A strain of rhizopus arrhizus ra-01 screened from lignite

Abstract

The present application belongs to the technical field of biodegradation, and particularly relates to a lignite degrading bacteria Rhizopus arrhizus RA-01 screened from lignite. Rhizopus arrhizus ), which is identified by ITS sequencing as Rhizopus arrhizus RA-01 (CGMCC No. 42237) and belongs to the genus Rhizopus. The strain has a significant degradation capacity for HNO3 pretreated lignite, and the degradation rate reaches 71.7% within 5 days, and the A450 absorbance value is 0.690. The Rhizopus arrhizus RA-01 has application potential in lignite biodegradation, and provides a scientific basis for realizing clean utilization of low-rank coal.

Description

Technical Field

[0001] This invention belongs to the field of biodegradation technology, specifically relating to a lignite-degrading bacterium, Rhizopus oligosporus RA-01, screened from lignite. Background Technology

[0002] Lignite, a low-rank coal rich in lignin-like substances, has global reserves of up to 4 trillion tons, and its development and utilization have attracted widespread attention from researchers. However, due to its high moisture content, high ash content, and low calorific value, lignite suffers from low direct combustion efficiency and poor economic benefits. Common processing methods for lignite, such as gasification and pyrolysis, suffer from high energy consumption, complex equipment, and harsh processing conditions. Furthermore, decomposition at high temperatures generates large amounts of combustion gases (such as sulfides and nitrogen oxides), posing a serious threat to the environment. Compared to physicochemical techniques, lignite biodegradation technology is an environmentally friendly and effective method. Coal biodegradation technology utilizes microorganisms such as fungi, bacteria, and actinomycetes to dissolve, degrade, liquefy, or gasify coal to obtain clean fuels and other high-value-added chemicals. This process only needs to be carried out at room temperature and normal pressure, offering advantages such as mild reaction conditions, simple equipment requirements, low energy consumption, and high product utilization. The prospects for the clean utilization of coal, especially low-rank coal, are bright. However, the efficiency of existing microorganisms in degrading lignite is relatively low, so it is crucial to screen and cultivate microbial strains with higher degradation capabilities. Summary of the Invention

[0003] To address the problems existing in the prior art, the present invention provides a lignite-degrading bacterium, Rhizopus oligosporus RA-01, screened from lignite.

[0004] The technical solution of this invention is as follows:

[0005] A strain of Rhizopus RA-01 ( Rhizopus arrhizus It was deposited at the China General Microbiological Culture Collection Center on September 30, 2025, with accession number CGMCC No. 42237.

[0006] The present invention also includes the application of the above-mentioned Rhizopus RA-01 in the degradation of lignite.

[0007] The beneficial effects of this invention are as follows:

[0008] (1) Screening of endogenous strains of Rhizopus oligosporus RA-01 that degrade lignite from lignite ( Rhizopus arrhizus ).

[0009] (2) The strain Rhizopus RA-01 provided by this invention achieved a degradation rate of 71.7% (A450 absorbance 0.690) on lignite pretreated with HNO3 within 5 days. Attached Figure Description

[0010] Figure 1 The images show the changes in the culture medium of Rhizopus oligosporus RA-01 on day 0 (A) and day 4 (B) during the degradation of lignite;

[0011] Figure 2 Micromorphology and phylogenetic tree of Rhizopus oligosporus RA-01;

[0012] Figure 3 The changes in the degradation rate of oxidized lignite treated with Rhizopus RA-01;

[0013] Figure 4 The change of A450 in oxidized lignite treated with Rhizopus RA-01 over 8 days;

[0014] Figure 5 pH changes in oxidized lignite treated with Rhizopus RA-01. Detailed Implementation

[0015] Example 1

[0016] Coal-degrading strain Rhizopus oligosporus RA-01 ( Rhizopus arrhizus Separation and identification of )

[0017] (1) Screening of coal-degrading bacteria

[0018] One g of fresh, unsterilized 80-120 mesh lignite (from Gongyi, Henan, China) was used as the inoculum and added to 150 mL of minimum salt medium. The medium was then incubated for 3 days at 150 rpm / min and 30°C using a constant temperature shaking incubator to enrich coal-degrading microorganisms. The minimum salt medium consisted of (per liter): 1 g K₂HPO₄, 0.2 g KH₂PO₄, 1 g NaCl, 0.002 g CaCl₂·2H₂O, 0.005 g boric acid, 1 g (NH₄)₂SO₄, 0.5 g MgSO₄·7H₂O, 0.001 g CuSO₄, 0.01 g ZnSO₄·7H₂O, 0.001 g MnSO₄, 0.01 g FeSO₄·7H₂O, and 3 g glucose. The bacterial suspension was then diluted sequentially with sterile water to a concentration of 10 g / L. -1 10 -2 and 10 -3 A suspension.

