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CN-122012321-A - Separation and identification and application of alcaligenes faecalis IDOH3 for efficiently degrading indole

CN122012321ACN 122012321 ACN122012321 ACN 122012321ACN-122012321-A

Abstract

The invention discloses separation and identification and application of an alkaline manure bacterium IDOH for efficiently degrading indole, wherein the preservation number of the strain IDOH3 is GDMCC NO.65566. The strain of the invention can completely degrade 100 mg/L indole in 3 hours under the optimal conditions of pH 7, 37 ℃ and 200 r/min through separation, identification and functional verification, and can maintain the degradation activity in the range of pH 3-9 and temperature 10-50 ℃. In addition, strain IDOH has degradation capability to aromatic compounds such as phenol and 2-methylindole. Experiments show that the degradation rate of the strain IDOH of the invention to indole in sterilized and unsterilized pig manure wastewater is 99.13% and 74.59%, respectively, and the strain IDOH is suitable for bioremediation of indole pollutants in livestock and poultry cultivation wastewater and other environments.

Inventors

  • DENG DUN
  • Weng Guangying
  • MA XIANYONG
  • SONG MIN
  • RONG TING
  • LIU ZHICHANG

Assignees

  • 广东省农业科学院动物科学研究所

Dates

Publication Date
20260512
Application Date
20260129

Claims (9)

  1. 1. A strain for efficiently degrading indole is characterized in that the strain is classified and named as alcaligenes faecalis (ALCALIGENES FAECALIS) IDOH and is preserved in the Guangdong province microorganism strain collection, and the preservation number is GDMCC NO.65566.
  2. 2. A method for separating strain according to claim 1, wherein the method comprises the steps of diluting fresh pig manure with sterile normal saline, uniformly coating the diluted pig manure on a solid inorganic salt culture medium with indole as a sole carbon source, culturing the solid inorganic salt culture medium in a constant temperature incubator, picking single colony from a flat plate after colony grows out, culturing the colony in the liquid inorganic salt culture medium, coating the plate again after culturing, and finally separating the strain grown with indole as the sole carbon source in a mode of repeatedly coating the flat plate.
  3. 3. The method for separating strains according to claim 2, wherein the screening process comprises diluting 10 g fresh pig manure with 100 ml sterile normal saline, diluting 10 times, uniformly coating on solid inorganic salt culture medium with indole as sole carbon source, culturing the solid inorganic salt culture medium in a 37 ℃ constant temperature incubator for 1-2 days, picking single colony from the flat plate after colony grows out, culturing in liquid inorganic salt culture medium at 37 ℃ and rotating speed of 170 r/min, coating again after culturing for 1-2 days, and finally separating the strain growing with indole as sole carbon source by 5 times of repeated flat plate coating.
  4. 4. A method for identifying a strain according to claim 1, comprising extracting bacterial DNA, PCR-amplifying the 16S rRNA gene of the strain IDOH using a universal primer for the 16S rRNA gene as an amplification primer, further homology-aligning the 16S rRNA gene sequences, and constructing a phylogenetic tree of the strain IDOH 3.
  5. 5. The method for identifying a strain according to claim 4, wherein the universal primers include an upstream primer 27F 5'-AGAGTTTGATCCTGGCTCAG-3' and a downstream primer 1492R 5 '-GGTTAC-CTTGTTACGACTT-3'.
  6. 6. The method for identifying a strain according to claim 4, wherein the 16S rRNA gene sequence is aligned with the closest sequence in the GenBank database by using BLAST, and the phylogenetic tree of the strain IDOH is constructed by the adjacency method by using MEGA 11 software.
  7. 7. The method according to claim 4, further comprising inoculating the strain IDOH to LB medium to obtain a bacterial liquid, taking a part of the bacterial liquid, and observing the cell morphology of the strain IDOH3 by a scanning electron microscope.
  8. 8. The method for identifying a strain according to claim 7, comprising inoculating IDOH of the strain to 100mL LB medium, culturing at 37 ℃ and 200 r/min for 24 h to obtain a bacterial liquid, taking 2 mL bacterial liquid, centrifuging the bacterial liquid at 8000 r/min for 5min, adding glutaraldehyde aqueous solution into the precipitate for fixing 2h, dehydrating with 30%, 50%, 70%, 90%, 95% and 100% gradient ethanol, taking 2 mL sample, and observing the cell morphology of the strain IDOH3 by a scanning electron microscope after critical point drying and ion gold sputtering of the obtained sample.
  9. 9. The method according to claim 1, wherein the strain IDOH is used for degrading indole in pig manure wastewater.

