Method for rapidly screening and identifying functional bacteria

Abstract

The invention discloses a method for rapidly screening and identifying functional bacteria, which comprises the following steps of: performing phenotype primary screening, function inspection and genotype determination, determining bacteria screening raw materials (S1), determining a functional culture medium (S2), determining functional indexes and a detection method (S3), performing phenotype primary screening (S4), performing phenotype secondary screening (S5) and performing genotype final screening (S6), thereby realizing the screening of the functional bacteria. The strain screening efficiency and the strain function reliability are remarkably improved, the cost is reduced, and the method is suitable for the fields of strain screening and microbial agent development.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial screening, and in particular to a method for rapid screening and identification of functional bacteria, which is applicable to the fields of strain screening and bacterial agent development. Background Art

[0002] Functional bacteria are specialized strains that possess specific functions, can solve specific problems, or achieve specific goals. Screening for functional bacteria involves isolating and selecting these specific functional bacteria from raw materials, preserving them, and reusing them repeatedly. Current patented microbial screening technologies face significant technological gaps. Traditional screening methods are often limited to single-dimensional verification, manifesting as a dual-track "phenotype-gene" approach. At the phenotypic screening level, mainstream patents still rely on colony morphology (edge regularity, color differentiation) or single biochemical indicators (such as ammonia nitrogen conversion rate) as core screening criteria, resulting in false positive rates as high as 30%. In the field of genetic testing, although NGS technology has enabled broad-spectrum detection of pathogenic microorganisms, existing patents only use it as a supplementary verification tool, without establishing screening models that integrate phenotypic data. This fragmented screening approach results in strain development cycles as long as 18-24 months.

[0003] The present invention optimizes the traditional method of only selective culture medium or gene identification by integrating phenotypic screening with gene function analysis, and can accurately, efficiently and economically screen out excellent strains with target functions, thus solving the above problems. Summary of the Invention

[0004] The purpose of the present invention is to solve the problems of low screening efficiency or poor reliability in traditional bacteria screening schemes.

[0005] The technical solution of the present invention to solve the above technical problems includes the following steps:

[0006] S1: Determine the environmental habitat or medium where the functional bacteria may exist, and obtain the screening materials in a targeted manner;

[0007] S2: Determine the specific culture medium X1 and detection method for functional bacteria based on the function of the target bacteria;

[0008] S3: Prepare the screened bacterial material into a bacterial suspension, inoculate it onto a plate of X1 medium for cultivation, pick the colonies grown on the plate individually, and preserve them to obtain the primary screening strains;

[0009] S4: Inoculate the selected individual strains into X1 liquid culture medium, and detect the increase or decrease of indicators directly related to the concentration changes of products and substrates in the metabolic pathway regulated by the target function. Select the best mid-screen strains based on the changes in the indicator values;

[0010] S5: Use gene identification methods such as 16s rRNA and whole genome detection to identify the species or genes of the strains finally screened out, and compare them with the database. Strains that match the species or functional gene fragments are the target functional bacteria.

[0011] Beneficial effects of the present invention:

[0012] This approach optimizes traditional methods involving selective culture media or genetic identification. Phenotypic screening eliminates non-target strains, reducing genetic testing costs while ensuring that selected strains express the desired function. Subsequently, genetic testing is performed on strains demonstrating phenotypic expression, accurately identifying strains with functional genes at the genetic level. Combining these two approaches overcomes the limitations of traditional methods, enabling the accurate and cost-effective selection of high-quality strains with the desired function. This approach provides a highly effective microbial agent development solution for nitrogen resource utilization in organic solid waste composting, with significant environmental application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is a flow chart of a method in an embodiment of the present invention; Figure 2 It is the nitrogen source culture medium used in the examples; Figure 3 is 10 -3 Growth of colonies on culture dishes at different dilution multiples; Figure 4 The growth of the strains was screened for phenotypic screening; Figure 5 The ammonia assimilation ability of the preferred strain; Figure 6 The growth ability of the preferred strain. DETAILED DESCRIPTION

[0014] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the technical solutions of the present invention are described clearly and completely below in conjunction with specific embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0015] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0016] The present invention discloses a method for rapid screening and identification of ammonia assimilating bacteria, which mainly comprises the following steps: (1) Determining the source of ammonia assimilated bacteria by habitat Ammonia-assimilating bacteria are commonly found in livestock and poultry manure and during composting, and they generally promote aerobic composting and help preserve nitrogen in the compost. Therefore, livestock and poultry manure was selected as the raw material for screening ammonia-assimilating bacteria.

[0017] (2) Determine the culture medium and characteristic detection method of ammonia assimilating bacteria Ammonia-assimilating bacteria primarily convert ammonia nitrogen into amino acids for storage. In addition to the nutrients normally required for bacterial life, they require large amounts of ammonia nitrogen. Therefore, a culture medium containing ammonium nitrogen as the sole nitrogen source is selected. Characteristic detection methods measure ammonia nitrogen, specifically the rate of consumption of substrates in the ammonia assimilation metabolic pathway.

[0018] (3) Sampling The original livestock and poultry manure samples were collected from the composting site, placed in sampling bags, and sealed and stored in a -20°C refrigerator.

