CN115011589A - Method for separating in-situ functional microorganisms by using reverse SIP-metagenomics - Google Patents

Abstract

The invention discloses a method for separating in-situ functional microorganisms by utilizing reverse SIP-metagenomics. The method mainly comprises the following steps: the method comprises the steps of carrying out in-situ exploration on degradation functional microorganisms in PAHs contaminated soil by adopting a DNA-SIP technology, identifying the functional microorganisms according to the results of DNA-SIP and high-throughput sequencing, then carrying out metagenome sequencing and analysis on heavy-layer DNA to obtain genomes of identified functional bacteria, analyzing the utilization and resistance characteristics of the strains in C source, vitamins and mineral elements, setting a specific culture medium to separate the microorganisms in the contaminated environment, and finally comparing the SIP-metagenome exploration result with an indoor culture separation experiment result to confirm whether the separated strains are the functional microorganisms which participate in pollutant degradation in situ. The technology of the invention can effectively separate and identify the microorganism with the function of degrading the polycyclic aromatic hydrocarbon in situ, and has wide application prospect.

Description

Method for separating in-situ functional microorganisms by using reverse SIP-metagenomics

Technical Field

The invention relates to the technical field of biology, in particular to a method for separating in-situ functional microorganisms by utilizing reverse SIP-metagenomics.

Background

With the rapid development of modern industry, the pollution is increasingly serious. Various persistent organic pollutants, such as Polycyclic Aromatic Hydrocarbons (PAHs), etc., are generally present in industrial wastes. Bioremediation is a low-cost and environment-friendly PAHs pollution remediation mode (Wilson, 1993; Harayama,1997), and microorganisms can degrade PAHs in the environment so as to achieve the remediation purpose. To explore the homing of PAHs in the environment, several culture-based identification methods were able to identify and isolate microorganisms that were able to degrade them individually. To date, many functional microorganisms have been isolated which degrade PAHs, mostly from strains of Paenibacillus (dane et Al, 2002), Burkholderia (Juhasz et Al, 1997), Stenotrophomonas (Juhasz et Al, 2000), Acinetobacter (Al-hadithii et Al, 2017), Alcaligenes (Kim et Al, 2005), mycobacter (Lease et Al, 2011), arthromobacter (Samanta et Al, 2002), Flavobacterium (Samanta et Al, 2002), Vibrio (samania et Al, 2002), eudomas (Van Hamme et Al, 2003), cyclophycus (Wong et Al, 2002), Sphingomonas (Zo et Al, 2008). The culture-dependent method provides clues for determining the types, functional genes and metabolic characteristics of degrading bacteria. However, in real life situations, only a few functional contaminant-degrading microorganisms can be isolated (Amann et al, 1995; Li et al, 2017); moreover, it is doubtful whether the isolated functional microorganism can function in a natural habitat. Since most microorganisms present in the natural environment are not culturable, moreover, this method greatly underestimates the prokaryotic microbial diversity (Oren,2004), does not explain the complex interactions between individuals within the microbial community in the natural environment, nor does it reflect the true situation of degradation of pollutants by functional microorganisms in contaminated environments (Li et al, 2019; Li et al, 2020). Therefore, it is difficult to reveal the ecological function of indigenous microorganisms using laboratory culture hair. In contrast, the use of non-culturable methods makes it possible to evaluate the metabolic response of functional microorganisms without the need for laboratory culture and to directly link their properties to functions in the natural habitat.

Stable Isotope Probe (SIP) technology enables the combination of stable isotope labeling technology with molecular biological means such as high-throughput sequencing, TRFLP, etc., by adding stable isotope labeled target compounds to environmental samples: ( ¹³ C or ¹⁵ N), analysis of biomarkers (such as DNA, RNA, etc.) labeled with stable isotopes using molecular biological means allows determination of microorganisms with degradative properties in environmental samples, in particular non-culturable microorganisms in the environment (Jiang et al, 2015). This technique can bypass the requirement of isolated culture and directly link microbial communities and functions (Dumont and Murrell, 2005). Currently, various PAHs degrading functional bacteria have been successfully identified by applying SIP technology (Singleton et al, 2007; Jones et al, 2008; Jones et al, 2011; Gutierrez et al, 2013; Song et al, 2016).

