CN115466690A - Novel geobacillus strain as well as culture method and application thereof - Google Patents
Novel geobacillus strain as well as culture method and application thereof Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention relates to a novel geobacillus strain and a culture method and application thereof. A species of Geobacillus, identified as Geobacillus thermophilus CX412, is called Geobacillus sp.CX412, is isolated from the soil around hot springs formed by volcanic eruption and has the preservation number of CGMCC No.22926, and is identified to belong to a typical new species. The geobacillus thermophilus new strain provided by the invention can grow and propagate in a high-temperature aerobic environment (70-90 ℃), and is an extreme thermophilic strain. The new strain has good thermal stability, can realize high-strength metabolism of polysaccharide and amino acid in a high-temperature environment, can be applied to aerobic fermentation of perishable organic matters to produce organic fertilizers, treat easily degradable organic high-temperature wastewater and the like, and has wide market prospect.
Description
Technical Field
The invention relates to the field of organic waste treatment, in particular to a novel geobacillus strain and a culture method and application thereof.
Background
As is well known, in the treatment of perishable organic wastes and high-temperature organic wastewater, there are many problems, for example, the perishable organic wastes have the problems of low degree of decomposition and incomplete degradation of perishable substances under the action of conventional aerobic bacteria; in the actual treatment process of the high-temperature production wastewater, because the proper temperature of the conventional treatment bacteria is 25-35 ℃, the high-temperature production wastewater can be treated only after the easily degradable organic high-temperature wastewater and normal-temperature water are mixed and cooled, so that the treatment capacity is greatly increased and the treatment efficiency is reduced.
The strains of Geobacillus are all from high-temperature environments such as hot springs, volcanoes, compost and the like, so that the strains and produced spores have extremely high heat resistance. The bacillus can form spores which can survive for several hours at 120-140 ℃.
Thermophilic bacteria have many advantages in practical applications: (1) the generation time of the thermophilic bacteria is short, thereby shortening the production period of synthesizing secondary metabolites. (2) The fermentation temperature is higher, can realize the thorough inactivation of harmful pathogenic bacteria, has reduced the possibility of polluting miscellaneous fungus. (3) The cooling cost in the large-scale fermentation process or water treatment process is reduced, and the energy consumption required for maintaining the optimal growth temperature of the extreme thermophilic bacteria is basically as much as that required for cooling the mesophilic bacteria. (4) As the temperature rises, the viscosity of the medium decreases, which reduces the energy required for agitation and allows better mixing of the medium. (5) At higher temperatures, most inorganic salts and organic molecules increase ionization and increase solvency, thereby favoring the production of more biomass and product quantities. (6) The enzyme generated by the thermophilic bacteria has the advantages of low enzyme activity, high stability and the like at normal temperature.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a novel Geobacillus strain as well as a culture method and application thereof. The thermophilic bacteria is used for perishable organic wastes and easily degradable organic high-temperature waste water, so that the technical bottleneck can be solved.
The purpose of the invention can be realized by the following technical scheme:
a new Geobacillus species identified as a Geobacillus species of Geobacillus thermodenitrificans CX412, designated Geobacillus sp.CX412 with a accession number of CGMCC No.22926.
Further, the new strain belongs to risk class 1, and the ribosomal DNA of the strain has SEQ ID No.1. In other words, geobacillus sp.CX412 is a new species of Geobacillus, and the 16S rRNA sequence thereof is shown in SEQ ID NO.1.
Further, the novel bacterial species are capable of producing biogenic substances in the presence of amino acids and polysaccharides and are capable of growing under thermophilic conditions at 70-90 ℃.
A method for culturing the new Geobacillus strain comprises the following steps: diluting the soil around the hot spring, coating the diluted soil on a screening culture medium, and performing screening culture to obtain a new strain; then inoculating the purified new strain into a fermentation culture medium for fermentation culture.
