Enrichment method of anaerobic nitrate reducing microorganisms and application thereof

Abstract

The invention relates to an enrichment method of anaerobic nitrate reduction microorganisms and application thereof, and belongs to the technical field of microorganisms. According to the enrichment method disclosed by the invention, active cells in a solid sample are extracted by utilizing a Nycodenz density gradient centrifugation method, and then the active cells are inoculated into an anaerobic nitrate culture medium to carry out directional enrichment on anaerobic nitrate reducing microorganisms. According to the method, active cells can be efficiently enriched, the influence of a matrix is effectively reduced, and meanwhile, compared with a conventional cell enrichment technology, the efficiency is higher, and the cost is lower.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a method for enriching anaerobic nitrate-reducing microorganisms and its application. Background Technology

[0002] Current research on functional microorganisms mainly focuses on two aspects: one is the isolation and purification of functional microorganisms using pure culture techniques; the other is metagenomics, which can obtain massive amounts of DNA data without culturing. However, both methods have drawbacks. While the former can obtain single strains, its throughput is low and experimental procedures are complex. Microorganisms in ecosystems do not act individually but exhibit community effects, thus pure culture techniques have reached a bottleneck. Although metagenomics can discover a large number of new, unculturable species and explain problems from a community perspective, its results depend on sequencing depth and the computing power of supercomputers. To comprehensively interpret all microbial community information in a habitat (sequencing depth greater than 200 Gb), a significant amount of analysis and computation time and sequencing costs are required. Furthermore, metagenomics only characterizes the structure of microbial communities and cannot verify community function, so it needs to be combined with other metagenomic technologies, thus multiplying research costs.

[0003] Compared to aerobic systems, the isolation and culture of microorganisms in anaerobic systems are more rigorous, requiring multiple steps such as anaerobic culture and roll tube separation to enrich, culture, and purify the microbial community. Furthermore, unlike the homogeneity of liquid samples, obtaining the complete microbial community from solid samples is more challenging. Therefore, the enrichment of solid samples under anaerobic conditions remains a challenge. Summary of the Invention

[0004] The purpose of this invention is to provide a method for enriching anaerobic nitrate-reducing microorganisms and its application. This invention utilizes Nycodenz density gradient centrifugation to extract viable cells from solid samples, which are then inoculated into anaerobic nitrate culture medium for targeted enrichment of anaerobic nitrate-reducing microorganisms. The enriched products are then characterized using physicochemical parameters and metagenomic methods to efficiently obtain the target bacterial population while reducing research costs.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for enriching anaerobic nitrate-reducing microorganisms, comprising the following steps: (1) Microbial cells were isolated from solid samples to obtain cell suspensions; (2) Take the cell suspension and inoculate it into anaerobic nitrate-reducing medium and culture for 8-12 days to obtain the G1 generation culture; (3) Take the G1 generation culture and inoculate it into the anaerobic nitrate-reducing medium for 2-3 generations to obtain anaerobic nitrate-reducing microorganisms.

[0006] Preferably, the separation method in step (1) is Nycodenz density gradient centrifugation.

[0007] Preferably, the initial live cell concentration in the cell suspension in step (1) is ≥10,000 cells / mL.

[0008] Preferably, the volume ratio of the cell suspension to the anaerobic nitrate-reducing culture medium in step (2) is 1:100-200; The culture temperature is 20–25°C.

[0009] Preferably, the volume ratio of the G1 generation culture to the anaerobic nitrate-reducing medium in step (3) is 1:50 to 100; The subculture temperature is 20–25°C, and the culture time for each generation is 10–14 days.

[0010] Preferably, the anaerobic nitrate reduction culture medium described in steps (2) to (3) uses water as a solvent and includes the following components at the following concentrations: KNO3 0.5–1.5 g / L, K2HPO4 0.2–1.0 g / L, Na2HPO4 0.2–1.0 g / L, MgSO4·7H2O 0.1–0.5 g / L, CaCl2·2H2O 0.01–0.02 g / L, and CH3COONa 0.5–1.5 g / L.

[0011] This invention also provides the application of the enrichment method in the enrichment of active cells of anaerobic nitrate-reducing microorganisms.

