CN117106678A - Enrichment culture of marine organism film bacteria and method for obtaining genome thereof - Google Patents

Enrichment culture of marine organism film bacteria and method for obtaining genome thereof Download PDF

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CN117106678A
CN117106678A CN202311376480.7A CN202311376480A CN117106678A CN 117106678 A CN117106678 A CN 117106678A CN 202311376480 A CN202311376480 A CN 202311376480A CN 117106678 A CN117106678 A CN 117106678A
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张伟鹏
崔涵
丁维
范燊
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Abstract

The invention belongs to the technical field of marine microorganism detection, and particularly relates to enrichment culture of marine biofilm bacteria and a genome acquisition method thereof. The invention establishes a new culture medium formula for enrichment culture of marine microorganisms, comprises non-carbon source components and 69 selected effective carbon sources, and designs culture conditions and metagenome sequencing schemes, thereby successfully culturing marine biofilm flora in a laboratory or obtaining 535 bacterial genomes with high integrity. The method can efficiently culture and obtain the microbial community with complex and stable structure from a plurality of doors and the bacterial genome with higher abundance in the community, thereby providing basis and guidance for further downstream development of microbial resources and improvement of culture methods.

Description

Enrichment culture of marine organism film bacteria and method for obtaining genome thereof
Technical Field
The invention belongs to the technical field of marine microorganism detection, and particularly relates to enrichment culture of marine biofilm bacteria and a genome acquisition method thereof.
Background
The current bacterial culture method is generally suitable for bacteria with specific metabolic capability, such as ammonia oxidizing bacteria, phosphate solubilizing bacteria and the like, has narrower coverage and poor universality, and cannot realize broad-spectrum culture of various bacteria in complex communities. The reasons for this technical problem are mainly the following three aspects: 1) The enrichment medium has single carbon source, for example, glucose is only used as the carbon source, and the complex and various carbon source types in the environment cannot be represented; 2) No or only a single trace element is added, which is not beneficial to the culture of bacteria with higher requirement on trace elements; 3) The influence of culture condition factors such as pH fluctuation on the growth of microorganisms is not considered except nutrient factors of the culture medium.
In addition, the current bacterial culture method does not have a method for acquiring genome information after corresponding enrichment, and the acquired genome information is incomplete. The reasons are as follows: on the one hand, the current bacterial culture method is established before the popularization and application of the third-generation DNA sequencing, and the second-generation DNA sequencing is directly carried out, and long contigs cannot be obtained due to the short reading length, so that high-quality genomes (MAGs) with high integrity and low pollution degree cannot be obtained; on the other hand, due to the limitations of bioinformatics technology, the prior art is built without software to achieve mixed assembly of second and third generation DNA sequences.
Disclosure of Invention
In order to solve the technical problems, the invention provides an enrichment culture method of marine biofilm bacteria and a genome acquisition method thereof, which can efficiently culture to obtain a microbial community with a complex and stable structure and obtain a high-quality bacterial genome of the community.
The invention is realized by the following technical scheme:
the enrichment culture method of the marine organism film bacteria comprises the following steps:
(1) Sampling: and collecting a marine biofilm sample.
Preferably, the marine biofilm sample is from a rock surface submerged in seawater, preferably in a marine area where human activity has a low impact.
(2) Sample treatment: centrifuging the collected sample to remove supernatant to obtain bacterial precipitate, washing the bacterial precipitate, re-suspending in sterile seawater, filtering to obtain biofilm bacterial cells, standing and preserving.
Preferably, the centrifugal speed is 5000-6000rpm, and the time is 5-6min;
the purpose of washing the bacterial precipitate is to remove carbon source nutrition, and the washing liquid is PBS;
the sterile sea water is prepared by dissolving artificial sea salt in ultrapure water for sterilization, wherein the addition amount of the artificial sea salt is 30g/L;
the filtration adopts a 3 mu m cellulose ester membrane filter, and can filter fungi, algae and other particles;
the standing preservation temperature is 2-8 ℃ and the time is 14-18h, so as to consume the residual carbon source nutrition.
(3) Preparing a culture medium: the culture medium comprises carbon source substances and non-carbon source substances, the types and the addition amounts of the non-carbon source substances are shown in table 1, the types of the carbon source substances are any one of substances in table 2, and the addition amount is 1g/L, so that 69 culture mediums corresponding to 69 carbon source substances in table 2 are respectively prepared.
Preferably, the pH of the medium is 6.5-7.5;
the culture medium also comprises HEPES, and the addition amount is 1mol/L, so as to stabilize the pH value in the growth process of the microbial community.