[0019] (4) Preparation and preservation of nitrogen source culture medium Prepare a solid medium for screening ammonia-assimilating bacteria. The main components of the medium are 0.5% glucose, 0.01% ferric ammonium citrate, 0.025% ammonium chloride, 0.05% magnesium sulfate heptahydrate, 0.05% sodium chloride, 0.01% manganese chloride tetrahydrate, 0.1% potassium dihydrogen phosphate, 0.32% potassium dihydrogen phosphate, and 1.5% agar. Sterilize and store in a refrigerator at 45°C.

[0020] (5) Preliminary culture of ammonia assimilating bacteria Take 10g of chicken manure in a 100mL conical flask, add 100mL of sterile deionized water, dissolve it by shaking, and place it in a constant temperature shaking table at 37℃ and 170 r·min. -1 Oscillate for 30 minutes under the same conditions to prepare the bacterial stock solution, which was diluted several times to 10% of the stock solution. -5 From the original, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Take 100 μL of the original solution from each centrifuge tube of the diluted concentration, inoculate it into the nitrogen source solid culture medium by evenly spreading the plate and culture it. Make three parallels for each dilution concentration.

[0021] (6) Isolation of ammonia assimilation bacteria According to the degree of dispersion of the colonies, the clarity of the colonies and the number of colonies, the appropriate dilution factor (10 in this embodiment) is selected. -3 The dilution multiple was recorded in the culture dishes. Single colonies with different morphologies (size, color, shape, transparency, and whether the edges were neat, etc.) were taken from the culture dishes and inoculated into solid culture media respectively. A total of 22 strains were taken and separated.

[0022] (7) Isolation and purification of ammonia assimilation bacteria The bacteria in different culture dishes were separated by a four-zone streak method in nitrogen source culture medium for multiple times until pure bacteria were obtained.

[0023] (8) Preservation of bacterial strains The obtained pure bacteria were inoculated into 50 mL centrifuge tubes, 40 mL of culture medium was added, and the tubes were placed in a constant temperature shaking incubator at 37 ° C and 170 r min. -1 The mixture was shaken under the conditions of 40 ° C for 30 minutes to prepare a bacterial solution, which was placed in a bacterial incubator and expanded and cultured at 37 ° C for 2 days. It was then mixed with glycerol in a 1:1 ratio and stored in a bacterial culture tube and placed in a -80 ° C refrigerator.

[0024] (9) Screening of ammonia assimilation bacteria The obtained pure bacteria were inoculated into 50 mL centrifuge tubes, 40 mL of culture medium was added, and the tubes were placed in a constant temperature shaking incubator at 37 ° C and 170 r min. -1 The mixture was shaken under the conditions of 40 °C for 30 min to prepare a bacterial solution, which was placed in a bacterial incubator and cultured at 37 °C. The ammonia nitrogen content (using Nash reagent spectrophotometry) and OD600 value were detected at 24 h, 48 h, and 72 h, respectively, as shown in Table 1.

[0025] Table 1

[0026] Five strains with better ammonia assimilation phenotypes were selected, namely ①5, ③5, ③7, ②4, and -2②1.

[0027] (10) Identification of the gene lineage of ammonia assimilating bacteria The sample was inoculated into 1-2 mL of liquid culture medium and cultured overnight. The sample was sent for 16S rRNA detection to identify the species. The whole genome was sent for testing and the functional genes were obtained using Prokka software through the whole genome bioinformatics analysis process. The functional genes were annotated using Diamond software combined with KEGG and NCBI NR databases using Prokka software to screen for carriers. gdhA、glnA The strains were further analyzed for core genes such as GDH and GS-GOGAT pathway integrity through KEGG analysis. The database was compared to verify whether they had genes for ammonia assimilation.

[0028] (11) Screening was performed to obtain strains carrying ammonia assimilation genes and having the best ammonia assimilation performance.

[0029] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for rapid screening and identification of functional bacteria, characterized in that: The following steps are involved: Determination of screening raw materials (S1), determination of functional culture medium (S2), determination of functional indicators and detection methods (S3), initial phenotypic screening (S4), secondary phenotypic screening (S5), and final genotypic screening (S6).

2. The method according to claim 1, characterized in that S1, determine the environment or medium where the functional bacteria may exist, and obtain the screening raw materials in a targeted manner.

3. The method according to claim 1, characterized in that S2. Determine the specific culture medium X1 and detection method for functional bacteria based on the function of the target bacteria.

4. The method according to claim 1, characterized in that S3, prepare the screened bacterial raw material into a bacterial suspension, inoculate it onto a plate of X1 medium for cultivation, pick the colonies grown on the plate separately, preserve them, and obtain the primary screening strains.

5. The method according to claim 1, characterized in that S4, inoculate the screened individual strains into X1 liquid culture medium respectively, detect the increase or decrease of indicators directly related to the target function, and select the mid-screen strains with good effects based on the changes in the indicator values.

6. The method according to claim 1, characterized in that S5. Use gene identification methods such as 16s rRNA and whole genome detection to identify the species or genes of the strains screened out in the final screening, and compare them with the database. Strains that match the species or functional gene fragments are the target functional bacteria.

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