Although various PAHs degrading strains have been successfully identified in natural environments such as soil and seawater by the DNA-SIP technology (Jeon et al, 2003; Jones et al, 2011; Martin et al, 2012; Gutierrez et al, 2013; Regonne et al, 2013), few researchers have succeeded in separating and culturing the strains for repair in actual environments (Jeon et al, 2003; Li et al, 2017), and meanwhile, the genetic information and the metabolic information of functional microorganisms cannot be analyzed. The metagenome sequencing technology can acquire genome information of certain microorganisms with high abundance in a complex environment so as to acquire genetic and metabolic information of the microorganisms. If SIP and metagenomic technology are combined, genetic and metabolic information of functional microorganisms which act in situ can be obtained very possibly. This information then provides us with information on the functional microorganisms' carbon source, microorganism, trace element utilization, and resistance to a certain class of substances. Until now, no researchers have combined SIP and metagenome technology to separate and culture functional microorganisms for assisting the PAHs degradation in an in-situ environment.

Therefore, a reverse SIP-metagenome method is provided for the first time to assist in screening microorganisms with in-situ PAHs degradation function in the polluted soil.

Disclosure of Invention

In view of the deficiencies of the prior art, it is an object of the present invention to provide a method for isolating in situ functional microorganisms using reverse SIP-metagenomics. The technology is based on SIP-metagenome technology, the 13C-DNA genome data is deeply excavated, the genome of the functional degradation microorganism is successfully obtained, the heredity and metabolism information of the functional bacteria is analyzed, the specific culture medium is reversely arranged, and the specific culture medium is separated and cultured.

The technical scheme adopted by the invention is as follows:

a method for isolating in situ functional microorganisms using reverse SIP-metagenomics, comprising the steps of:

(1) adding sterile water into the soil polluted by the polycyclic aromatic hydrocarbon, ¹² C-PHE or ¹³ C-PHE, shake culturing in dark place, adding ¹² C-PHE or ¹³ C-PHE pollutes the microcosm culture system of the soil, then collect the sample of the microcosm culture system and carry on DNA extraction;

(2) ultracentrifugation is carried out on the extracted DNA to obtain DNA solutions with different buoyancy densities from top to bottom, isopropanol and glycogen are used for precipitating each layer of DNA, the DNA precipitate is washed by ethanol, and then is dissolved by double-purified water and is stored;

(3) performing high-throughput sequencing on DNA of each layer of sample, performing OTU classification according to sequence similarity, dividing species classification information, and adding by comparison ¹² C-PHE and ¹³ selecting OTU capable of representing functional microorganism species according to abundance change of OTU of different separation layers in C-PHE polluted soil;

(4) to pair ¹³ Performing metagenome sequencing on the DNA of the C heavy layer, further mining data by using a metagenome binning technology, acquiring genome information of all microorganisms of the heavy layer, respectively splicing the genome information of the microorganisms, comparing the spliced genome information with the functional microorganisms represented by the OTU, and further confirming that the spliced genome information is from the functional bacteria represented by the OTU, thereby acquiring the identified workGenome information of the competent bacteria;

(5) analyzing the genome information of the identified functional bacteria, setting a specific culture medium by analyzing the utilization and resistance characteristics of the strain in carbon sources, vitamins and mineral elements, and separating out the functional bacteria in the polycyclic aromatic hydrocarbon-polluted soil by using the specific culture medium;

(6) and purifying, identifying, confirming and preserving the separated functional bacteria, and comparing the SIP-metagenome exploration result with the indoor culture separation experiment result to confirm whether the separated bacterial strain is the functional microorganism participating in pollutant degradation in situ.

Preferably, in the step (3), the OTUs are classified according to sequence similarity, specifically, the sequences with similarity greater than 97% are classified as the same OTU.

Preferably, in the step (4), the spliced genome information is compared with the functional microorganism represented by OTU, specifically, the alignment is performed according to gyrB gene sequence information.

We propose a reverse SIP-metagenome technology for the first time, the technology is based on the SIP-metagenome technology, the 13C-DNA genome data is deeply excavated, the genome of the degradation functional microorganism is successfully obtained, the inheritance and metabolism information of the functional bacteria is analyzed, the specific culture medium is reversely set, and the specific culture medium is separated and cultured.

Drawings

FIG. 1 is a schematic diagram of functional microorganism isolation by the reverse SIP-metagenome method.

FIG. 2 shows the phylogenetic information of OTU5 functional bacteria.

FIG. 3 shows phylogenetic information based on the gyrB gene.

FIG. 4 shows the analysis of genetic and metabolic information of OTU5 functional strains.