Non-carbon source substances in the medium described in Table 1
Carbon source substances in the Medium described in Table 2
(4) Enrichment culture: inoculating the biofilm bacterial cells obtained in the step (2) into the culture mediums prepared in the step (3), wherein each culture medium is provided with two culture conditions of oxygen culture and anaerobic culture, standing and culturing under dark conditions until biofilm is formed, collecting biofilm bacterial cells, freezing with liquid nitrogen, and storing for subsequent DNA extraction.
Preferably, the culture temperature is 25-26 ℃, the culture time is 20-30 days, and the bacterial density OD is collected when the biofilm bacteria are collected 600 0.8-1.2, and the preservation temperature of the thalli is less than or equal to-80 ℃;
the gas environment of aerobic culture is 80% of nitrogen and 20% of oxygen, and the gas environment of anaerobic culture is nitrogen;
the nitrogen is high-purity nitrogen with the purity of 99.999 percent.
The acquisition method of the marine organism membranous bacteria genome is realized by metagenome sequencing and metagene component box technology, and specifically comprises the following steps:
s1, sequencing the collected biofilm thalli by an Illumina sequencing technology, obtaining 138 samples, respectively obtaining 20G data amount by each sample, and obtaining MAGs by sequencing data through a binning software Maxbin (v2.2.6);
s2, dividing MAGs with the integrity of more than 80% and the pollution of more than 5% obtained in the step S1 into boxes by using Metabat (v0.32.5);
s3, further separating MAGs with the integrity of more than 80% and the pollution of more than 5% obtained in the step S2 into boxes by using Concoct (v1.1.0);
s4, respectively mixing the biological film thalli cultured by four carbon sources of alcohol, amino acid, organic acid and sugar in the table 2 into 4 samples, carrying out Nanopore sequencing, and reassembling the long-reading long sequence obtained by sequencing and MAGs with the integrity of more than 80% and the pollution of less than 5% obtained by the steps S1, S2 and S3 in a box, thereby obtaining the high-quality non-redundant MAGs with the integrity of more than 90% and the pollution of less than 5%.
The recombinant assembly in step S4 specifically includes the steps of:
s41, constructing an original MAG as an index, mapping an Illumina sequence to the original MAG index by using Bowtie2, and extracting a matched sequence by using Samtools (v 1.11);
s42, performing BLASTN search on the Nanopore data aiming at the original MAG, and reserving a matching sequence with more than 99% of identity;
s43, in the single genome mode, the Illumina and Nanopore sequences on a given MAG match are cross-assembled using software SPades (v3.12.0).
The high quality non-redundant MAGs have an average nucleotide similarity (ANI, average nucleotide identity) of less than 99%.
Genome quality was detected by software CheckM (v1.1.2).
Compared with the prior art, the invention has the following beneficial effects:
1. the culture medium formula disclosed by the invention contains a plurality of dissolved organic carbon and a plurality of vitamin components which are abundantly existing in the marine water body, and meanwhile, a pH buffer solution is used. Through testing the culture capacity of more than 100 carbon sources, the finally available biomass is used as an index, and 69 carbon sources are finally obtained as unique carbon sources to perform enrichment culture on the biofilm community. These carbon sources can be divided into four groups according to their chemical nature: alcohols, amino acids, organic acids and carbohydrates, the use of these carbon sources enables efficient diversion of bacterial genomes in the metagenome at different carbon sources. The invention uses multiple vitamin components, while other culture medium components, such as nitrogen source, have simple components and can eliminate other unnecessary component influences. In addition, HEPES buffer solution is added into the culture medium, and the buffer system has a good effect of stabilizing the fluctuation of pH in the enrichment culture process.
2. According to the invention, two culture conditions of oxygen and anaerobic are correspondingly arranged on each culture medium of the carbon source, and the microbial community obtained by culture under each oxygen condition is independently subjected to secondary metagenomic sequencing, so that the obtained file is very beneficial to the subsequent two-dimensional metagenomic component boxes. In addition, after the microbial community samples enriched and cultured by the same type of carbon source culture medium are combined, the third-generation DNA sequencing is performed, a long-reading long sequence is obtained, and the integration of the second-generation and third-generation data is obviously improved for the genome integrity in the box separation process.
Drawings
FIG. 1, a technical roadmap of the invention;
FIG. 2, a structure of a phylum/class horizontal microbial community enriched in culture according to the present invention, in which the bacteria are ranked according to the top 30 of abundance, and the bacteria not at the top 30 are others;
FIG. 3, distribution of gate/line level numbers of 535 high quality genomes assembled after enrichment culture according to the invention.