[0019] The sterilized potato dextrose agar (PDA: 200 g potato, 20 g glucose, 20 g agar, and 1000 mL distilled water) was then poured into sterile petri dishes. After cooling and solidification, the dishes were inverted and left to stand for 24 hours until no colonies formed before use. (From 10...) -1 10 -2and 10 -3 1 µl of each diluted solution was dropped onto the surface of a sterile agar plate and spread evenly with a sterile spreader. After spreading, the culture medium was inverted and incubated in a 30°C biochemical incubator. Once fungal spores grew, they were streaked and separated, and then passaged to purify the fungus.

[0020] The coal sample was crushed and ground to 40-80 mesh. 10 g of the coal sample was added to a 500 mL solution containing 2 mol / L HNO3 and incubated at room temperature for 48 h. After the coal reacted with nitric acid, distilled water was added and stirred. The mixture was allowed to stand until the coal precipitated, and the supernatant was discarded. A suitable amount of distilled water was added, and a saturated aqueous solution of sodium carbonate was added in small amounts several times with rapid stirring until bubbles were generated. This process was repeated 4-5 times until no more bubbles were generated. The mixture was allowed to stand until the coal precipitated, and the supernatant was discarded. A suitable amount of distilled water was added to wash the solution until the pH was neutral. The coal sample was dried at 60℃ for 48 h to constant weight, and then autoclaved at 121℃ for 20 minutes for later use.

[0021] (2) Microbial degradation of lignite experiment

[0022] Rhizopus oligosporus RA-01 isolated from sterile PDA plates were inoculated and cultured at 30℃ for 4 days. Then, 0.5g of sterile oxidized lignite (80-120 mesh) was evenly sprinkled on the surface of the petri dish and placed in a constant temperature incubator at 30℃. Two control groups were set up in this experiment. One group was to evenly sprinkle sterile oxidized lignite on PDA plates without Rhizopus oligosporus RA-01 to determine whether the culture medium would degrade the coal. The other control group was to inoculate Rhizopus oligosporus RA-01 on PDA plates without adding lignite.

[0023] Figure 1 This image shows the effect of Rhizopus oligosporus RA-01 on the biosolubilization of lignite before and after treatment. It was found that brown droplets formed on the surface of the solid culture medium after treatment with Rhizopus oligosporus RA-01, and the reverse side of the medium showed obvious staining, changing from light yellow to dark brown. This indicates that Rhizopus oligosporus RA-01 can decompose complex macromolecular organic matter in coal into soluble small molecules, demonstrating its good biodegradation effect on coal.

[0024] (3) Preservation of coal-degrading bacteria

[0025] The selected fungal strains were inoculated onto solid slant agar plates and cultured in a 30°C biochemical incubator. After the strains had grown sufficiently, they were stored in a 4°C refrigerator. Every month, the strains were transferred to fresh slant agar plates for preservation.

[0026] (4) Identification of coal-degrading bacteria

[0027] On a clean bench, 50-100 mg of fungal mycelium was taken and appropriately dispersed using a glass grinder. 20 μL of RNase A was added, followed by 100 mg of glass beads. The mixture was shaken on a high-speed shaker for approximately 30 min. Then, a fungal genomic DNA extraction kit was used to extract the DNA, yielding high-quality genomic DNA. The nucleotide sequence is shown in SEQ ID NO.1. PCR amplification was performed using universal primers ITS 1 and ITS 4 targeting the ITS region. The nucleotide sequences of primers ITS 1 and ITS 4 are shown in SEQ ID NO.2 and SEQ ID NO.3. An ABI-3730XL sequencer was used with a 25 μL amplification program: 95°C for 5 min, 95°C for 30 s, 50°C for 30 s, 72°C for 1 min 30 s, for 35 cycles, followed by a final extension at 72°C for 10 min. Finally, the sequencing results were compared using BLAST in the NCBI database, and the extracted DNA sequences were compared. Figure 2 As shown in the figure. Developmental tree construction confirmed that this strain belongs to *Rhizopus oligorhizopus*.

