Prussella sp. And application thereof

Abstract

The invention relates to a Prussella sp. And application thereof, and belongs to the technical field of microorganisms. The method comprises the following steps: firstly, screening and separating a strain of Plesteria sp. RL-1 from intestinal tracts of worker termites of saw termites, and preserving the Plesteria sp. RL-1 in China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.32481; the Prussella reevesii RL-1 can grow on an inorganic salt culture medium with alkali lignin as a unique carbon source, and has the capacity of degrading alkali lignin and decolorizing dye; experiments prove that the Princisteria RL-1 can rapidly and efficiently degrade alkali lignin, the degradation rate reaches up to 88% or above, the Princisteria RL-1 can adapt to wide temperature and pH ranges and can directly degrade crop straw such as rice straw, obtained metabolites can promote plant growth, and the Princisteria RL-1 can be applied to resource application of agricultural waste and has a wide application prospect. And wastewater treatment in industries such as papermaking and printing and dyeing.

Description

Technical Field

[0001] The invention relates to Priesteria and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Agricultural waste is one of the most abundant renewable resources in the world, but most of it has not been fully and rationally utilized, which not only causes a waste of resources but also brings serious environmental pollution. Among them, lignin has a high molecular weight dense structure, covalently binds to hemicellulose through side chains and tightly covers cellulose, which greatly reduces the hydrolysis efficiency of macromolecules such as cellulose and is the main obstacle to the bioconversion of lignocellulose. In addition, the pulp and paper industry produces a large amount of lignin by-products, and most of the lignin is still directly discharged with wastewater in the form of "black liquor". Therefore, efficient degradation and conversion of lignin is of great significance for improving resource utilization, reducing environmental pollution, and promoting economic development.

[0003] The degradation methods of lignin mainly include physical and chemical methods and biological treatment. Although physical and chemical methods such as high temperature, radiation, and organic solvents can achieve the degradation of lignin, they have high requirements for equipment, high cost, and are prone to produce secondary pollutants. The biological method of using microorganisms to degrade lignin does not require high temperature and high pressure conditions, and has the advantages of being green, environmentally friendly, and easy to operate. At present, the most thorough research on the biodegradation of lignin is fungi (such as white rot fungi and brown rot fungi), but the application of fungi in lignin degradation is limited by the long pretreatment cycle. Compared with fungi, bacteria have the advantages of rapid growth, strong adaptability, and easy genetic manipulation, and their potential in lignin degradation has gradually attracted attention.

[0004] Bacteria that have been reported to be able to degrade lignin include Pseudomonas, Klebsiella, Rhodococcus, Streptomyces, Enterobacter, Sphingomonas, Arthrobacter and Bacillus. However, the degradation efficiency of lignin by the strains currently obtained is relatively low, among which the lignin degradation rate of most (90%) strains is less than 50%, and other carbon sources (such as glucose) need to be added to promote growth. The ability to adapt to the environment is also relatively weak, for example: the growth pH is generally between 6.0 and 7.5. Therefore, it is urgent to find a microbial strain with strong adaptability to pH and temperature for degrading lignin, which is of great significance to the resource utilization of solid waste and the treatment of wastewater. Summary of the invention

[0005] In view of some problems existing in the prior art, the present invention provides a Priesteria and application thereof, which provide high-quality resources for the resource utilization of solid wastes and the application of microorganisms in industrial wastewater treatment.

[0006] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0007] The present invention provides a Priestia sp., which is classified and named as Priestia sp. RL-1, has a preservation number of CGMCC No. 32481, and is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on November 4, 2024.

[0008] Wherein, the Priesteria RL-1 is derived from the saw-like termite.

[0009] Furthermore, the Priesteria RL-1 was isolated from the intestines of wood-eating saw-saw termites collected from the tropical rain forest of Xishuangbanna, and has the following morphological and physiological and biochemical characteristics: using conventional bacterial physiological and biochemical identification methods and microscopic observation, the bacteria are Gram-positive rod-shaped bacteria, aerobic, and oxidase-positive.

