Thermophilic bacteria Geobacillus sp.Cys63 and application thereof

Abstract

The invention discloses a thermophilic bacterium Geobacillus sp.Cys63 and application thereof, the thermophilic bacterium Geobacillus sp.Cys63 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20242661. According to the present invention, the thermophilic bacteria Geobacillus sp.Cys63 has strong heat resistance, and the optimum growth temperature can achieve 75 DEG C; the bacillus subtilis has the advantages that the bacillus subtilis has a strong capability of producing high-temperature hydrolase, can secrete amylase, protease and esterase under a high-temperature condition, has a capability of dissolving insoluble phosphorus, and can intensively decompose starch, fat and protein substances in organic solid wastes; the temperature of a compost body in the composting process can be obviously improved, and the harmless degree and the quality of a compost product are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental microorganisms, and in particular relates to a thermophilic bacterium Geobacillus sp.Cys63 and an application thereof. Background Art

[0002] With the continuous improvement of my country's urbanization level, the amount of sewage and wastewater has also increased year by year. As a byproduct of sewage treatment plants, the amount of residual sludge generated has also increased sharply. Sludge has the dual attributes of "pollution" and "resource". Sludge is enriched with toxic and harmful substances such as pathogenic bacteria and heavy metals in sewage. At the same time, there are a large number of organic matter containing nitrogen and phosphorus that is rich in nutrients. Following the principle of "reduction, stabilization, harmlessness, and resource utilization", the sludge is composted through high-temperature aerobic fermentation technology to convert the residual sludge into fertilizer or soil conditioner. While avoiding sludge from polluting the environment, the sludge fermentation products can also improve soil fertility.

[0003] Temperature is an important factor affecting the sludge composting process, which directly affects the efficiency of composting and the quality of the final product. Generally, the temperature of the pile rises rapidly to the highest temperature in the early stage and maintains in the high temperature period (≥50℃) for a period of time. After that, the temperature begins to decrease and reaches the cooling stage and the maturity stage. Maintaining the high temperature period helps to accelerate the humification process, shorten the composting cycle, improve the degradation efficiency of antibiotics and resistance genes, and remove pollutants. However, in the traditional composting process, the composting temperature exceeds 70℃, which will inhibit the activity of almost all microorganisms, making it difficult for the subsequent composting process to run stably. Therefore, it is of great significance to isolate and screen thermophilic bacteria (optimal growth temperature > 60℃) and apply them to sludge composting. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a thermophilic bacterium Geobacillus sp.Cys63 and its application.

[0005] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0006] A thermophilic bacterium Geobacillus sp.Cys63, which is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO:M20242661, a deposit date of November 28, 2024, and a deposit address of Wuhan University, Wuhan, China.

[0007] The thermophilic bacterium Geobacillus sp.Cys63 includes the gene sequence shown as SEQ ID NO.1.

[0008] The present invention also provides a bacterial agent, which contains the thermophilic bacterium Geobacillus sp. Cys63.

[0009] The present invention also provides an application of the thermophilic bacterium Geobacillus sp.Cys63 or the bacterial agent in sludge composting treatment.

[0010] As a further improvement, the thermophilic bacteria Geobacillus sp. Cys63 is inoculated into the sludge compost material for aerobic fermentation.

[0011] As a further improvement, according to (0.5~1.5)×10 6 CFU / g 物料 The thermophilic bacteria Geobacillus sp.Cys63 was inoculated into the sludge compost material at a concentration of.