[0012] The present invention has the following technical effects and advantages: The enrichment method of this invention, while retaining viable cells by using Nycodenz density gradient centrifugation, efficiently enriches anaerobic nitrate-reducing microbial communities in solid samples. This method can effectively reduce the influence of the matrix on the enrichment results, and is more efficient and lower in cost than conventional cell enrichment techniques, thus showing certain commercial potential. Attached Figure Description

[0013] Figure 1 The results show the concentrations of nitrate, nitrite, and ammonium in each anaerobic nitrate enrichment system. Figure 2 The composition of the microbial community in each solid sample and each enriched cell sample; Figure 3This section describes the functional and metabolic status of MAGs in the enrichment system. Here, A represents the number of bins in the sample, MQ bins represent medium-quality bins, and HQ bins represent high-quality bins; both are collectively referred to as MAGs (metagenomically assembled genomes). B represents the metabolic pathways of nitrate reduction-related MAGs. Detailed Implementation

[0014] This invention provides a method for enriching anaerobic nitrate-reducing microorganisms, comprising the following steps: (1) Microbial cells were isolated from solid samples to obtain cell suspensions; (2) Take the cell suspension and inoculate it into anaerobic nitrate-reducing medium and culture for 8-12 days, preferably 10 days, to obtain the G1 generation culture; (3) Take the G1 generation culture and inoculate it into the anaerobic nitrate reduction medium for 2 to 3 generations, preferably 2 generations, to obtain anaerobic nitrate reduction microorganisms.

[0015] In this invention, the separation method in step (1) is the Nycodenz density gradient centrifugation method.

[0016] In this invention, the initial live cell concentration in the cell suspension in step (1) is ≥10,000 cells / mL, preferably 10,000 cells / mL.

[0017] In this invention, the volume ratio of the cell suspension and the anaerobic nitrate reduction culture medium in step (2) is 1:100 to 200, preferably 1:150; The culture temperature is 20-25℃, preferably 24℃.

[0018] In this invention, the volume ratio of the G1 generation culture and the anaerobic nitrate reduction medium in step (3) is 1:50 to 100, preferably 1:50; The temperature for the subculture is 20–25°C, preferably 24°C; the culture time for each generation of subculture is 10–14 days, preferably 10 days.

[0019] In this invention, the anaerobic nitrate reduction culture medium described in steps (2) to (3) uses water as a solvent and includes the following components at the following concentrations: KNO3 0.5-1.5 g / L, preferably 1.0 g / L; K2HPO4 0.2-1.0 g / L, preferably 0.5 g / L; Na2HPO4 0.2-1.0 g / L, preferably 0.5 g / L; MgSO4·7H2O 0.1-0.5 g / L, preferably 0.2 g / L; CaCl2·2H2O 0.01-0.02 g / L, preferably 0.01 g / L; CH3COONa 0.5-1.5 g / L, preferably 1.0 g / L.

[0020] This invention also provides the application of the enrichment method in the enrichment of active cells of anaerobic nitrate-reducing microorganisms.

[0021] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0022] The solid samples of this invention are antimony-contaminated soil, saline-alkali soil, and weathered rock wall samples from caves. The collection sites are Xikuangshan in Lengshuijiang City, Hunan Province, the Hetao Plain Saline-alkali Experimental Area in Inner Mongolia Autonomous Region, and the weathered rock wall of Heshangdong in Yichang City, Hubei Province, respectively. The DNeasy PowerSoil kit (catalog number: 12888-100) and DNeasy PowerWater kit (catalog number: 1490-1000-NF) used in the reagents of this invention were purchased from Qiagen GmbH, Germany; the NEBNext Ultra DNA library preparation kit (catalog number: NEB#E7103) was purchased from New England Biolabs, USA; and the SYTO 9 green fluorescent nucleic acid dye was purchased from Thermo Fisher Scientific, USA. In the instruments used in this invention, the CytoFLEX SRT flow cytometer was purchased from Beckman Coulter, Inc., USA; the NanoDrop 2000 spectrophotometer was purchased from Thermo Fisher Scientific, Inc., USA; and the Epoch microplate reader was purchased from BioTek, Inc., USA.