[0010] The present invention also provides a product containing the Priesteria.

[0011] The sequence information of the 16S rRNA fragment of Priesteria RL-1 of the present invention is shown in SEQ ID No: 1.

[0012] The present invention also provides a product containing the Priesteria.

[0013] The present invention also provides the use of the Priesteria or the product in agricultural waste biodegradation and wastewater treatment.

[0014] Furthermore, the application includes lignin degradation, and the agricultural waste includes crop straw, corn straw, rice straw or wheat straw.

[0015] Furthermore, the application includes decolorization or degradation of dyes; wherein the dyes include: basic red, reactive blue, aniline blue or naphthol green.

[0016] The present invention also provides the use of the Priesteria or the product in promoting plant seed germination and plant growth.

[0017] Furthermore, the plant includes rice.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The Priestia sp. RL-1 provided by the present invention has a highly efficient degradation effect on lignin. Compared with other bacteria that can degrade lignin (Pseudomonas, Klebsiella, Rhodococcus, Streptomyces and Enterobater, etc.), the bacterium has a fast growth rate and high degradation efficiency. The degradation rate of lignin by most other bacteria is less than 50% after more than 168 hours, while the degradation rate of lignin by the bacterium can reach 49.6% in 72 hours, and the degradation rate of lignin in 168 hours is as high as more than 88.7%. In addition, the culture medium is simple in composition and has strong environmental adaptability, and can grow in the range of pH 4.0 to 12.0 and temperature 15 to 40°C.

[0020] (2) The present invention also provides the use of the Priestia sp. RL-1 in degrading crop straw and dye pollutants; the Priestia sp. RL-1 of the present invention not only has a degradation rate of 20.5% for rice straw, 22.9% for wheat straw, and 18.2% for corn straw; it also has a good degradation effect on the dyes of basic red, reactive blue, aniline blue and naphthol green, and has a strong broad-spectrum property.

[0021] (3) The metabolites of crop straw degradation by Priesteria RL-1 provided by the present invention can promote the germination rate of rice seeds to reach 80%, which is significantly higher than that of the group treated with clean water; at the same time, compared with the group treated with clean water, the rice plants increased by 63.3% after being treated with the metabolites of crop straw degradation by Priesteria RL-1.

[0022] The Priesteria RL-1 provided by the present invention can be applied to solid waste resource utilization and environmental protection fields such as papermaking and textile wastewater treatment, and its high-efficiency degradation performance can be used to solve the problem that lignin-related biomass is difficult to degrade in industry and agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a scanning electron micrograph of Priestia sp.RL-1 strain.

[0024] Figure 2 This is the phylogenetic tree diagram of Priestia sp.RL-1 strain constructed based on the 16S r RNA gene.

[0025] Figure 3 This is a graph showing the degradation efficiency of Priestia sp.RL-1 strain on lignin.

[0026] Figure 4These are scanning electron micrographs of alkali lignin before and after degradation by Priestia sp.RL-1 strain; Figure A shows alkali lignin before degradation by Priestia sp.RL-1 strain, and Figure B shows alkali lignin after degradation by Priestia sp.RL-1 strain for 7 days.

[0027] Figure 5 It is the degradation efficiency of Priestia sp.RL-1 strain on alkali lignin under different pH conditions.

[0028] Figure 6 This is a diagram showing the decolorization effect of Priestia sp.RL-1 strain on basic red, reactive blue, aniline blue and naphthol green dyes.

[0029] Figure 7 The growth-promoting effect of the metabolites of straw degradation by Priestia sp. RL-1 on rice seeds; in the figure, from left to right are the blank control group, sterile group and RL-1 group. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the technical solution of the present invention, the preferred embodiments of the present invention are described in detail below, but the following embodiments do not limit the protection scope of the present invention.

[0031] In the embodiments of the present invention, those that are not described in detail are all completed by conventional experimental methods, and those processes involved in the embodiments that are not described in detail are all understandable and easily implementable by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore are not described in detail again.

