CN115895975A - Acinetobacter ruffii and application thereof in degradation of coal gangue - Google Patents
Acinetobacter ruffii and application thereof in degradation of coal gangue Download PDFInfo
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- CN115895975A CN115895975A CN202211675178.7A CN202211675178A CN115895975A CN 115895975 A CN115895975 A CN 115895975A CN 202211675178 A CN202211675178 A CN 202211675178A CN 115895975 A CN115895975 A CN 115895975A
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Abstract
The application discloses a Acinetobacter ruffii strain and application thereof in degrading coal gangue, and relates to the technical field of Acinetobacter ruffii. The Acinetobacter rufii (Acinetobacter lwoffii) XAQ2297 is preserved in China center for type culture Collection (CCTCC NO: M20221438) at 9/15/2022, and the preservation address is Wuhan university in Wuhan city, hubei province. The strain belongs to gram-negative aerobic bacteria, can degrade coal gangue in a short time, and has no toxic action on human and livestock; the strain can effectively decompose organic matters in the coal gangue into humic acid and other inorganic matters, and can be prepared into a biological fertilizer and solve the environmental problem caused by stacking the coal gangue.
Description
Technical Field
The application relates to the technical field of acinetobacter luffaensis, in particular to acinetobacter luffaensis and application thereof.
Background
The coal gangue is solid waste discharged in the coal mine construction, coal mining and processing processes, and is solid waste with the largest discharge amount, the largest occupied area and serious pollution in various industrial waste residues. It forms a dust particle, and if the dust particle is inhaled into the lung by a human body, various diseases can be caused, and even cancer can be caused; large particles can remain in the nasal cavity of a person, causing nasal infections that, if introduced into the eye, can cause various eye diseases. In the open-air stacking process, partial substances are dissolved after rainwater leaching, and form surface runoff with rainwater to enter soil, surface water or underground water, so that the soil, surface water or underground water is polluted, and the aquatic ecological environment is damaged.
Disclosure of Invention
Therefore, some strains can effectively degrade the coal gangue in the biological treatment research of the coal gangue, a strain capable of effectively decomposing the coal gangue is separated and purified from the coal gangue powder, the similarity of the strain and Acinetobacter ruffiae (NCBI comparison result) is up to 99% through identification, and the strain is named as Acinetobacter ruffii (Acinetobacter lwoffi) XAQ2297, the strain can effectively decompose organic matters in the coal gangue into humic acid and other inorganic matters, and the strain can be prepared into a fertilizer and can solve the environmental problem caused by stacking the coal gangue. The bacteria can utilize nutrient components in the coal gangue to secrete metabolites such as polysaccharide, the polysaccharide is adhered to the surface of the coal gangue and gradually forms a biological membrane, the formed biological membrane decomposes and converts minerals, and formed mineral ions are continuously released outwards to form fertilizer components. Part of organic matters of the coal gangue are converted into humic acid, and the humic acid can load nutrient elements, slowly release nutrients, retain water and fertilizer; secondly, the organic colloid can improve the granular structure of the soil; thirdly, heavy metal and harmful substances are adsorbed, and disease resistance, low temperature resistance and salt and alkali resistance are achieved.
Therefore, the embodiment of the application at least discloses the following technical scheme:
the embodiment of the application discloses Acinetobacter rufoenii (Acinetobacter lwoffii) XAQ2297 which is preserved in China center for type culture Collection in 2022, 9 and 15 days, with the preservation number of CCTCC NO: M20221438, and the preservation address of the Acinetobacter rufoenii is Wuhan university in Wuhan city, hubei province.
Further, acinetobacter ruffiensis (Acinetobacter lwoffi) XAQ2297 is isolated from coal gangue.
Further, the acinetobacter rufoenii strain is positive in catalase, negative in oxidase, negative in methyl red test and negative in nitrate reduction; no hydrogen sulfide and no indole are produced; no power is supplied; the sugars are not fermented.
The 16S rRNA gene sequence of the acinetobacter rufoenii is shown as SEQ ID NO. 3.