FIG. 5 shows the composition of functional bacteria isolated from MMM (a) and TCM (b) culture media.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

The schematic diagram of the method for separating in-situ functional microorganisms by using reverse SIP-metagenomics provided by the invention is shown in figure 1.

Example 1

1. Sample collection

The contaminated soil used in the experiment was collected from the victory oil field in Shandong province, sampled and stored at 4 ℃ for development in SIP experiments.

2. Microcosmic culture system

In the experiment, 5g of soil and 20mL of sterile water were added to a 150mL serum bottle, and then unlabelled phenanthrene (F) (M) was added to the bottle by syringe ¹² C-PHE) or ¹³ C-phenanthrene (C) ¹³ C-PHE) (99%, Cambridge Isotrope Laboratories, Inc., Tewksbury, MA, USA) to a final concentration of 10mg/L phenanthrene, sealed with a rubber stopper and aluminum cap. All treatment groups of the experiment included: control group without phenanthrene addition, sterile control group with sterilized soil, and addition ¹² C-PHE or ¹³ C-PHE soil samples. Three parallel groups were set up for all experiments. Placing the mixture in an incubator at 25 ℃ and shaking out of the sun for culture. To ensure sufficient oxygen in the culture system, the serum bottles were opened in a clean bench for about 1 hour per day. The experimental time was 6 days, after 6 days samples were collected for chemical analysis and DNA extraction.

3. DNA extraction and ultracentrifugation

Strong soil DNA extraction kit Using MOBIO (A)

DNA Isolation Kit) total genomic DNA was extracted from the different treatment samples (Tillett and Neilan,2000), three parallel groups were set up for each sample. After DNA extraction, DNA concentration was measured by ND-1,000UV-Vis UV-visible spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and then stored at-20 ℃.

The extracted DNA was ultracentrifuged, 5. mu.g of DNA was added to a Tris-EDTA (TE, pH 8.0)/cesium chloride solution, the refractive index of the solution was measured using an AR200 type digital refractometer (Reichert, Inc., USA), and the refractive index was brought to 1.4020 by adding an appropriate amount of TE or TE/cesium chloride solution. The solution was transferred to a Quick-Seal ultracentrifuge tube (13X 51mm,5.1mL, Beckman Coulter) and after the tube was mass-balanced, it was heat-sealed with a tube topper (Cordless Quick-Seal tube topper, Beckman). The sealed tubes were placed in a Beckman ultracentrifuge (Optima L-100XP, Beckman corporation) and ultracentrifuged at 20 ℃ for 48h at 178000 g. After centrifugation, the tube was carefully removed and the DNA solution was separated into 14 layers using a Beckman layering apparatus to obtain DNA solutions with different buoyancy densities from top to bottom. The refractive index of each layer of the solution was measured with an AR200 digital display refractometer and calculated to convert it to a Buoyant Density value (BD). Purification of the DNA Each layer of DNA was precipitated with isopropanol and glycogen, followed by two washes with 70% volume fraction ethanol, and then DNA was dissolved in deionized water, using methods reported in the literature (Li et al, 2017 b). The purified DNA solution was stored in a freezer at-80 ℃ until use.

4. High throughput sequencing and analysis

Specific primers with 12 bases were synthesized to amplify the 16S rRNA V4 region of each layer of sample DNA according to the indicated sequencing region (Liu et al, 2014). The primers used were 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 806R (5 '-GGACTACHVGGGTWTCTAAT-3'). The PCR reaction (25. mu.L) included 12.5. mu.L of TaKaRa mix, 0.8. mu.L of forward primer 515F (10. mu. mol/L), 1.0. mu.L of DNA template, 0.8. mu.L of reverse primer 806R (10. mu. mol/L) and the balance sterile water. The PCR reaction conditions are as follows: 5min at 94 ℃; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, and circulation for 30 times; extension was carried out at 72 ℃ for 10 min. 3 replicates were set for each sample and a sterile water negative control was set for each PCR reaction. After the PCR is finished, the PCR products of the same sample are combined, and the detection and verification are carried out by using 1.5% agarose gel electrophoresis under the detection condition of 5V/cm for 20 min. PCR products were recovered by Gel cutting using an Agarose Gel DNA Fragment Recovery Kit Ver 2.0(TaKaRa), and the recovered target DNA fragments were dissolved in 30. mu.L of sterile ultrapure water after elution with TE buffer. Referring to the results of agarose gel electrophoresis, the PCR-recovered products in each layer were detected and quantified using the Qubit 2.0(ThermoFisher Co.) or the GE NanoVue system (GE Healthcare Co.), and then equimolar mixed according to the sequencing requirements of the samples. The mixed DNA samples are placed in a freeze dryer for freeze drying, then are dissolved in sterile ultrapure water again, the DNA concentration of each sample is measured, and the samples are sent to a company for sequencing.