[0032] Example 1: Strain screening and identification

[0033] The Priesteria RL-1 of the present invention is separated and purified from the intestines of worker ants of Microcerotermes sp. collected from Xishuangbanna Tropical Botanical Garden of the Chinese Academy of Sciences in Yunnan Province.

[0034] The specific separation and purification methods are as follows:

[0035] Take 30 saw-termite workers and dissect them in a clean bench. First, use 75% alcohol to disinfect the surface of the saw-termite workers, then wash with sterile water, use sterile forceps and dissecting needles to pull out all the intestines of the saw-termite workers and place them in a sterilized 1.5mL EP tube. After all the saw-termite workers are dissected, add 500μL of PBS solution (pH 7.2, 50mmol) to the EP tube, and use a sterilized grinding rod to grind the intestines of the saw-termite workers in the tube and mix them. Dilute the intestinal homogenate with PBS for 10 -1 ~10 -6Times, take 100 μL from each gradient and spread it on a culture dish with solid culture medium using alkali lignin as the only carbon source, place it in a constant temperature incubator at 30°C for culture, and after the colonies grow, use an inoculation loop to pick a single colony for streak culture, repeat 3 to 5 times until a pure culture of the strain is obtained.

[0036] Among them, the formula of the solid culture medium with alkali lignin as the only carbon source is as follows: alkali lignin 2.0g, KH2PO41.0g, MgSO4 0.2g, K2HPO4 1.0g, (NH4)2SO4 2.0g, CaCl2 0.1g, FeSO4 0.05g, MnSO4 0.02g, CuSO4 0.001g, ZnSO4 0.001g, distilled water 1000mL, and 18g agar.

[0037] The main biological characteristics of the isolated and screened strains were identified by the following methods:

[0038] Isolate and preserve the screened strains: transfer the screened monoclonal strains to LB medium, culture them in a 30°C constant temperature incubator, and freeze the strains at -80°C after the culture is completed.

[0039] Morphological characteristics of bacteria: Figure 1 As shown, the selected bacterial colonies are rod-shaped, with a size of (1.4-1.6) μm × (3.7-5.5) μm, and some are arranged in chains. Gram-positive rod-shaped bacteria are aerobic, motile, and oxidase-positive.

[0040] Physiological and biochemical characteristics: Through the above-mentioned separation and screening process, it can be obtained that the screened strain can grow in a solid culture medium with alkaline lignin as the only carbon source. Furthermore, in this embodiment, the growth temperature of the bacteria and the pH value of the culture medium are adjusted. The experimental results show that the optimal growth temperature range of the screened strain is 15-40°C, preferably 30-37°C; the optimal growth pH range is 4-12, and the preferred pH value is 8-10. At the same time, in this embodiment, 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,6-dimethoxyphenol (DMP) are used as substrates, and spectrophotometry is used to detect that the screened strain has laccase and manganese peroxidase activity, and can secrete oxidase to degrade lignin.

[0041] The single colonies of the selected strains were further cultured in LB liquid medium overnight, and the genomic DNA of the strains was extracted using the Takara Bacterial Genomic DNA Extraction Kit (Cat. No. 9763), and the 16S rRNA fragment of the strain was amplified using the bacterial 16S rRNA universal primers synthesized by Shanghai Biotechnology (27F: 5'-AGAGTTTGATCMTGGCTCAG-3', 1492R: 5'-TACGGYTACCTTGTTACGACTT-3'). PCR reaction system (50 μL): 10×Ex Taq Buffer (20 mM Mg 2+ plus) 5μL, dNTP Mixture (2.5mM) 4μL, primers (20μmol / L) 1.0μL each, TaKaRa Ex Taq (5U / μL) 0.5μL, bacterial DNA 2μL, sterile water 36.5μL. PCR reaction conditions: 95℃4min; 95℃30s, 54℃45s, 72℃2min, 32 cycles in total; 72℃10min; 4℃ storage.