[0023] In the method of this invention, the gas chromatography method uses a TRACE 1600 gas chromatograph, which was purchased from ThermoFisher Scientific, USA.

[0024] Example 1: Enrichment of Anaerobic Nitrate-Reducing Microorganisms (1) Solid samples were collected from antimony-contaminated soil, saline-alkali soil and weathered rock walls of caves. Anaerobic nitrate-reducing microorganisms were isolated and enriched using Nycodenz density gradient centrifugation. Specifically, 2g of each solid sample was taken and 6mL of 1×PBS buffer containing 0.5% Tween-20 was added. After incubation at 4℃ and 400rpm for 20min, 6mL of Nycodenz solution with a density of 1.3g / mL was added to each solid sample. After centrifugation at 4℃ and 14000×g for 90min, the cell-containing component at the density gradient interface was collected. After centrifugation at 4℃ and 500×g for 55min, residual Nycodenz was completely removed to obtain the precipitate. The precipitate was resuspended in 2mL of 1×PBS buffer to obtain cell suspensions of each solid sample that could be used for subsequent analysis. (2) Take 1 mL of each cell suspension and mix thoroughly with 3 μL of SYTO 9 green fluorescent nucleic acid dye. Incubate at room temperature in the dark with shaking at 400 rpm for 1 h. Detect and quantify using a CytoFLEX SRT flow cytometer. Dilute each cell suspension to an initial viable cell concentration of 10,000 cells / mL. Take 1 mL of the diluted cell suspension and inoculate it into 150 mL of deoxygenated anaerobic nitrate-reducing medium. Incubate at 24℃ for 10 days until the logarithmic growth phase (OD2). 600nm =0.12), to obtain the G1 generation culture; take 50 mL of the G1 generation culture and concentrate it to 12 mL with 1×PBS buffer, then take 1 mL of the concentrated G1 generation culture and inoculate it into 50 mL of anaerobic nitrate-reducing medium for two subcultures, set the culture temperature at 24℃, and culture for 10 days each time to obtain anaerobic nitrate-reducing microorganisms; wherein: The anaerobic nitrate reduction medium was prepared using water as a solvent and included 1.0 g KNO3, 0.5 g / L K2HPO4, 0.5 g / L Na2HPO4, 0.2 g / L MgSO4·7H2O, 0.01 g / L CaCl2·2H2O, and 1.0 g / L CH3COONa, with the pH adjusted to 7.0.

[0025] Experimental Example 1: Determination of Nitrate, Nitrite and Ammonium Concentrations The method described in Example 1 was used to isolate and enrich anaerobic nitrate-reducing microorganisms from solid samples of antimony-contaminated soil, saline-alkali soil, and weathered rock walls of caves. After each subculture, the culture was centrifuged at 8000 rpm for 5 min, and the supernatant and enriched cell pellet were separated. The NO3 content in each enrichment system was measured using an Epoch microplate reader. - NO2 - and NH4 + The concentration of N2O in the nitrate reduction system was determined by gas chromatography. The results are as follows: Figure 1 As shown.

[0026] The results showed that anaerobic nitrate-reducing microorganisms isolated from antimony-contaminated soil, saline-alkali soil, and weathered cave wall solid samples were capable of nitrate reduction, and these microbial groups achieved a nitrate reduction efficiency of 100%, generally reducing NO3 by the sixth day. - The concentration then falls below the detection limit, and the process ultimately produces N2O through denitrification.