The embodiment of the application discloses a preparation for degrading coal gangue, which comprises acinetobacter ruffiensis.
Further, the formulation is a solid or liquid formulation.
The embodiment of the application discloses a method for degrading coal gangue, which comprises the step of adding the coal gangue into an inorganic salt liquid culture medium containing Acinetobacter ruffii as claimed in any one of claims 1 to 3 and culturing for 79h.
Further, the inorganic salt medium contains 3g/L (NH) 4 ) 2 SO 4 ，0.5g/L KH 2 PO 4 ，1.26g/L Na 2 HPO 4 ·12H 2 O，0.54g/L MgSO 4 ·7H 2 O，0.05g/L FeCl 3 ·6H 2 O，0.03g/L FeSO 4 ·7H 2 O，0.015g/L MnSO 4 ·H 2 O，0.024g/L ZnSO 4 ·7H 2 O，0.366g/L CoCl 2 ·6H 2 O。
Compared with the prior art, the application has at least one of the following beneficial effects:
the Acinetobacter xylinum (Acinetobacter lwoffii) XAQ2297 for degrading the coal gangue belongs to gram-negative aerobic bacteria, can degrade the coal gangue in a short time, and has no toxic action on people and livestock; the strain can effectively decompose organic matters in the coal gangue into humic acid and other inorganic matters, and can be prepared into a biofertilizer and solve the environmental problem caused by stacking the coal gangue.
Drawings
FIG. 1 is a morphological diagram of Acinetobacter ruffensis (Acinetobacter lwoffii) XAQ2297 colonies provided in the examples of the present application.
Fig. 2 is a phylogenetic tree of Acinetobacter ruffeae (Acinetobacter lwoffii) XAQ2297 provided in an embodiment of the present application.
FIG. 3 is a graph showing the growth of A.ruphtalens (Acinetobacter lwoffii) XAQ2297 provided in the examples of the present application.
Fig. 4 is a scanning electron microscope image of coal gangue after Acinetobacter rufoenii (Acinetobacter lwoffii) XAQ2297 soaks the coal gangue provided in the embodiment of the present application.
Fig. 5 is a graph of humic acid content in inorganic salt solution after Acinetobacter iwoffii (Acinetobacter lwoffi) XAQ2297 degrades coal gangue, which is provided in the embodiment of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of and not restrictive on the broad application. Reagents not individually specified in detail in this application are conventional and commercially available; methods which are not specified in detail are all customary experimental methods and are known from the prior art.
It should be noted that the terms "first", "second", and the like in the description and claims of the present invention and in the drawings are used for distinguishing similar objects, and do not necessarily have to be used for describing a specific order or sequence or have a substantial limitation on technical features thereafter. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in other sequences than those illustrated or described herein. Moreover, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but includes other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
1. Collection of samples for strain screening
The samples adopted in the experiment are mainly derived from coal gangue. The coal gangue is taken from a mine dump in Huangbaizhen mining area of Uhai city.
2. Culture medium
The culture medium and the formula are LB culture medium and inorganic salt culture medium.
LB culture medium: 10g/L sodium chloride, 10g/L peptone, 5g/L yeast extract, and finally pH 7 adjusted with NaOH or HCl.
The inorganic salt medium contains 3g/L (NH) 4 ) 2 SO 4 ，0.5g/L KH 2 PO 4 ，1.26g/LNa 2 HPO 4 ·12H 2 O，0.54g/L MgSO 4 ·7H 2 O，0.05g/L FeCl 3 ·6H 2 O，0.03g/LFeSO 4 ·7H 2 O，0.015g/L MnSO 4 ·H 2 O，0.024g/L ZnSO 4 ·7H 2 O，0.366g/LCoCl 2 ·6H 2 O。
3. Screening of coal gangue degrading bacteria
Soaking 10g of coal gangue in 50mL of inorganic salt culture medium for 24 hours; taking supernatant by using plate streaking, inoculating the supernatant into an LB solid culture medium, and culturing for 3d in a constant temperature incubator at 30 ℃; selecting the microorganisms growing on the LB culture medium into a 50mL liquid inorganic salt culture medium for culture, adding 0.5g coal gangue, and carrying out shake culture for 24h under the conditions that the temperature is 30 ℃ and the rotating speed is 220 rpm; then 10mL of the culture solution after shaking culture is transferred to a conical flask containing 50mL of fresh inorganic salt culture medium for further shaking for 24h, and the shaking period is 10. And inoculating the final domesticated bacterium liquid into an LB culture medium, culturing for 24 hours in a constant-temperature incubator at 30 ℃, selecting a single strain, and performing streaking purification for multiple times to obtain a purified bacterium, namely the coal gangue degrading bacterium.