[0042] The PCR product was sent to a sequencing company for sequencing. The sequencing results were compared with the NCBI nucleic acid database by Blast homology, and a phylogenetic tree was constructed using the neighbor-joining method (e.g. Figure 2 As shown in the figure, it can be seen that the strain is in the same branch as Priestia megaterium and has the closest relationship. The strain RL-1 was identified as the genus Priestia and named Priestia sp.RL-1.

[0043] The sequence information of the 16S rRNA fragment of the Priestia sp.RL-1 is shown in SEQ ID No: 1:

[0044]

[0045] Example 2: Degradation ability of Priestia sp. RL-1 on alkali lignin

[0046] The single colony of Priestia sp. RL-1 screened in Example 1 was inoculated into LB medium and cultured overnight at a temperature of 37° C. and a rotation speed of 180 rpm. When the absorbance of the bacterial solution at 600 nm reached about 1.0, 10 mL of the bacterial solution was centrifuged and washed with distilled water, and then resuspended with 10 mL of a liquid inorganic salt medium containing alkali lignin. The resuspended solution of the bacteria was then inoculated into 200 mL of a liquid inorganic salt medium containing alkali lignin (the formula was the same as that of the solid medium with alkali lignin as the sole carbon source described in Example 1, except that the agar was removed) at an inoculation amount of 5% (volume ratio), and cultured with shaking at a temperature of 37° C., pH 8.0, and a rotation speed of 180 rpm for 24-168 h. The obtained culture solution was used for subsequent detection.

[0047] At the same time, the liquid inorganic salt culture medium containing alkali lignin before inoculation was used as the control culture medium to detect the degradation ability of the bacteria on lignin. The concentration of alkali lignin before and after degradation by the strain was characterized by measuring the COD value in the solution.

[0048] First, the culture solution was centrifuged at 12000r / min for 10min to filter out the bacteria, and the supernatant was diluted 80 times as a sample for COD determination. The COD was determined by potassium dichromate determination method (national standard method, conventional method), and the sample was digested by a multi-functional digestion instrument (Taizhou weighted JQ-102). Potassium dichromate solution was added to the solution obtained after digestion, and the remaining potassium dichromate was back-titrated by ammonium ferrous sulfate. The COD value was calculated using the national standard method (HJ828-2017) according to the amount of potassium dichromate consumed. The lignin degradation rate was calculated according to the following formula: Degradation rate (%) = (Ai-Af) / Ai*100

[0049] Wherein, Ai: COD value in the culture medium without inoculation of Priesteria RL-1; Af: COD value in the culture medium with inoculation of Priesteria RL-1.

[0050] The experimental results show that Figure 3 As shown, the Priestia sp.RL-1 strain can reach a lignin degradation rate of 49.6% after being cultured in the lignin culture solution for 72 hours; and the lignin degradation rate is as high as over 88.7% after being cultured for 168 hours.

[0051] Furthermore, scanning electron microscopy was used to detect the physical structural changes of lignin before and after treatment with Priesteria RL-1. Figure 4As shown in the figure, the lignin that has not been treated with Priestia sp. RL-1 has a dense and complete structure, is in block shape, and has a smooth surface; the lignin treated with Priestia sp. RL-1 is loose and porous, and the particle size is significantly smaller than that of the lignin before treatment. This shows that Priestia sp. RL-1 can destroy the microstructure of alkali lignin by secreting extracellular enzymes to achieve the purpose of degradation.

[0052] Example 3: Degradation ability of Priestia sp. RL-1 on alkali lignin under different pH conditions

[0053] A single colony of Priestia sp. RL-1 obtained in Example 1 was inoculated into LB medium and cultured overnight at a temperature of 37° C. and a rotation speed of 180 rpm. When the absorbance of the bacterial solution at 600 nm reached about 1.0, 55 mL of the bacterial solution was centrifuged and washed with distilled water, and then resuspended with the same volume of liquid inorganic salt medium containing alkali lignin (the formula is the same as the solid medium with alkali lignin as the sole carbon source described in Example 1, only the agar is removed), and then 10 mL of the resuspended bacterial solution was transferred to 200 mL of liquid inorganic salt medium containing alkali lignin with pH values ​​of 4.0, 6.0, 8.0, 10.0 and 12.0 (the formula is the same as the solid medium with alkali lignin as the sole carbon source described in Example 1, only the agar is removed, and different pH values ​​are adjusted with 0.1 M NaOH or HCl), and cultured with shaking at a temperature of 37° C. and a rotation speed of 180 rpm for 7 days, and the obtained culture solution was used for subsequent detection.