[0027] Experiment Example 2: Metagenomic Analysis Genomic DNA was extracted from 1g of solid samples and 1mL of enriched cell samples using the DNeasy PowerSoil kit and the DNeasy PowerWater kit, respectively. The concentration and purity of each genomic DNA sample were determined using a NanoDrop 2000 spectrophotometer. Libraries were constructed from the qualified genomic DNA using the NEBNext Ultra DNA library preparation kit. The constructed libraries were sequenced using the Illumina NovaSeq 6000 platform, with a sequencing depth of 6Gb for each solid sample and 2Gb for each enriched cell sample. Sequencing data were processed using Trimmomatic (V.0.36) software for primer removal, removal of sequences with more than 5 "N"s, and low-quality sequence filtering to obtain high-quality sequences. Each sample was assembled into contigs using MEGAHIT (V.1.1.3) software. Contigs with a sequence length <500bp were filtered out. Binning templates (MetaBAT2 and MaxBin2) from the metaWRAP pipeline were used to obtain bin sets. The bin sets were merged into a final bin set using the Bin_refinement module. Aggregation and deduplication were performed using dRep software (-comp 50 -con 10 options). CheckM software was used to evaluate the integrity, contamination and heterogeneity of each Bin set. Medium-quality Bin sets (integrity > 50% and contamination < 10%) and high-quality Bin sets (integrity > 90% and contamination < 5%) were retained as MAGs. Genome Taxonomy Database (GTDB) (release 93) was used as the benchmark database, and GTDB-tk software was used to compare genomic taxonomic information. The functions of each sequence in MAGs were predicted using Prodigal (V.2.6.3) software, yielding the DNA and protein sequences of the functional genes. The protein sequences encoded by these functional genes were aligned with the NCBI nr database (release 20200128), eggNOG, and KEGG databases. Functional annotation was performed using DIAMOND blastp software, with an e-value threshold of 10. -5 Relative abundance statistics were performed using BWA-MEM software, and the results are as follows: Figures 2-3 As shown.

[0028] Metagenomics results showed that, compared to direct metagenomic sequencing of raw samples, sequencing of enriched samples using metagenomics improved the efficiency of obtaining high-quality MAGs (metagenomically assembled genomes) by 10-20%, while reducing experimental and sequencing costs, demonstrating the feasibility of the targeted enrichment method described in this invention. Furthermore, the microbial community structures of the three different soil samples treated by the enrichment method described in this invention tended to be similar, and were predominantly... Pseudomonas To dominate Pseudomonas Its genomic metabolic pathways also show that it has a complete nitrate reduction and denitrification pathway.

[0029] As can be seen from the above embodiments, the present invention provides a method for enriching anaerobic nitrate-reducing microorganisms and its application. The enrichment method of the present invention enriched anaerobic nitrate-reducing microbial communities in different environmental samples and found that anaerobic nitrate-reducing microbial communities from soil environments of different sources have similar community structures. These results indicate that the synergistic effect of microorganisms can maintain the stability of the microenvironment.

[0030] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for enriching anaerobic nitrate-reducing microorganisms, characterized in that, Includes the following steps: (1) Microbial cells were isolated from solid samples to obtain cell suspensions; (2) Take the cell suspension and inoculate it into anaerobic nitrate-reducing medium and culture for 8-12 days to obtain the G1 generation culture; (3) Take the G1 generation culture and inoculate it into the anaerobic nitrate-reducing medium for 2-3 generations to obtain anaerobic nitrate-reducing microorganisms.

2. The enrichment method according to claim 1, characterized in that, The separation method described in step (1) is the Nycodenz density gradient centrifugation method.

3. The enrichment method according to claim 2, characterized in that, The initial live cell concentration in the cell suspension in step (1) is ≥10,000 cells / mL.

4. The enrichment method according to claim 1, characterized in that, The volume ratio of the cell suspension and the anaerobic nitrate-reducing medium in step (2) is 1:100-200; The culture temperature is 20–25°C.

5. The enrichment method according to claim 1, characterized in that, The volume ratio of the G1 generation culture to the anaerobic nitrate-reducing medium in step (3) is 1:50-100; The subculture temperature is 20–25°C, and the culture time for each generation is 10–14 days.

6. The enrichment method according to claim 5, characterized in that, The anaerobic nitrate reduction medium described in steps (2) to (3) uses water as a solvent and includes the following components at the following concentrations: KNO3 0.5-1.5 g / L, K2HPO4 0.2-1.0 g / L, Na2HPO4 0.2-1.0 g / L, MgSO4·7H2O 0.1-0.5 g / L, CaCl2·2H2O 0.01-0.02 g / L, and CH3COONa 0.5-1.5 g / L.

7. The application of the enrichment method according to any one of claims 1 to 6 in the enrichment of active cells of anaerobic nitrate-reducing microorganisms.

Images