bacillus thiaminelolyticus SX-2 strain.

The novelty of the bacterial strain provided by the present invention is that it has the characteristic of simultaneously degrading cellulose and oil, and can withstand high temperature, and still has a relatively high level of oil degradation efficiency in a high-oil environment.

Description of drawings

Figure 1: Screening results of cellulose-degrading bacteria;

Figure 2: Culture morphology of the screened SX-2;

Figure 3: Growth status diagram of strain SX-2 under different salinity gradients;

Fig. 4: The graph of the growth status of bacterial strain SX-2 under different pH;

Figure 5: Comparison of the changes in the oil degradation rate of the strain SX-2 under different oil concentrations.

Detailed ways

The strain screening of the present invention, the effect of tolerance to environmental pressure and its application effect in degrading cellulose and oil will be described in detail below in conjunction with the examples.

Example 1: Screening of thermophilic cellulose degrading strains

Main medium:

Cellulose-degrading bacteria medium (CMC, g/L): CMC-Na 10.0, ammonium sulfate 4.0, magnesium sulfate heptahydrate 0.5, potassium dihydrogen phosphate 2.0, peptone 1.0, agar 20.0, pH=6.5.

Congo red cellulose medium: purchased directly and reserved.

Luria-Bertani (LB) medium (g/L): sodium chloride 10.0, tryptone 10.0, yeast extract 5.0, add distilled water to 1 L, and adjust its pH to 7.2.

Oil-degrading bacteria screening medium (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.

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

Specific experimental methods:

Take 10g of soil samples from the food waste dumping area of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences into a 250ml Erlenmeyer flask, add 90ml of sterile water in a shaker at 180r/min at 30°C, and shake fully for 30min to obtain a suspension. Gradually dilute to 10 ^-7 , choose a dilution of 10 ^-5 -10 ^-7 and spread them on LB solid medium plates, CMC solid medium plates and Congo red cellulose solid medium plates respectively, 3 for each gradient Plate, apply 200 μL of diluted sample suspension to each plate, seal and label the plate and place it in a 50°C incubator for 48 hours, pick different types of single colonies from different plates and divide them into three sections on the corresponding screening plates Separation and purification were carried out by the line method. A single colony was selected and inoculated in LB liquid medium, cultured on a shaker at 180 r/min at 50°C for 24 hours to obtain a single bacterial liquid, and stored in 25% glycerol at -80°C for future use.

Screening of cellulose-degrading bacteria: Culture isolated single bacteria in LB medium and shake flasks to obtain bacterial liquid, take 20 μL of bacterial liquid to fully wet the filter paper placed on the Congo red cellulose medium plate, place 3 on each plate filter paper sheets, set as 3 in parallel. Seal the plate and place it in an incubator at 50°C for 1 to 2 hours, then invert it after the filter paper is dry. After culturing for 72 hours, the ability of the strain to degrade cellulose was preliminarily judged according to the plate hydrolysis circle.

Screening of oil-degrading bacteria: spot each single bacterium on its corresponding screening plate, and evenly spot 3 drops of 10 μL bacterial solution on each plate, making 3 parallels. ℃ incubator for 72 hours, by comparing the ratio (D/d) of the diameter of the transparent circle (D) to the diameter of the colony (d), initially estimate the degradability of the strain to various substances, and select the strain with a stronger degradation ability, that is, a larger ratio to filter the results.

Strain identification: identify the species of the purified strains, extract DNA from the screened strains, use bacterial 16SrRNA for PCR amplification, perform agarose gel electrophoresis on the PCR stock solution, and send the stock solution with bands to the sequencing company for sequencing To obtain the 16S rRNA sequence of the target strain, carry out similarity comparison in the BLAST database and combine the morphological characteristics, physiological and biochemical characteristics of the strain to identify the genus of the strain.

Experimental results:

1. Screening results of cellulose-degrading bacteria

Cellulose-degrading bacteria are single bacteria isolated from soil samples using different medium plates. A total of 8 single strains were isolated. The single bacteria were inoculated into Congo red cellulose medium for primary screening. According to the red precipitate around the colony The color depth of the circle (Figure 1) can preliminarily judge the degradation ability of the strain, and the degradation results are shown in Table 1.

Table 1: Results of primary screening of oil-degrading bacteria

2. Screening results of oil-degrading bacteria

Through the screening medium plate of oil-degrading bacteria, 7 strains of single bacteria were preliminarily isolated from the soil sample, and the degradation ability of the 7 strains was tested, the diameter of the hydrolysis circle and the diameter of the colony were measured, and the comparison of the ratio showed that 5 strains were stronger. strains, the degradation results are shown in Table 2.

Table 2: Results of primary screening of oil-degrading bacteria

3. Identification results of strains

According to the comparison of the sequencing results and the comprehensive analysis of the morphological characteristics of the strains (Figure 2), the genus of the screened strains was determined. The strain CG-3 was identified as Aneurinibacillus thermoaerophilus and named: Aneurinibacillus thermoaerophilus SX-2.