[0012] As a further improvement, according to 10 6 CFU / g 物料 The thermophilic bacteria Geobacillus sp.Cys63 was inoculated into the sludge compost material at a concentration of.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The thermophilic bacterium Geobacillus sp.Cys63 of the invention has strong heat resistance, and the optimum growth temperature reaches 75 DEG C; it has strong ability to produce high-temperature hydrolases, can secrete amylase, protease and esterase under high temperature conditions, and especially has the ability to dissolve insoluble phosphorus, and can strengthen the decomposition of starch, fat and protein substances in organic solid waste; its phosphorus dissolving activity shows that it has the ability to convert insoluble phosphorus in a pile into soluble phosphorus, and can promote the absorption of phosphorus by plants and promote plant growth when compost products are used in the later stage; the strain can significantly increase the temperature of the pile in the composting process, and improve the harmlessness degree and quality of the compost products. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 is the phylogenetic tree of Cys63 strain based on 16S rRNA;

[0017] Figure 2is the optimal growth temperature of the Cys63 strain;

[0018] Figure 3 It is the hydrolysis circle formed by Cys63 on different screening plates;

[0019] Figure 4 This is the temperature change curve of the compost pile in the small-scale composting test inoculated with Cys63 strain. DETAILED DESCRIPTION

[0020] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0021] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0022] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0023] Example 1 Screening of Cys63 strain

[0024] Hot spring samples were collected from Reshui Town, Chenzhou City, Hunan Province. The samples were cultured and enriched using Cys medium. The culture temperature was gradually increased to 75°C, and the enriched liquid was gradient diluted and separated. The samples were cultured at 70°C, and single colonies grew on Cys solid medium. After multiple streaking, a pure single colony was obtained, named Cys63. The colony morphology is round, light yellow, opaque, with neat edges and smooth surface.

[0025] The 16S rRNA gene of Cys63 strain was amplified using primers 27F and 1492R. After NCBI Blast analysis, the closest relative of Cys63 was Geobacillus sp. JC106. The 16S rRNA gene of Cys63 strain was used together with the 16S rRNA genes of other strains of Geobacillus included in the database to construct a phylogenetic tree, such as Figure 1 As shown, the Cys63 strain was clustered with Geobacillus sp. JC106, and the Cys63 strain was identified as Geobacillus sp.

[0026] After sequencing, the 16S rDNA sequence of Cys63 is shown as SEQ ID NO.1.

[0027] SEQ ID NO.1:

[0028]

[0029] Example 2 Performance determination of Cys63 strain

[0030] 1. Thermophilic ability

[0031] The Cys63 strain was shaken and cultured at different temperatures. The Cys medium used was composed of 4.0 g yeast extract, 6.0 g hydrolyzed casein, 3.0 g soluble starch, 3.0 g NaCl, 0.27 g MgCl2·6H2O, 0.025 g CaCl2, 0.01 g FeSO4·7H2O, 100 μL trace elements (12.0 g / L Na2MoO4·2H2O, 1.0 g / L VOSO4·xH2O, 5.0 g / L MnCl2, 0.6 g / L ZnSO4·7H2O, 0.15 g / L CuSO4·5H2O, 8.0 g / L CoCl2·6H2O, 0.2 g / L NiCl2·6H2O), initial pH 7.5, and 1000 mL distilled water.

[0032] After shaking culture for 16 h, the OD 600 The absorbance value at Figure 2 As shown, the Cys63 strain grows well between 60℃ and 75℃, and its optimal growth temperature is 75℃, which means it is an extremely thermophilic microorganism.

[0033] 2. Ability to produce high temperature hydrolase and phosphate solubilizing activity

[0034] The Cys63 strain was inoculated onto a selection medium containing starch, skimmed milk powder, tributyrin and tricalcium phosphate, respectively. The culture medium was as follows:

[0035] Starch hydrolysis medium: soluble starch 10g / L, peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, low acyl gellan gum 10g / L, agar powder 5.0g / L, 0.015% trypan blue dye solution;

[0036] Protein hydrolysis medium: 1.5% skim milk powder, peptone 10.0g / L, beef extract powder 3.0g / L, sodium chloride 5.0g / L, gellan gum 10.0g / L, agar powder 5.0g / L;

[0037] Esterase screening medium: 4% polyvinyl alcohol was used to emulsify tributyrin at a volume ratio of 3:1, and added to NB medium (peptone 10.0 g / L, beef extract powder 3.0 g / L, sodium chloride 5.0 g / L) at a ratio of 2%, and gellan gum 10.0 g / L and agar powder 5.0 g / L were added;

[0038] Phosphorus solubilizing activity assay plate: glucose 10 g / L, Ca3(PO4)2 5 g / L, (NH4)2SO4 0.1 g / L, NaCl 0.2 g / L, MgSO4·7H2O 0.25 g / L, KCl 0.2 g / L, gellan gum 10.0 g / L, agar powder 5.0 g / L.