The single colony morphology of acinetobacter ruffiae on LB culture medium is shown in figure 1, and the single colony forms milky white colony which is round, has a raised surface, is smooth and sticky and is attached to the culture medium.
4. Strain identification
The physiological and biochemical identification of the strain is carried out according to the manual of identifying the system of common bacteria and the method of identifying the common bacteria. The strains obtained by result screening are positive in catalase, negative in oxidase, negative in methyl red test and negative in nitrate reduction; no hydrogen sulfide and no indole are generated; is unpowered; the sugars are not fermented.
16S rDNA sequencing and comparison:
the Bacterial DNA kit from OMEGA was used for DNA extraction. The primers are bacterial 16S rDNA amplification universal primer 16SF (27F) SEQ ID NO:1, AGAGAGTTTGATCTMTGGCTCAG, 16SR (1492R) SEQ ID NO:2 and GGTTACCTTGTTACGACTT, and the PCR amplification system comprises 12.5 muL of 2 xTaq PCR Master Mix, 1.0 muL of template, 1.0 muL of 16SF, 1.0 muL of 16SR and 9.5 muL of double distilled water in a total volume of 25 muL.
PCR reaction conditions of pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and cycle times of 30 times; final extension at 72 ℃ for 10min. And 5 mu L of the amplified 16S rDNA sequence product is taken for agarose gel electrophoresis detection, and after the electrophoresis is finished, the gel is taken out and put into a Bio-Rad gel imaging system for observing the amplification effect. And (3) sending the PLR amplification product to a biology (Shanghai) company for sequencing, performing homology comparison (Blast) on the sequenced sequence in NCBI, selecting a bacterial sequence with higher homology, performing comparison analysis by using MEUA6.0, and constructing a phylogenetic tree.
The sequence of the 16S rDNA is shown as SEQ ID NO. 3, the result of the evolutionary tree is shown as figure 2, and the bacteria obtained by screening are proved to be really Acinetobacter ruffii named as Acinetobacter ruffii (Acinetobacter lwoffii) XAQ2297 which is preserved in China center for type culture collection at 9 and 15 months 2022 with the preservation number of CCTCC NO. M20221438 and the preservation address of the bacteria is at Wuhan university, wuhan city, hubei province.
5. XAQ2297 characteristic of degrading coal gangue degrading bacteria
(1) Enrichment culture of XAQ2297 strain and soaking experiment for coal gangue
As shown in the growth curve (FIG. 3) of Acinetobacter ruffii XAQ2297, the strain cultured on LB medium for 24 hours was shake-cultured in 50mL of activated enrichment medium (10 g/L sodium chloride, 10g/L peptone, 5g/L yeast extract, pH 7.0, autoclaving at 121 ℃ for 30 min) for 24 hours. In order to preliminarily investigate the degradation effect of the bacterium on the coal gangue, a soaking experiment is carried out on the coal gangue. Taking 0.5g of coal gangue and 50mL of inorganic salt culture medium as CK, putting 0.5g of coal gangue and 5mL of logarithmic phase bacterial liquid into 50mL of inorganic salt culture medium as an experimental group, soaking for 48h at room temperature, and drying; and (3) performing a scanning electron microscope on the raw coal gangue, the coal gangue soaked by the inorganic salt, the bacterial liquid and the coal gangue soaked by the inorganic salt.