High throughput sequencing was performed using the Miseq PE300 platform from Illumina. The original sequence obtained by sequencing is subjected to quality control, low-quality base and linker-contaminated sequences are removed, and reads are sequentially inverted to obtain a high-quality sequence for subsequent biological analysis (Magoc and Salzberg, 2011). The method comprises the steps of performing sequence splicing by using Mothur to obtain a target sequence, finally performing flow analysis by QIIME (quality identity), classifying sequences with similarity larger than 97% as the same OTU (operational Taxonomic Unit), and dividing species classification information (Caporo et al, 2009; Desantis et al, 2006; Edgar, 2010; Mcdonald et al, 2012; Werner et al, 2012). Comparative addition ¹² C-phenanthrene and ¹³ and (3) analyzing the relative abundance of each OTU in each layer after the C-phenanthrene contaminated soil sample is centrifuged by combining the buoyancy density of each separation layer. Culturing the sample for a period of time, adding ¹³ The DNA corresponding to certain functional microbial communities in the C-phenanthrene contaminated soil sample is enriched in the heavy layer, so that the relative abundance of the communities in the heavy layer is obviously more than that of the added community ¹² High in C-phenanthrene-treated group. OTUs with abundances greater than 0.5% were selected for subsequent analysis in this study. Finally, by comparative analysis ¹² C-phenanthrene and ¹³ the abundance of each OTU in different layers of the C-phenanthrene-treated group was varied, and 1 OTU (OTU _5) representing a functional microorganism species belonging to Achromobacter sp. According to BLAST (National Center for Biotechnology Information, Bethesda, Md., USA), OUT has the highest similarity with Achromobacter sp.2789STDY5608615, with a similarity as high as 99.7%. Phylogenetic information of degrading functional microorganisms was determined according to MEGA (Tamura et al, 2007), see fig. 2.

5. Functional bacterium genome obtained by SIP-metagenome method

To pair ¹³ Performing metagenomic analysis on the C-DNA, further mining data by using a metagenomic binning technology to obtain 1Genomic information of 3 microorganisms. The genome information of these microorganisms was joined together (table 1), and the joined genome information was compared with the functional microorganisms represented by OTU, thereby confirming that the joined genome information was derived from the functional bacteria represented by OTU 5. Here we used the gyrB gene to further confirm that the genome is from OTU5 functional bacteria, and the phylogenetic information is shown in FIG. 3.

TABLE 1.13 genomic information spliced in C-DNA

6. Functional bacteria genomic analysis based on SIP-genome acquisition

The genome Bin 10 obtained above was analyzed, and as shown in fig. 4, the strain contained some relevant genes such as vitamins (e.g., vitamin B1, B2, B3, B6, B7, B12, and lipoic acid) and minerals (e.g., Ca, Cu, Mn, Zn, Co, and Ni); in addition, the presence of the aminoglycoside antibiotic resistance protein in the genome suggests that the strain may be resistant to aminoglycoside antibiotics. Therefore, we modified the conventional medium (tables 2-5) by supplementing 10mL/L vitamin stock solution, 10mL/L mineral stock solution and 5mg/L streptomycin, an aminoglycoside antibiotic, to isolate OTU-functional bacteria that degrade polycyclic aromatic hydrocarbons in situ in the soil.

TABLE 2 conventional Medium (TCM)

TABLE 3 SIP-metagenome-revised Medium (MMM)

TABLE 4 vitamin addition information

TABLE 5 mineral addition information

7. Isolation and culture of functional microorganisms of interest

To compare the separation effect of the amended medium (MMM). According to the SIP results, we also used Traditional Culture Medium (TCM) to separate the microorganisms.

The formula compositions of MMM and TCM are shown in the above table 2-5, and the preparation method comprises the steps of adding the components into water, stirring and mixing uniformly, and sterilizing to obtain the product.