[0054] At the same time, the liquid inorganic salt medium containing alkali lignin before inoculation was used as the control culture medium to detect the degradation ability of the bacteria on lignin. The concentration of alkali lignin before and after the strain degradation was characterized by measuring the COD value in the solution (the determination and calculation of COD were the same as in Example 2).

[0055] The experimental results show that Figure 5 As shown, Priestia sp.RL-1 strain can grow in lignin medium with pH values ​​of 4.0, 6.0, 8.0, 10.0 and 12.0, and has the ability to degrade lignin. The lignin degradation efficiency is the highest (more than 80%) when the pH value is 8.0, the lowest (17.6%) when the pH value is 4.0, and the lignin degradation rate is about 40% under the conditions of pH values ​​of 6, 10, and 12. It can be seen that Priestia sp.RL-1 has good environmental adaptability.

[0056] Example 4: Decolorization ability of Priestia sp. RL-1 on various dyes

[0057] The Priestia sp. RL-1 single colony obtained in Example 1 was inoculated into LB medium and cultured overnight. When the absorbance of the bacterial solution at 600 nm reached about 1.0, the bacterial solution was dipped with an inoculation loop and streaked onto solid LB medium containing 50-100 mg / L of basic red, reactive blue, aniline blue and naphthol green. The culture was placed in a 30°C incubator and cultured for 1 to 3 days. The decolorization was observed. The results are shown in Table 1. Figure 6 As shown. Figure 6 It can be seen that after Priestia sp.RL-1 strain grows on the above four dye solid culture media for a period of time, the dyes can be almost completely decolorized. Among them, aniline blue decolorizes the fastest, which can be completely decolorized within 24 hours, and basic red is slightly slower, generally decolorizing within 1-3 days, indicating that this bacterium can secrete extracellular enzymes to decolorize and degrade it.

[0058] Example 5: Degradation of crop straw by Priestia sp. RL-1

[0059] The Priestia sp. RL-1 single colony obtained in Example 1 was inoculated into LB medium and cultured overnight at a temperature of 37° C. and a rotation speed of 180 rpm. When the absorbance of the bacterial solution at 600 nm reached about 1.0, 10 mL of the bacterial solution was centrifuged and washed with distilled water, and then resuspended with 10 mL of liquid inorganic salt medium with crop straw as the sole carbon source, and then the bacterial suspension was inoculated into 200 mL of liquid medium with crop straw as the sole carbon source according to an inoculation amount of 5% (volume ratio), and cultured for 7 days under the conditions of a culture temperature of 37° C. and a rotation speed of 180 rpm. The degradation rate of crop straw was determined by weight loss method, the culture solution was centrifuged at 6000 r / min for 10 min, the supernatant was removed, the precipitate was washed with water, and dried in an oven at 80° C. to constant mass, and the degradation rate of crop straw was calculated by the following formula, which was repeated 3 times.

[0060] Crop straw degradation rate (%) = (W0-W1) / W0×100%

[0061] Where: W0 is the mass of crop straw in the culture medium before inoculation, g; W1 is the mass of crop straw remaining after drying after culture, g.

[0062] Among them, the formula of the liquid culture medium with crop straw as the only carbon source is as follows: 25g of dried straw powder (passed through a 20-mesh sieve), 1.0g of KH2PO4, 0.2g of MgSO4, 1.0g of K2HPO4, 2.0g of (NH4)2SO4, 0.1g of CaCl2, 0.1g of peptone, and 1000mL of distilled water.