The structure of the coal gangue after the bacterial liquid and the inorganic salt are soaked is shown in figure 4. As can be seen from FIG. 4, the surfaces of the coal gangue soaked by the bacteria liquid and the inorganic salt are looser than the surfaces of the gangue soaked by the raw coal gangue and the inorganic salt, and the decomposition effect of the microorganisms on the coal gangue is intuitively explained.
(2) Coal gangue degradation experiment with bacterial liquid
And (3) taking the content of humic acid in the degraded coal gangue solution as an evaluation index of the degradation effect, wherein the higher the concentration of the humic acid is, the better the degradation effect is. Wherein, the humic acid is measured by a potassium dichromate method.
The strain cultured on LB medium for 24h is taken to be cultured in 50mL activation enrichment medium for 24h with shaking. Taking 0.5g of coal gangue and 50mL of inorganic salt culture medium as CK, putting 0.5g of coal gangue and 5mL of bacterial liquid in logarithmic phase into 50mL of inorganic salt culture medium as an experimental group, respectively culturing for 2, 4 and 6 days in a shaking table with the culture temperature of 30 ℃ and the rotating speed of 220rpm, and numbering; taking supernatant fluid on days 2, 4 and 6 to determine the content of humic acid.
The content of humic acid in the solution after coal gangue degradation is shown in figure 5. After acinetobacter rufoenii is degraded for 2 days under the conditions that the temperature is 30 ℃, the inoculation amount is 5mL and the pH value is 7, the content of humic acid after degradation is measured to be 85.1mg/L and is 66.2mg/L higher than that of CK humic acid which is 18.9 mg/L; after 4 days of degradation, the content of humic acid after degradation is 97.2mg/L, which is 81.4mg/L higher than that of CK humic acid 15.8 mg/L; after 6 days of degradation, the humic acid content after degradation is measured to be 92.0mg/L, which is 73.4mg/L higher than that of CK humic acid content of 18.6 mg/L. The effect is best after the acinetobacter ruffii is degraded for 4 days.
The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.
Claims (8)
1. Acinetobacter rufoenii (Acinetobacter lwoffii) XAQ2297 is preserved in China center for type culture Collection in 2022, 9 and 15 days, with the preservation number of CCTCC NO:
m20221438, the preservation Address is Wuhan university in Wuhan city, hubei province.
2. Acinetobacter xylinum according to claim 1, isolated from coal gangue.
3. Acinetobacter ruffei according to claim 1, which strain is catalase positive, oxidase negative, methyl red test negative, nitrate reduction negative; no hydrogen sulfide and no indole are produced; is unpowered; the sugars are not fermented.
4. The acinetobacter xylinum according to claim 1, wherein the 16S rRNA gene sequence is shown in SEQ ID NO 3.
5. A biological agent for degrading coal gangue, comprising acinetobacter rufoenii according to any one of claims 1 to 3.
6. The formulation of claim 3, which is a solid or liquid formulation.
7. A method for degrading coal gangue, comprising adding the coal gangue to an inorganic salt liquid culture medium comprising acinetobacter ruffeae of any of claims 1-3, and culturing for 79h.
8. The method of claim 7, wherein the mineral salts medium comprises 3g/L (NH) 4 ) 2 SO 4 ，0.5g/L KH 2 PO 4 ，1.26g/L Na 2 HPO 4 ·12H 2 O，0.54g/LMgSO 4 ·7H 2 O，0.05g/L FeCl 3 ·6H 2 O，0.03g/L FeSO 4 ·7H 2 O，0.015g/LMnSO 4 ·H 2 O，0.024g/L ZnSO 4 ·7H 2 O，0.366g/L CoCl 2 ·6H 2 O。
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| CN110951626A (en) * | 2019-12-27 | 2020-04-03 | 北京林业大学 | Penicillium oxalicum capable of decomposing coal gangue |
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| WO2022160922A1 (en) * | 2021-01-28 | 2022-08-04 | 山西大学 | Solid waste-based porous material, preparation method therefor and method for ecological restoration of coal gangue mountains |
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