The screened Aneurinibacillus thermoaerophilus SX-2 was deposited in the General Microorganism Center of China Committee for the Collection of Microorganisms on May 17, 2021, with the preservation number CGMCC No.22533.

Example 2: Effects of salinity and pH on thermophilic cellulose degrading bacteria Aneurinibacillus thermoaerophilusSX-2

Main medium:

Luria-Bertani (LB) medium (g/L): sodium chloride 10.0, tryptone 10.0, yeast extract 5.0, add distilled water to 1 L, and adjust its pH to 7.2.

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

The specific experimental method is as follows:

Tolerance test of salinity: Inoculate the target strain with 10% inoculation amount in LB liquid medium with NaCl concentration of 10, 50, 100, 150, 200g/L respectively, set 3 parallels for each strain, and inoculate at 50°C Cultivate on a shaker at 180r/min for 24 hours, measure the OD ₆₀₀ value of the bacterial solution, and then judge the growth status of the target strain in the medium with different salt concentration gradients.

Acid-base tolerance test: Inoculate the target strain with 10% inoculum amount in LB liquid medium with pH of 3, 5, 9, and 11 respectively, set up 3 parallels, and culture on a shaker at 50°C at 180r/min After 24 hours, measure the OD ₆₀₀ value of the bacterial solution, and then judge the tolerance of the target strain to different pH.

Statistical processing All statistical analyzes were performed using SPSS 20.0 statistical software package. Measurement data are expressed as mean ± standard deviation (x ± s).

Experimental results:

1. Growth status of Aneurinibacillus thermoaerophilus SX-2 under different salinity gradients

The cellulose-degrading bacteria Aneurinibacillus thermoaerophilus SX-2 screened above was tested for tolerance to salinity, and the OD ₆₀₀ was used as an indicator of strain growth to test the tolerance of the strain to a high-salinity environment. The specific results are shown in Figure 3. When the NaCl concentration is 10g/L, the strain grows well, and its OD ₆₀₀ value in 24 hours can reach 1.428.

2. Growth status of Aneurinibacillus thermoaerophilus SX-2 at different pH

The cellulose-degrading bacteria Aneurinibacillus thermoaerophilus SX-2 screened in the above experiments were cultured in LB liquid medium with different pH for 24 hours, and the tolerance of the strain to pH was determined according to OD ₆₀₀ . As shown in Figure 4. Through this study, it was found that this strain can grow well in an environment with a pH of 5, and the growth condition is better than that at a pH of 7. Under the conditions of pH 5 and 7, the 24-hour OD ₆₀₀ values were 1.650 and 1.428, respectively. When the pH values were 3 and 5, the strains also showed signs of growth, but the growth conditions were not good, and the average OD ₆₀₀ values in 24 hours were 0.297 and 0.287, respectively.

Example 3: The application effect of the thermophilic cellulose-degrading bacteria Aneurinibacillus thermoaerophilus SX-2 on the degradation of cellulose and oil

Main medium:

Filter paper medium (g/L): ammonium sulfate 4.0, magnesium sulfate heptahydrate 0.5, potassium dihydrogen phosphate 2.0, peptone 1.0, filter paper 1%, pH=6.5-7.0.

Re-screening medium for oil-degrading bacteria (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 3.0ml, pH=7.2～7.4.

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

The specific experimental method is as follows:

Small test of cellulose-degrading bacteria: Inoculate the bacterial liquid Aneurinibacillus thermoaerophilus SX-2 into the filter paper medium, calculate the degradation rate of the strain on the filter paper and quantitatively measure the degradation ability of the strain, so as to select efficient degrading bacteria. Inoculate to filter paper medium for shaking flask culture, set up three parallels and one group of control group CK without inoculation, and measure the change of filter paper weight after culturing at 180r/min at 50°C for 3 days under exactly the same conditions.

Filter paper degradation rate = (CK filter paper weight - treated filter paper weight) / CK filter paper weight

Determination of filter paper quality: spread the gauze over the mouth of a 1L beaker, pour the culture solution through the gauze into the beaker, the filter paper debris is filtered and left on the gauze, wash the Erlenmeyer flask with distilled water for 1 or 2 times, and the washing liquid remains on the gauze. Slowly pour it on the filter paper scraps on the filter paper scraps, wash off the bacterial solution of the culture solution remaining in the filter paper scraps, and the filter residue is basically white. Gently squeeze out the remaining moisture from the filter paper residue, place it in a petri dish and vacuum-dry it in a vacuum drying oven at 105°C until the mass does not change, and measure the mass of the remaining filter paper.

Small test of oil degradation effect: Inoculate the screened strain Aneurinibacillus thermoaerophilus SX-2 into the oil degradation small test shake flask medium as the bacterial agent treatment group, and set a group of CK as a blank control, the initial oil concentration is 3g/L, At the same time, the two treatment groups were placed in the same environment at 180r/min, cultured at 50°C, and the change of oil content was measured for 48h.