Further, the screening culture medium comprises the following base materials in parts by mass: 7-9 parts of tryptone, 6-8 parts of casein, 2-4 parts of glucose, 4-6 parts of sodium chloride, 1-3 parts of disodium hydrogen phosphate, 9-11 parts of dehydrated calf brain soaking powder and 14-16 parts of agar, wherein the pH value is 7.0-7.4.
Further, the fermentation medium comprises the following base materials in parts by mass: 4-6 parts of bovine albumin, 4-6 parts of tryptone, 4-6 parts of casein, 4-6 parts of glucose, 4-6 parts of soybean meal papain digest, 5-7 parts of sodium chloride, 1-3 parts of disodium hydrogen phosphate, 4-6 parts of dehydrated calf brain extract powder and 14-16 parts of agar, wherein the pH value is 7.2 +/-0.4.
Further, the temperature of the screening culture is 70-75 ℃, and the time is 20-72h; the temperature of the fermentation culture is 70-90 ℃, preferably 80 ℃, pH =6.8-7.6, preferably pH =7.2-7.4. The above-mentioned cultivation is carried out under aerobic conditions.
The application of the new bacillus cereus strain is used for aerobic fermentation of perishable organic wastes.
The application of the new Geobacillus strain is used for treating high-temperature wastewater containing a large amount of easily degradable organic matters.
The perishable organic wastes and the easily degradable organic high-temperature wastewater are secondary resources. Wherein the secondary resources comprise sewage sludge, livestock and poultry manure and high-temperature wastewater containing easily degradable organic matters. Wherein the strains produce biogenic substances which promote the degradation of perishable organic matters and synthesize abundant humus; the strain grows under thermophilic condition, which promotes the degradation and sedimentation of easily degradable organic high-temperature wastewater.
Further, the temperature range of the high-temperature wastewater is 70-90 ℃.
Compared with the prior art, the invention provides a new strain Geobacillus thermophilus (Geobacillus sp.) which is separated from soil around a hot spring, can promote aerobic fermentation of perishable organic matters to produce organic fertilizers, can treat easily degradable organic high-temperature wastewater and the like, and has wide market prospect.
Bacterial culture Collection notes
The Geobacillus thermophilus CX412 disclosed by the invention is preserved in China general microbiological culture Collection center (CGMCC) at 19.7.2021, and the preservation address is as follows: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101. the preservation number of the strain is as follows: CGMCC No.22926. The classification designation of the strain is Geobacillus sp.
Drawings
FIG. 1 is a pure culture colony of strain CX412 on a medium;
FIG. 2 is a 16S rRNA phylogenetic tree of strain CX 412;
FIG. 3 is an alignment of the sequence similarity of strain CX412 to existing strains;
FIG. 4 shows the results of ANI analysis of strain CX412 and the model strain.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.
A new Geobacillus strain is identified as a Geobacillus sp.CX412 strain with the preservation number of CGMCC No.22926. The new strain belongs to risk level 1, and the ribosome DNA of the strain has SEQ ID No.1. The new strain can produce biogenic substances in the presence of amino acids and polysaccharides, and can grow under thermophilic conditions of 70-90 deg.C.
The culture method comprises the following steps: diluting the soil around the hot spring, coating the diluted soil on a screening culture medium, and performing screening culture to obtain a new strain; then inoculating the purified new strain into a fermentation culture medium for fermentation culture. The screening culture medium comprises the following base materials in parts by mass: 7-9 parts of tryptone, 6-8 parts of casein, 2-4 parts of glucose, 4-6 parts of sodium chloride, 1-3 parts of disodium hydrogen phosphate, 9-11 parts of dehydrated calf brain soaking powder and 14-16 parts of agar, wherein the pH value is 7.0-7.4. The fermentation medium comprises the following base materials in parts by mass: 4-6 parts of bovine albumin, 4-6 parts of tryptone, 4-6 parts of casein, 4-6 parts of glucose, 4-6 parts of soybean meal papain digest, 5-7 parts of sodium chloride, 1-3 parts of disodium hydrogen phosphate, 4-6 parts of dehydrated calf brain extract powder and 14-16 parts of agar, wherein the pH value is 7.2 +/-0.4. The temperature of the screening culture is 70-75 ℃, and the time is 20-72h; the temperature of fermentation culture is 70-90 deg.C, and pH =6.8-7.6.