Detailed Description
The invention is further described below in connection with examples, all materials used in the examples and comparative examples being commercially available, except as specifically indicated.
Example 1
The enrichment culture method of the marine organism film bacteria comprises the following steps:
(1) Sampling: collecting a marine organism film sample;
collecting a marine biofilm sample in an intertidal zone of a Qingdao wheat island sea area, wherein the sampling point is positioned at a position leading to open sea, and the influence of human activities is low; at the time of collection, a biofilm was scraped from the rock surface immersed in seawater with a sterile cotton swab and the collected sample was immediately transferred to the laboratory.
(2) Sample treatment: centrifuging the collected sample to remove supernatant to obtain bacterial precipitate, washing the bacterial precipitate, re-suspending in sterile seawater, filtering to obtain biofilm bacterial cells, standing and preserving;
the centrifugal speed is 6000rpm, and the time is 5min;
the purpose of washing the bacterial precipitate is to remove carbon source nutrition, and the washing liquid is PBS;
the sterile sea water is prepared by dissolving artificial sea salt in ultrapure water for sterilization, wherein the addition amount of the artificial sea salt is 30g/L;
the filtration adopts a 3 mu m cellulose ester membrane filter, and can filter fungi, algae and other particles;
the standing preservation temperature is 4 ℃ and the time is 16 hours, so as to consume the rest carbon source nutrition.
(3) Preparing a culture medium: the culture medium comprises carbon source substances and non-carbon source substances, the types and the addition amounts of the non-carbon source substances are shown in table 1, the types of the carbon source substances are any one of substances in table 3, and the addition amount is 1g/L, so that 69 culture mediums corresponding to 69 carbon source substances in table 3 are respectively prepared;
the pH of the culture medium is 6.5-7.5;
the culture medium also comprises HEPES, and the addition amount is 1mol/L, so as to stabilize the pH value in the growth process of the microbial community.
Carbon source substances and numbers in the medium shown in Table 3
(4) Enrichment culture: respectively inoculating the biofilm bacterial cells obtained in the step (2) into the culture mediums prepared in the step (3), wherein each culture medium is provided with two culture conditions of oxygen culture and anaerobic culture, standing and culturing under dark conditions until a biofilm is formed, collecting biofilm bacterial cells, freezing with liquid nitrogen, and storing for subsequent DNA extraction;
the culture temperature is 25 ℃, the culture time is 25 days, and the bacterial density OD when the biofilm bacterial cells are collected 600 1.0, and storage temperature of-80 ℃;
the gas environment of aerobic culture is 80% of nitrogen and 20% of oxygen, and the gas environment of anaerobic culture is nitrogen; the nitrogen is high-purity nitrogen with the purity of 99.999 percent.
The acquisition method of the marine organism membranous bacteria genome is realized by metagenome sequencing and metagene component box technology, and specifically comprises the following steps:
s1, sequencing the collected biofilm thalli by an Illumina sequencing technology, obtaining 138 samples, respectively obtaining 20G data amount by each sample, and obtaining MAGs by sequencing data through a binning software Maxbin (v2.2.6);
s2, dividing MAGs with the integrity of more than 80% and the pollution of more than 5% obtained in the step S1 into boxes by using Metabat (v0.32.5);
s3, further separating MAGs with the integrity of more than 80% and the pollution of more than 5% obtained in the step S2 into boxes by using Concoct (v1.1.0);
s4, respectively mixing the biological film thalli cultured by four carbon sources of alcohol, amino acid, organic acid and sugar in the table 2 into 4 samples, carrying out Nanopore sequencing, and reassembling the long-reading long sequence obtained by sequencing and MAGs with the integrity of more than 80% and the pollution of less than 5% obtained by the steps S1, S2 and S3 in a box, thereby obtaining the high-quality non-redundant MAGs with the integrity of more than 90% and the pollution of less than 5%.
The recombinant assembly in step S4 specifically includes the steps of:
s41, constructing an original MAG as an index, mapping an Illumina sequence to the original MAG index by using Bowtie2, and extracting a matched sequence by using Samtools (v 1.11);
s42, performing BLASTN search on the Nanopore data aiming at the original MAG, and reserving a matching sequence with more than 99% of identity;
s43, in the single genome mode, the Illumina and Nanopore sequences on a given MAG match are cross-assembled using software SPades (v3.12.0).
The high quality non-redundant MAGs have an average nucleotide similarity (ANI, average nucleotide identity) of less than 99%.
Genome quality was checked by software CheckM (v1.1.2), and the data analysis described above was performed on the linux system installed in this experiment.