[0028] The strain was biopreserved, and the preservation information is as follows:

[0029] Strain name: Rhizopus RA-01;

[0030] Classification and nomenclature: Rhizopus septemlobus Rhizopus arrhizus ;

[0031] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0032] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0033] Deposit date: September 30, 2025;

[0034] Accession number: CGMCC No.42237.

[0035] Experimental Example 1

[0036] (1) Degradation rate of coal-degrading bacteria

[0037] First, the selected Rhizopus oligorhizoides RA-01 was inoculated from the preserved slant culture medium onto PDA medium using a sterile inoculation loop and activated for 48 h. Then, 0.9% physiological saline was added to form a bacterial suspension for later use. Twenty-four 100 mL Erlenmeyer flasks were washed and dried. 75 mL of PDA medium was added to each flask, and the flasks were sterilized by moist heat at 121℃ for 20 min. Rhizopus oligorhizoides RA-01 was then inoculated into each flask and cultured in a constant temperature shaker at 30℃ and 150 rpm for 4 days. 1 g of sterile nitric acid was added to oxidize the coal, and the culture was continued with constant temperature shaking. Simultaneously, two control groups were set up. The first control group was inoculated with only *Rhizopus oligosporus* RA-01 culture medium, without the addition of sterilized oxidized lignite samples. The second control group was not inoculated with *Rhizopus oligosporus* RA-01, but the culture medium was supplemented with sterilized oxidized lignite. Samples were taken daily, and the lignite samples were separated from the culture medium. Residual lignite on the mycelial surface was washed with distilled water to separate residual lignite from the mycelium. The collected residual lignite was dried at 80°C to constant weight. Three replicates were performed for each group. The results were then analyzed using the formula... The degradation rate of the coal sample is calculated using the formula, where η is the degradation rate, m0 is the original mass of the coal sample (g), and m1 is the residual mass of the coal sample after biodegradation (g).

[0038] Degradation rate analysis revealed that Rhizopus RA-01 was able to dissolve 15.6% of lignite on day 1. Figure 3 Subsequently, the degradation rate of lignite continued to increase, reaching a peak of 71.6% on day 5. From day 6 to 8, the degradation rate of lignite stabilized at 71.7%, while the degradation rate of the control group without Rhizopus RA-01 inoculation was less than 4%.

[0039] (2) Changes in A450 liquid culture medium for coal-degrading bacteria and pH

[0040] The specific experimental method for assessing the degree of lignite degradation using the A450 value is as follows: 5 mL of sample was taken from the liquid culture medium every 1 day, and the sample was centrifuged at 10,000 rpm for 10 min. The pH of the supernatant was measured, and the supernatant was diluted 30 times. The absorbance was measured at 450 nm using a UV-Vis spectrophotometer. Three replicates were set up for each group of experiments.

[0041] The changes in A450 of the degradation solution are as follows Figure 4 As shown, when the degradation time was 5 days, the A450 of the medium inoculated with Rhizopus RA-01 and with added oxidized lignite reached 0.690, while the A450 values ​​of the control group inoculated only with Rhizopus RA-01 and the medium inoculated without Rhizopus RA-01 but with added lignite remained at low values ​​(0.01-0.06) throughout the degradation process.

[0042] like Figure 5As shown, the pH of the culture medium containing *Rhizopus oligosporus* RA-01 decreased from an initial pH of 6.0 to 4.92, and then steadily increased over time, reaching a maximum of 6.9 on day 8. This is similar to the pH change in the culture medium containing oxidized lignite inoculated with *Rhizopus oligosporus* RA-01, where the initial pH rapidly decreased from 6.0 to 4.57 on day 1, and then rose to 7.1 after the degradation reaction was complete. In the control group containing only sterilized lignite, the pH decreased from 6.0 to approximately 5.38 on day 1, and remained stable at around 5 on day 8. The decrease in culture medium pH after inoculation with *Rhizopus oligosporus* RA-01 is attributed to the production of pyruvate and lactic acid during microbial growth due to the consumption of carbon sources, and is also related to the acidic nature of lignite. This further demonstrates that *Rhizopus oligosporus* RA-01 can effectively degrade lignite, thereby achieving the goal of clean coal production.

Claims

1. A lignite-degrading bacterium, Rhizopus oligosporus RA-01, screened from lignite ( Rhizopus arrhizus ), characterized in that, The preservation information for *Rhizopus oligorhizoides* RA-01 is as follows: it was deposited on September 30, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 42237; its classification name is *Rhizopus oligorhizoides*. Rhizopus arrhizus .

2. The application of Rhizopus RA-01 as described in claim 1 in the degradation of lignite.

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