[0063] The crop straw used in the culture medium includes rice straw, corn straw or wheat straw, which are respectively used in the experiments.

[0064] Experimental verification has shown that Priestia sp. RL-1 can directly degrade crop straw that has not undergone any chemical pretreatment, and has a degrading effect on rice straw, corn straw and wheat straw. After strain RL-1 treated crop straw for 7 days, the degradation rate of rice straw reached 20.5%, the degradation rate of wheat straw reached 22.9%, and the degradation rate of corn straw reached 18.2%.

[0065] Example 6: Effect of the metabolites of Priestia sp. RL-1 degrading straw on plant growth

[0066] A pot experiment was conducted to test plant growth promotion using rice seeds (Oryza sativa) as materials (purchased from the market).

[0067] A single colony of Priestia sp. RL-1 was inoculated into LB medium and cultured overnight at a temperature of 37° C. and a rotation speed of 180 rpm. When the absorbance of the bacterial solution at 600 nm reached about 1.0, 60 mL of the bacterial solution was centrifuged and washed with distilled water, and then resuspended with an equal volume of an inorganic salt medium with crop straw as the sole carbon source. The bacterial suspension was inoculated into 1.2 L of a liquid inorganic salt medium with rice straw as the sole carbon source (the formula is the same as the liquid medium with crop straw as the sole carbon source in Example 5, wherein the dried straw powder uses rice straw powder) according to an inoculation amount of 5% (volume ratio), and the culture was shaken and cultured at a temperature of 37° C. and a rotation speed of 180 rpm for 7 days. The culture solution was centrifuged at 10000 r / min for 10 min, and the supernatant was retained.

[0068] Rice seeds were soaked in deionized water for 8 h, a certain amount of vermiculite was placed in each flowerpot, 10 swollen seeds were selected and planted in the pots, and three groups were set up. They were irrigated every day with clean water (recorded as the blank control group), supernatant of rice straw that had not been treated with bacteria (recorded as the sterile group), and supernatant of the product after rice straw was treated with Priestia sp. RL-1 for 7 days (recorded as the RL-1 group), 200 mL each time, and the seeds were allowed to germinate and grow at room temperature for 6 days.

[0069] The results are as follows Figure 7 As shown, two days after sowing, the rice seeds irrigated with the supernatant of the product of Priestia sp. RL-1 after 7 days of rice straw treatment had the highest germination rate, reaching 80%, while the clear water group had only 30%, and the germination rate of the rice straw supernatant group without bacteria treatment was 50%. There were also significant differences in plant height. Compared with the clear water group, the plant height of the supernatant group of the product of Priestia sp. RL-1 after 7 days of rice straw treatment increased by 63.3%, and compared with the straw supernatant group without bacteria treatment, the plant height increased by 38.5%. This shows that the metabolites of Priestia sp. RL-1 degrading rice straw can promote the germination and growth of rice seeds.

Claims

1. A Priesteria sp., characterized in that The Priestia sp. is named as Priestia sp. RL-1 and is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC No.32481.

2. The Priesteria according to claim 1, characterized in that The Priesteria RL-1 is derived from worker ants of the saw-like termite.

3. The Priesteria according to claim 1, characterized in that The sequence information of the 16S r RNA fragment of Priesteria RL-1 is shown in SEQ ID No:

1.

4. A product containing Priesteria according to any one of claims 1 to 3.

5. Use of the Priesteria described in any one of claims 1 to 3 or the product described in claim 4 in agricultural waste biodegradation and wastewater treatment.

6. The use according to claim 5, characterized in that: The application includes lignin degradation, and the agricultural waste includes crop straw, corn straw, rice straw or wheat straw.

7. The use according to claim 5, characterized in that: Such applications include dye decolorization or degradation.

8. The use according to claim 7, characterized in that: The dye includes: basic red, reactive blue, aniline blue or naphthol green.

9. Use of the Priesteria described in any one of claims 1 to 3 or the product described in claim 4 in promoting plant seed germination and plant growth.

10. The use according to claim 9, characterized in that: The plants include rice.

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