By the method of the present invention (FIG. 1), a structurally complex, stable microbial community is cultivated, which is composed of microorganisms from a plurality of doors, as shown in FIG. 2 (corresponding data of FIG. 2 are shown in Table 4 and Table 5, wherein suffix-1 represents an aerobic cultivated sample, suffix-2 represents an anaerobic cultivated sample), and which can be preserved in glycerol tubes for a long period of time for resuscitative and cultivation. By the method of the invention we obtained 535 high quality (integrity >90%, potential contamination <5%, below) genomes of bacteria (FIG. 3). Analysis showed that these genomes were distributed in 13 different bacterial phylum, 68 different bacterial families, 156 different bacterial genera, with higher diversity. The acquisition of these genomes has a positive effect on the subsequent analysis of the development of the responding bacteria, the mining of their valuable metabolites.
TABLE 4 door/class level top 30 microbial community abundance table for aerobic culture
TABLE 5 door/class level top 30 microbial community abundance table for anaerobic enrichment culture
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Comparative example 1
Comparative example 1 enrichment culture of marine biofilm bacteria and genome acquisition were performed by conventional methods, as follows:
(1) Sampling: collecting biofilm growing on rock and artificial material plates (artificial material plates are placed for 12-30 days) in offshore areas of three oceans (Pacific, indian, atlantic), obtaining 101 biofilm samples in total, storing the biofilm samples in ice cubes and rapidly transferring to a laboratory, and storing at-80 ℃;
(2) DNA extraction and sequencing: the DNA of the biofilm was extracted (Qiagen kit, germany) and Illumina sequencing was performed, 115±39 million sequences per biofilm metagenome;
(3) Metagene component box: the contigs (contigs) after metagenomic data assembly (software MEGAHIT, v 1.0.2) were subjected to binning analysis. The first step uses Bowtie2 to map individual metagenome reads to corresponding contigs to generate coverage profiles to reflect the abundance of each contig in the original metagenome; subsequently, the contigs were separated into different genomes by MaxBin2 (v 2.0) using the maximum Expectation-Maximization (EM) with probability (minimum probability of 0.8); the obtained sequences were then further binned with Metabat (v0.24.1) and finally mass-detected for the MAG obtained using CheckM (v0.9.4).
By the method, a high-quality genome of 51 strains of bacteria is finally obtained.
In comparative example 1, there were 101 biofilm samples in total, and the total number of MAGs obtained was 479, of which the number of high-quality MAGs was only 51; in example 1, there were 138 biofilm samples in total, 535 MAG's were counted, and the gate levels and mass results corresponding to the genomes obtained in example 1 and comparative example 1 are shown in Table 6. In the cultivation method, comparative example 1 the present invention example 1 adopts a method of enriching and cultivating a biofilm with a unique carbon source by collecting a biofilm on an artificial material plate and an intertidal zone rock; in the genome acquisition method, comparative example 1 only performs Illumina sequencing to acquire metagenome data, and in the invention, in example 1, a Nanopore sequencing technology is adopted, and genome quality is improved by recombining Illumina and Nanopore results.
In summary, the method for enriching the biofilm by the carbon source and the recombinant assembly method combining the two sequencing technologies of Illumina and Nanopore can obtain enough genomes with high quality in a small amount of biofilm samples, and can provide more high-value information for later analysis of microbial genomes.