The method mainly comprises the following steps: from the above-mentioned addition ¹² 1mL of sample was taken in SIP treatment of C-phenanthrene, spread-separated by dilution plating, and separated by MMM and TCM with 1.5% agar added by mass fraction. And (3) placing the coated flat plate at 25 ℃ for culturing for 4d, growing obvious single colonies on the surface of the culture medium, selecting various different single colonies according to the characteristics of the colony, such as morphology size, transparency, color and the like, and carrying out streak purification on the corresponding culture medium flat plate. If single colonies of different types can still grow on the streaked and purified plate, the single colonies are picked out and streaked again until single colonies of different characteristics cannot be observed on the same plate.

100 strains with phenanthrene degradation function were screened separately in TCM and MMM media (FIG. 5). And (3) selecting the purified single bacteria, dropping the single bacteria into a liquid culture medium, culturing to a logarithmic phase, mixing the bacteria liquid and sterile glycerol, subpackaging the mixture into 2mL freezing tubes (the mass fraction of the glycerol is 15%), and placing the tubes in a refrigerator at the temperature of-80 ℃ for long-term storage. According to the experimental result, the microorganisms related to in-situ degradation functional microorganisms (Achromobacter sp.) can be effectively separated by MMM; however, with the conventional culture medium TCM, although the types of the separated microorganisms become large, the target functional microorganisms cannot be separated.

In order to further confirm that the separated strain is a functional microbial strain which acts in situ, 16S rRNA and GyrB gene sequencing is carried out on the obtained strain, the nucleotide sequence of the 16S rRNA is shown as SEQ ID NO.1, and the nucleotide sequence of the GyrB gene is shown as SEQ ID NO.2, and the gene similarity of the genes of the separated strain and the genes of the obtained functional bacteria is 100 percent, so that the feasibility of separating the in-situ degradation functional microorganisms by the reverse SIP-metagenomic method is further verified.

The above are only preferred embodiments of the present invention, and it should be noted that the above preferred embodiments should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and should be considered to be within the scope of the invention.

Sequence listing

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Claims (3)

1. A method for separating in-situ functional microorganisms by reverse SIP-metagenomics, comprising the steps of:

(1) adding sterile water into the soil polluted by the polycyclic aromatic hydrocarbon, ¹² C-PHE or ¹³ C-PHE, shake culturing in dark place, adding ¹² C-PHE or ¹³ C-PHE pollutes the microcosm culture system of the soil, then collect the sample of the microcosm culture system and carry on DNA extraction;

(2) ultracentrifugation is carried out on the extracted DNA to obtain DNA solutions with different buoyancy densities from top to bottom, isopropanol and glycogen are used for precipitating each layer of DNA, the DNA precipitate is washed by ethanol, and then is dissolved by double-pure water and is stored;

(3) performing high-throughput sequencing on DNA of each layer of sample, performing OTU classification according to sequence similarity, dividing species classification information, and adding by comparison ¹² C-PHE and ¹³ selecting OTU capable of representing functional microorganism species according to abundance change of OTU of different separation layers in the C-PHE contaminated soil;

(4) to pair ¹³ Performing metagenome sequencing on the DNA of the C heavy layer, further mining data by using a metagenome binning technology, acquiring genome information of all microorganisms of the heavy layer, respectively splicing the genome information of the microorganisms, comparing the spliced genome information with the functional microorganisms represented by the OTU, and further confirming that the spliced genome information is from the functional bacteria represented by the OTU, thereby acquiring the genome information of the identified functional bacteria;

(5) analyzing the genome information of the identified functional bacteria, setting a specific culture medium by analyzing the utilization and resistance characteristics of the strain in carbon sources, vitamins and mineral elements, and separating out the functional bacteria in the polycyclic aromatic hydrocarbon-polluted soil by using the specific culture medium;

(6) and (3) purifying, identifying, confirming and preserving the separated functional bacteria, and comparing the SIP-metagenome exploration result with the indoor culture separation experiment result to confirm whether the separated bacterial strain is the functional microorganism participating in the degradation of the polycyclic aromatic hydrocarbon pollutants in situ.

2. The method for separating in situ functional microorganisms according to claim 1, wherein in step (3), the OTUs are classified according to sequence similarity, specifically, the sequences with similarity greater than 97% are classified as the same OTU.

3. The method for separating in situ functional microorganisms using reverse SIP-metagenome according to claim 1, wherein in step (4), the spliced genome information is compared with the functional microorganisms represented by OTU, specifically, according to gyrB gene sequence information.

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