Description

Separation and identification and application of alcaligenes faecalis IDOH3 for efficiently degrading indole Technical Field The invention relates to the technical field of microorganisms, in particular to separation and identification and application of an alkaline manure bacterium IDOH for efficiently degrading indole. Background How to reduce the pollution of livestock and poultry raising odor to the environment to the greatest extent becomes a great problem which must be faced and solved in the sustainable development of the Chinese industry. Wherein, livestock and poultry breeding wastewater such as pig manure wastewater is taken as a main livestock and poultry breeding odor source. The main odor components of the odor substances of livestock and poultry raising wastewater such as pig manure wastewater are ammonia gas, sulfides, volatile fatty acid, indole and the like, wherein indole is a main odor pollutant in the livestock and poultry raising wastewater, is a metabolite of nitrogen-containing compounds such as hindgut microorganism fermentation protein and amino acid, is a typical nitrogen pollutant, has extremely strong manure odor, and causes environmental pollution and public health problems which are always focus problems of attention of the raising industry. Therefore, how to reduce the odor pollution of indole and the like discharged by the livestock and poultry breeding industry is a technical problem to be solved in the livestock and poultry breeding process. Disclosure of Invention (One) solving the technical problems Aiming at the defects of the prior art, the invention provides separation, identification and application of the alcaligenes faecalis IDOH3 for efficiently degrading indole, and the strain IDOH3 can efficiently degrade indole in pig manure wastewater. (II) technical scheme In order to solve the technical problems, the invention provides a first technical scheme that a bacterial strain for efficiently degrading indole is classified and named as alcaligenes faecalis (ALCALIGENES FAECALIS) IDOH, and is preserved in the Guangdong province microorganism strain preservation center with the preservation number of GDMCC NO.65566. The method comprises the steps of diluting fresh pig manure with sterile normal saline, uniformly coating the diluted fresh pig manure on a solid inorganic salt culture medium with indole as a unique carbon source, culturing the solid inorganic salt culture medium in a constant temperature incubator, picking single colonies from a flat plate to culture the single colonies in the liquid inorganic salt culture medium after colonies grow out, coating the flat plate again after the culture, and finally separating the bacterial strain growing with indole as the unique carbon source in a repeated flat plate coating mode. The screening process comprises diluting 10 g fresh pig manure with 100 ml sterile physiological saline, uniformly coating on a solid inorganic salt culture medium with indole as a sole carbon source after dilution for 10 times, culturing the solid inorganic salt culture medium in a 37 ℃ constant temperature incubator for 1-2 days, picking single bacterial colonies from a flat plate to culture in a liquid inorganic salt culture medium after bacterial colonies grow out, culturing at 37 ℃ at the rotating speed of 170 r/min, coating again after culturing for 1-2 days, and finally separating bacterial strains growing with indole as the sole carbon source through a 5-time repeated flat plate coating mode. In order to solve the technical problems, the invention provides a third technical scheme that the method for identifying the bacterial strain in the first technical scheme comprises the steps of extracting bacterial DNA, taking a general primer of a 16S rRNA gene as an amplification primer, carrying out PCR (polymerase chain reaction) amplification on the 16S rRNA gene of the bacterial strain IDOH, further carrying out homology comparison on the 16S rRNA gene sequence, and constructing a phylogenetic tree of the bacterial strain IDOH 3. Preferably, the universal primers include an upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', and a downstream primer 1492R: 5 '-GGTTAC-CTTGTTACGACTT-3'. Preferably, the above-described method for identifying a strain specifically includes homology alignment of the 16S rRNA gene sequence with the most similar sequence in GenBank database using BLAST, and construction of phylogenetic tree of strain IDOH by an adjacent method using MEGA 11 software. Preferably, the method for identifying the strain further comprises inoculating the strain IDOH to LB medium for culturing to obtain a bacterial liquid, taking part of the bacterial liquid, and observing the cell morphology of the strain IDOH by using a scanning electron microscope. Preferably, the method for identifying the bacterial strain specifically comprises inoculating the bacterial strain IDOH to a 100 mL LB culture medium, culturing at 37 ℃ and 200 r/min for 24 h to