The new strain is used for aerobic fermentation of perishable organic wastes or for treating high-temperature wastewater containing a large amount of easily degradable organic matters. The temperature range of the high-temperature wastewater is 70-90 ℃.
Examples
Isolation and purification of Geobacillus thermophilus sp.CX412
The Geobacillus sp.CX412 is obtained by sampling hot spring soil in Tengchong City of Yunnan province, domesticating, separating and purifying. The method comprises the following specific steps:
preparing physiological saline (0.85) with sterile distilled water%) 10g of sample soil was diluted to 10 -5 Coating on a screening culture medium for culturing at 70 ℃; wherein, the formula of the screening culture medium is as follows: 8g of tryptone, 7g of casein, 3g of glucose, 5g of sodium chloride, 2g of disodium hydrogen phosphate, 10g of dehydrated calf brain soaking powder and 15g of agar, wherein the pH value is 7.0-7.4, and 1000ml of deionized water. Carrying out aerobic culture at 75 ℃ for 48h on a screening culture medium, picking out a single colony, and carrying out three-zone streaking separation and purification for 5 times to obtain the separated and purified pure culture bacteria.
Inoculating the activated pure culture bacteria into a fermentation culture medium suitable for thallus reproduction, and performing reproduction culture at 80 ℃ for 60 hours; wherein, the formula of the fermentation medium is as follows: 5g of bovine albumin, 5g of tryptone, 5g of casein, 5g of glucose, 5g of soybean meal papain digest, 6g of sodium chloride, 2g of disodium hydrogen phosphate, 5g of dehydrated calf brain extract powder, 15g of agar, pH value of 7.2 +/-0.4 and 1000ml of deionized water. A strain numbered CX412 is preferably selected from the group shown in FIG. 1.
Identification of Geobacillus thermophilus sp.CX412
The strain CX412 is subjected to whole genome sequencing by Shanghai worker to obtain a whole genome sequence. The phylogenetic tree is shown in fig. 2.
The determined CX 412S rRNA sequence is compared with the existing strain sequence in NCBI and EzBiocluod (FIG. 3), strain 16S rRNA sequence is selected according to similarity to construct phylogenetic tree, as shown in FIG. 2, CX412 is close to the genetic distance of Geobacillus and is independent branch in phylogenetic tree, geobacillus is determined, and is known as Paragene toebi NBRC 107807 according to similarity (99.59%), similar model strain complete genome is downloaded from NCBI, then JSpecies WS (http:// jspecies. Ribohost.com/jspecbank) and EzBiocluod are compared, and Average Nucleotide similarity (Average Nucleotide hybridization, ANI) (FIG. 4) and DNA hybridization value (DDH) are analyzed. ANI is an indicator of the relatedness of two genomes compared at the nucleotide level. ANI is defined as the average base similarity between homologous fragments of two microbial genomes and is characterized by a high degree of discrimination between closely related species. Compared with the traditional DDH, the ANI index is simple and time-saving in calculation, and is beneficial to building a structural database, so that the follow-up research of bioinformatics is facilitated.
In this experiment, the ANI of strain CX412 with strain Geobacillus sp.wch70 was 92.08%, the ANI with model strain, parameobacillus toebi NBRC 107807 was 91.35%, and the new cut-off value was 95% lower, and genomic DNA-DNA homology hybridization (DDH) was performed with model strain, parameobacillus toebi NBRC 107807: DNA Homology (DDH) was 35.5% [32.1-39% ], and DDH was less than 70%, and thus, it was determined to be a novel species.