[0039] After culturing in a 70°C incubator for 18-48 hours, obvious hydrolysis circles appeared on the screening plates (such as Figure 3 ), indicating that the Cys63 strain has the ability to produce thermophilic amylase, protease and esterase, and has phosphate solubilizing activity.

[0040] The Cys63 strain has a strong ability to produce high-temperature hydrolases, which is beneficial to the degradation of macromolecular organic matter during the composting process, thereby promoting the temperature rise and maturity of the compost. Its phosphorus solubilizing ability shows that it has the potential to convert insoluble phosphorus in the soil into soluble phosphorus for plant absorption and promote plant growth, which has a promoting effect on improving the quality of compost products in the secondary fermentation stage.

[0041] The applicant has conducted a large number of comparative studies and has not yet found other strains of Geobacillus (e.g. Figure 1 Other strains in the strain have the activity of solubilizing insoluble phosphorus.

[0042] Example 3 Application of Cys63 strain in sludge composting

[0043] The extremely thermophilic strain Cys63 was expanded and cultured (Cys medium, culture temperature 65-70°C) for 10 5 CFU / g 物料 , 10 6 CFU / g 物料 and 10 7 CFU / g 物料 The concentrations were inoculated into the sludge composting materials, and the control group was inoculated with the same volume of sterilized culture medium. The temperature during the composting process was detected in real time, such as Figure 4 As shown, according to 10 6 CFU / g 物料 After Cys63 was inoculated into the compost at a concentration of , the duration of the compost in the high temperature period (≥50℃) was significantly increased compared with the control group, which was 1.9 times the duration of the control group. This shows that appropriate inoculation of Cys63 strains can prolong the high temperature period, contribute to the degradation of macromolecular organic matter and pollutants during sludge composting, improve the degree of harmlessness, and accelerate the compost humification process.

[0044] In summary, the present invention obtains an extremely thermophilic strain Geobacillus sp.Cys63, whose optimal growth temperature reaches 75°C. It has the ability to produce high-temperature amylase, protease, esterase and dissolve phosphorus. After exogenous inoculation of Geobacillus sp.Cys63 into sludge composting materials, the maintenance time of the compost in the high temperature period can be significantly prolonged, which helps to improve the harmlessness of sludge composting and improve the humification process.

[0045] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Therefore, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the scope of protection of the technical solution of the present invention.

Claims

1. A thermophilic bacterium Geobacillus sp.Cys63, characterized in that: It is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO:M20242661.

2. The thermophilic bacterium Geobacillus sp.Cys63 according to claim 1, characterized in that The thermophilic bacterium Geobacillus sp.Cys63 includes the gene sequence shown as SEQ ID NO.

1.

3. A bacterial agent, characterized in that: The bacterial agent comprises the thermophilic bacterium Geobacillus sp. Cys63 according to claim 1 or 2.

4. Use of the thermophilic bacterium Geobacillus sp. Cys63 according to claim 1 or 2, or the bacterial agent according to claim 3 in sludge composting treatment.

5. The use according to claim 4, characterized in that: The thermophilic bacteria Geobacillus sp. Cys63 is inoculated into the sludge compost material for aerobic fermentation.

6. The use according to claim 5, characterized in that: According to (0.5~1.5)×10 6 CFU / g 物料 The thermophilic bacteria Geobacillus sp.Cys63 was inoculated into the sludge compost material at a concentration of.

7. The use according to claim 6, characterized in that: Follow 10 6 CFU / g 物料 The thermophilic bacteria Geobacillus sp.Cys63 was inoculated into the sludge compost material at a concentration of.

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