TABLE 6 Gate level and quality results for genomes obtained in example 1 and comparative example 1
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Claims (9)

1. An enrichment culture method of marine biofilm bacteria is characterized by comprising the following steps: the method comprises the following steps:
(1) Sampling: collecting a marine organism film sample;
(2) Sample treatment: centrifuging the collected sample to remove supernatant to obtain bacterial precipitate, washing the bacterial precipitate, re-suspending in sterile seawater, filtering to obtain biofilm bacterial cells, standing and preserving;
(3) Preparing a culture medium: the culture medium comprises carbon source substances and non-carbon source substances;
the types and the addition amounts of non-carbon source substances in the culture medium are as follows: mgSO (MgSO) 4 0.489g/L、NH 4 Cl 0.25g/L、CaCl 2 0.1g/L、KNO 3 1g/L、KH 2 PO 4 0.272g/L、Na 2 HPO 4 0.284g/L、FeCl 3 0.004g/L、H 3 BO 3 0.0286g/L、MnCl 2 ·4H 2 O 0.0181g/L、ZnSO 4 ·7H 2 O 0.0222g/L、Na 2 MoO 4 ·2H 2 O 0.00039g/L、CuSO 4 ·5H 2 O 0.00079g/L；
The carbon source substance in the culture medium is any one of alcohol, amino acid, organic acid and sugar, and the addition amount is 1g/L, wherein the alcohol is any one of salicyl alcohol, dulcitol, D-mannitol, 1, 3-propanediol, erythritol, cholesterol, xylitol, maltitol, 1, 2-hexanediol, L-arabitol, L-sorbitol, ethanol, glycerol, amyl alcohol, inositol, D-arabitol and D-sorbitol; the amino acid is any one of L-tyrosine, D-tyrosine, L-cysteine, L-alanine, D-alanine, L-glutamic acid, D-glutamic acid, L-tryptophan, D-tryptophan, L-serine, D-serine, L-leucine, D-asparagine, D-glutamine, L-histidine, D-histidine, L-methionine, L-phenylalanine, D-phenylalanine, L-threonine and sarcosine; the organic acid is any one of acetic acid, octanoic acid, fumaric acid, D-malic acid, alpha-ketoglutaric acid, lactic acid, taurine, glyoxylic acid, glycolic acid, D-gluconic acid, glucuronic acid, pyruvic acid, oxaloacetic acid, citric acid and acrylic acid; the sugar is any one of L-glucose, L-fucose, D-arabinose, L-mannose, D-raffinose, D-xylose, L-galactose, D-fructose, L-rhamnose, inulin, soluble starch and hyaluronic acid;
69 culture mediums corresponding to 69 carbon source substances are prepared respectively;
(4) Enrichment culture: inoculating the biofilm bacterial cells obtained in the step (2) into the culture mediums prepared in the step (3), wherein each culture medium is provided with two culture conditions of oxygen culture and anaerobic culture, standing and culturing under dark conditions until biofilm is formed, collecting biofilm bacterial cells, freezing with liquid nitrogen, and storing for subsequent DNA extraction.
2. The enrichment culture method of marine biofilm bacteria according to claim 1, wherein: in the step (2), a 3 μm cellulose ester membrane filter was used for filtration.
3. The enrichment culture method of marine biofilm bacteria according to claim 1, wherein: in the step (2), the standing preservation temperature is 2-8 ℃ and the time is 14-18h.
4. The enrichment culture method of marine biofilm bacteria according to claim 1, wherein: in step (3), the pH of the medium is 6.5-7.5.
5. The enrichment culture method of marine biofilm bacteria according to claim 1, wherein: in the step (3), the culture medium further comprises HEPES, and the adding amount is 1mol/L.
6. The enrichment culture method of marine biofilm bacteria according to claim 1, wherein: in the step (4), the culture temperature is 25-26 ℃ and the culture time is 20-30 days.
7. The enrichment culture method of marine biofilm bacteria according to claim 1, wherein: in the step (4), the bacterial density OD at the time of collecting the biofilm cells 600 0.8-1.2.
8. A method for obtaining a genome of marine biofilm bacteria obtained by the enrichment culture method according to any of claims 1 to 7, characterized in that: the method comprises the following steps:
s1, sequencing the collected biofilm thalli by an Illumina sequencing technology, obtaining 138 samples, respectively obtaining 20G data amount by each sample, and obtaining MAGs from sequencing data by a binning software Maxbin;
s2, dividing MAGs with the integrity of more than 80% and the pollution of more than 5% obtained in the step S1 into boxes by using Metabat;
s3, further separating MAGs with the integrity of more than 80% and the pollution of more than 5% obtained in the step S2 into boxes by using a Conct;
s4, respectively mixing the biological film thalli cultured by four carbon sources of alcohol, amino acid, organic acid and sugar in the table 2 into 4 samples, carrying out Nanopore sequencing, and reassembling the long-reading long sequence obtained by sequencing and MAGs with the integrity of more than 80% and the pollution of less than 5% obtained by the steps S1, S2 and S3 in a box, thereby obtaining the high-quality non-redundant MAGs with the integrity of more than 90% and the pollution of less than 5%.
9. The method for obtaining a genome of marine biofilm bacteria according to claim 8, wherein: the recombinant assembly in step S4 specifically includes the steps of:
s41, constructing an original MAG as an index, mapping an Illumina sequence to the original MAG index by using Bowtie2, and extracting a matched sequence by using Samtools;
s42, performing BLASTN search on the Nanopore data aiming at the original MAG, and reserving a matching sequence with more than 99% of identity;
s43, in a single genome mode, using software SPades to cross-assemble the Illumina and Nanopore sequences on a given MAG match.
CN202311376480.7A 2023-10-24 2023-10-24 Enrichment culture of marine organism film bacteria and method for obtaining genome thereof Pending CN117106678A (en)

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