Application of Geobacillus thermophilus (Geobacillus sp.CX412)
Pig manure, chicken manure and slaughter sewage sludge are piled according to 200 kg/group, the proportion of aerobic fermentation microbial inoculum sold in the market is 10% in a control group, the proportion of microbial inoculum containing Geobacillus (Geobacillus sp.CX412) in an experimental group is 10%, high-temperature stacking fermentation is carried out for 21 days after inoculation, and two groups of three parallel samples are respectively taken before and after fermentation.
The organic matter degradation rate of the control group is 40%, and the content of soluble humus is 15-25mg/gVS; the organic matter degradation rate of the experimental group is 60% (50% improvement), and the content of soluble humus is 25-50mg/gVS. The new strain of the invention can promote the degradation of perishable organic matters and synthesize rich humus, and has wide market application prospect.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Sequence listing
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tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatggg cggtacaaag 1260
ggctgcgaac ccgcgagggg gagcgaatcc caaaaagccg ctctcagttc ggattgcagg 1320
ctgcaactcg cctgcatgaa gccggaatcg ctagtaatcg cggatcagca tgccgcggtg 1380
aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagcttg caacacccga 1440
agtcggtgag gtaacccgta agggagccag ccgccgaagg tggggcaagt gattggggtg 1500
aagtcgtaac aaggtagccg taccggaagg tgcggctgga tcacct 1546
Claims (10)
1. A new species of Geobacillus, characterized in that it is a species of Geobacillus species identified as Geobacillus sp.CX412 with the accession number CGMCC No.22926.
2. The novel strain of Geobacillus as claimed in claim 1, wherein the novel strain belongs to Risk class 1, and the ribosomal DNA of the strain has SEQ ID No.1.
3. A novel species of Geobacillus as claimed in claim 1 or 2, wherein the novel species is capable of producing biogenic material in the presence of amino acids and polysaccharides and is capable of growing under thermophilic conditions at 70-90 ℃.
4. A method for culturing a novel strain of Geobacillus as claimed in any one of claims 1 to 3, which comprises: diluting the soil around the hot spring, coating the diluted soil on a screening culture medium, and carrying out screening culture to obtain a new strain; then inoculating the purified new strain into a fermentation culture medium for fermentation culture.
5. The method for culturing a novel strain of Geobacillus as claimed in claim 4, wherein the screening culture medium comprises the following base materials in parts by mass: 7-9 parts of tryptone, 6-8 parts of casein, 2-4 parts of glucose, 4-6 parts of sodium chloride, 1-3 parts of disodium hydrogen phosphate, 9-11 parts of dehydrated calf brain soaking powder and 14-16 parts of agar, wherein the pH value is 7.0-7.4.
6. The method for culturing the novel geobacillus strain as claimed in claim 4, wherein the fermentation medium comprises the following base materials in parts by mass: 4-6 parts of bovine albumin, 4-6 parts of tryptone, 4-6 parts of casein, 4-6 parts of glucose, 4-6 parts of soybean meal papain digest, 5-7 parts of sodium chloride, 1-3 parts of disodium hydrogen phosphate, 4-6 parts of dehydrated calf brain extract powder and 14-16 parts of agar, wherein the pH value is 7.2 +/-0.4.
7. The method for culturing a novel strain of Geobacillus as claimed in claim 4, wherein the temperature for the screening culture is 70-75 ℃ and the time is 20-72h; the temperature of fermentation culture is 70-90 deg.C, and pH =6.8-7.6.
8. Use of a novel strain of Geobacillus as claimed in any one of claims 1 to 3 for aerobic fermentation of perishable organic waste.
9. Use of a novel strain of Geobacillus as claimed in any one of claims 1 to 3 for the treatment of high temperature wastewater containing large amounts of easily degradable organics.
10. The use of a novel species of Geobacillus as claimed in claim 9, wherein the temperature of the high temperature wastewater is in the range of 70-90 ℃.
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