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

Preparation of the standard curve of food waste oil: the research found that the maximum absorption peak of food waste oil is 225nm, so take seven 50ml volumetric flasks, use petroleum ether as solvent, and prepare 0.1, 0.2, 0.4, 0.5, 0.6, 0.8, The OD ₂₂₅ value of 1.0 mg/ml soybean oil solution of 7 concentrations was measured, and the standard curve was drawn with the soybean oil concentration as the abscissa and the absorbance value as the ordinate. There are three repeated samples for each concentration. According to this method, the standard oil concentration is taken as the abscissa and the absorbance OD ₂₂₅ is taken as the ordinate to make a standard curve for kitchen waste oil. Our research group has previously obtained a standard curve: Y=0.58893X+0.00548, R ² =0.99375.

Determination of oil content in the culture medium: transfer the shake flask culture medium into a 250ml separatory funnel, add 1ml concentrated hydrochloric acid to acidify, then add about 2% (m/v) NaCl, wash the shake flask with 5ml petroleum ether, and pour the lotion into In the separatory funnel, wash with 5ml petroleum ether repeatedly once. Shake the separatory funnel well and let it stand to make layers. After stratification, the water layer was moved to the Erlenmeyer flask, and the petroleum ether layer was moved into a 100ml ceramic crucible and covered with a lid to reduce the volatilization of petroleum ether (or moved into a small beaker and covered with a petri dish). The water layer in the Erlenmeyer flask was transferred to the separatory funnel again, and 10ml of petroleum ether was repeatedly extracted once. Add an appropriate amount of anhydrous sodium sulfate to the recovered petroleum ether, cover the lid after stirring, and let it stand for more than half an hour to ensure sufficient dehydration. Then transfer the petroleum ether to a 25ml volumetric flask, add petroleum ether dropwise to the volume, mix well, measure the OD ₂₂₅ value, and obtain the oil content of the medium according to the standard curve.

Degradation efficiency of different initial oil content: prepare oil degradation medium with soybean oil initial concentration of 3, 9, 15, 30, 45g/L respectively, inoculate the target strain in the oil degradation medium with 10% inoculation amount, and inoculate the oil degradation medium at 50°C The shaker cultured at 180r/min for 48h, the content of soybean oil in the medium was measured and the degradation rate was calculated.

Statistical processing All statistical analyzes were performed using SPSS 20.0 statistical software package. Measurement data are expressed as mean ± standard deviation (x ± s).

The experimental results are as follows:

1. The cellulose degrading bacteria strain degraded the filter paper small test

Inoculate the filter paper medium to measure the degradation rate of the filter paper, and quantitatively judge the strain's ability to degrade cellulose. According to the experimental results, compared with CK, the strain Aneurinibacillus thermoaerophilus SX-2 was cultivated on a shaker at 50°C and 180r/min for 3 days, and the remaining filter paper was collected, dried and weighed to calculate the degradation rate of about 32.9%. .

2. The strain was tested in shake flasks at a concentration of 3g/L oil

According to the standard curve of the oil petroleum ether solution drawn, the bacterial strain Aneurinibacillus thermoaerophilus SX-2 obtained by the preliminary screening was carried out in a shake flask test, and the degradation rate of the bacterial strain to the oil in the small test was measured in 48 hours, and the degradation ability of the bacterial strain was quantitatively judged. Preliminary calculation obtained its degradation rate of soybean oil in 48 hours: 53.4%.

3. The degradation rate of the degrading bacteria at different initial oil contents

Strain Aneurinibacillus thermoaerophilus SX-2 was cultured in media with different soybean oil concentrations, and the soybean oil content was measured after 48 hours to calculate the degradation rate of soybean oil. The degradation rates of the strains in medium with different soybean oil concentrations are shown in Figure 5. It is worth noting that the results show that the degradation rate of the strain Aneurinibacillus thermoaerophilus SX-2 is within the concentration range of the initial oil content of 3-30g/L, and its degradation rate increases with the increase of the oil concentration. When it is 30g/L, its degradation rate is as high as 69.6%. Even when the concentration is 45g/L, the degradation rate of soybean oil still maintains a degradation rate effect of 22.6%.

In summary, the strain Aneurinibacillus thermoaerophilus SX-2 of the present invention has not only cellulose degrading ability but also excellent oil degrading ability, and has good growth ability in acidic environment (pH5). With the increase of oil concentration, the degradation rate of this strain increases at any time, and when the oil content is as high as 30g/L, it still maintains a high oil degradation rate. The above results show that the strain Aneurinibacillus thermoaerophilus SX-2 of the present invention has an excellent effect in degrading cellulose and kitchen fat, and has good application and promotion value.

Claims (4)

1. A thermophilic aerophilic Bacillus thiaminolyticum, characterized in that the preservation number of the strain is CGMCC No.22533. 2. The application of the thermophilic aerothiamine bacillus described in claim 1 in the degradation treatment of oil. 3. The application of the thermophilic aerothiamine bacillus described in claim 1 in degrading and processing cellulose. 4. A fungal product for degrading oil and/or cellulose, characterized in that said product comprises the thermophilic aerophilic Bacillus thiaminolyticus according to claim 1.

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