Method for identifying and analyzing microorganisms and bacteriophages participating in perchlorate reduction in perchlorate polluted wastewater

Abstract

The invention discloses a method for identifying and analyzing microorganisms and bacteriophages participating in perchlorate reduction in perchlorate polluted wastewater. Microbial system development information capable of driving the ClO4 <-> reduction process in a culture system is identified by using a DNA-SIP technology, then the metabolic potential of ClO4 <-> reduction microorganisms is evaluated by using a box-separating assembly technology, and the species information of the bacteriophage and the auxiliary contribution of the bacteriophage to the ClO4 <-> reduction function microorganisms are mainly analyzed by combining the deep analysis of the bacteriophage. The method disclosed by the invention has great significance for cleaning the ClO4 <-> reduction function microbial group of sludge and driving the ClO4 <-> reduction process by the synergistic bacteriophage of the microbial group, and understanding the ClO4 <-> reduction function bacteria and key functional genes for driving the ClO4 <-> reduction by the synergistic bacteriophage of the ClO4 <-> reduction function bacteria.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a method for identifying and analyzing microorganisms and bacteriophages involved in perchlorate reduction in perchlorate-contaminated wastewater. Background Technology

[0002] Perchlorate (ClO4) - Perchlorate is a persistent oxidizing pollutant widely used in aerospace, fireworks, electroplating, and military industries. Due to its high water solubility and chemical stability, perchlorate exhibits strong mobility in hydrological systems, penetrating various environmental matrices (water, atmospheric particulate matter, soil-sediment systems, and the food chain), thus posing a bioaccumulation risk and providing pathways for human exposure. Mechanistically, perchlorate inhibits the physiological absorption of iodine ions in the human body, directly interferes with the synthesis of thyroid hormones and neural development, and has a potential cascade effect on overall growth.

[0003] Perchlorate removal methods include physical, chemical, and biological treatments. Traditional physical and chemical methods face challenges such as non-selective reaction pathways, high operating costs, and the generation of subsequent waste streams; while microbial reduction mediated by perchlorate-reducing bacteria (PeRB) under anoxic conditions has become a highly promising strategy due to its sustainability. Although research on microbial perchlorate reduction dates back to the early 20th century, our understanding of its phylogenetic diversity, metabolic potential, and the role of bacteriophages in the process remains insufficient. Currently, some PeRBs (such as...) are known... Dechloromonas , Azospira Perchlorate or chlorate (ClO3) can be removed by perchlorate reductase (Pcr). - Preliminary catalysis to chlorite (ClO2) - The chlorite superoxide dismutase (Cld) then breaks it down into non-toxic chloride ions (Cl). - Although the involvement of microorganisms in perchlorate reduction is well-known, bacteriophages, as the most abundant biological entity, encode accessory metabolic genes (vAMGs) that play a significant role in the reduction of ClO4. - The contribution of the reduction process is not yet clear, limiting the scope of research on ClO4. - A comprehensive understanding of biological reduction mechanisms is needed. Therefore, research on ClO4 is crucial. - ClO4 is involved in the polluted wastewater - The reduction of bacterial strains and the benefits of bacteriophages are of great significance in this biogeochemical process. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for identifying and analyzing microorganisms and bacteriophages involved in perchlorate reduction in perchlorate-polluted wastewater.

[0005] Another object of the present application is to identify the ClO4- reducing functional bacteria in wastewater by means of the stable isotope tracer-bin assembly-phage collaborative analysis platform, to reveal the key functional genes of the reduction process and the phage auxiliary benefits, and to provide an efficient and advanced technical method for subsequent identification of other pollutant conversion related microorganisms and phages.

[0006] The object of the present application is achieved by the following technical solutions: A method for identifying and analyzing microorganisms and phages involved in perchlorate reduction in perchlorate contaminated wastewater, comprising the following steps: (1) Collecting ClO4 - contaminated sludge, adding it to an inorganic salt culture medium, anaerobic culture until the sludge background organic matter is consumed, then dividing it into four groups of microcosm systems, adding 13 C-pe microcosm system to the first group 13 C-acetate and ClO4 - for culture, to mark and observe microorganisms with ClO4 - reduction ability; adding 12 C-pe microcosm system to the second group 12 C-acetate and ClO4 - for culture, to further confirm the microorganisms with ClO4 - reduction ability in the system, and exclude the interfering microorganisms in the first group due to other reasons existing in heavy components; adding 13 C-control microcosm system to the third group only for culture with acetate, to exclude microorganisms not involved in ClO4 - reduction; the fourth group 13 CPe-sterilized first sterilized sludge, then adding 12 C-acetate and ClO4 - for culture, as a sterile control; (2) Extracting the total DNA of the sludge microorganisms in the three groups of microcosm systems after culture, except 13 CPe-sterilized, adding the obtained DNA extract to CsCl solution for ultracentrifugation, and collecting the centrifugal components of each layer; (3) Determining the Buoyant Density (BD) value of each layer of centrifugal components, dividing the total DNA of soil microorganisms in the three groups of microcosm systems into 20-25 layers according to the BD value, removing CsCl in each layer, and performing q-PCR amplification on each layer of centrifugal components; taking the BD value of each layer of centrifugal components as the abscissa, and taking the functional genes 16S and pcrAPlot the q-PCR amplification values ​​on the ordinate; select 1-3 strata with higher q-PCR amplification values ​​from the heavy or light components of the three micro-universe systems for high-throughput sequencing of the V4-V5 region of the 16S rRNA gene. (4) After denoising the obtained 16S rRNA V4-V5 region sequencing data, it was compared with the existing 16S rRNA database to identify each precise sequence variant (ASV, AmpliconSequence Variant). Subsequently, the abundance of each ASV in the three micro-universe systems was analyzed. Each ASV corresponds to a different 16S rRNA sequence, that is, to a different microorganism. (5) Exclusion 13 In the C-control group, precise sequence variant groups (ASVs) that are significantly enriched in heavy components, due to 13 No ClO4 was added to the C-control group. - This indicates that these ASVs enriched in heavy components are for other assimilation. 13 Microorganisms involved in C-acetic acid production, rather than those involved in ClO4 production. - Microorganisms that reduce metabolism; (6) In 13 The ASVs enriched in the heavy components of the CPe group are those with ClO4 - Reducing microorganisms; these ASVs represent microorganisms that have undergone ClO4 formation in the microcosm system. - The process of restoration 13 In the CPe group, microorganisms assimilated the substrates in the system. 13 C-acetic acid, 13 C makes its DNA "heavier," from 13 The light components of the CPe group "moved" to 13 The heavy components of the CPe group, and ASVs with the above characteristics, are judged to contain ClO4. - Microorganisms with reducing power; (7) After identifying the microorganisms and bacteriophages involved in perchlorate reduction in the above-mentioned perchlorate-contaminated wastewater, different components of microorganisms were obtained, and the first group was established. 13 The CPe microcosm system metagenomic library of heavy component DNA was used to obtain raw sequencing reads. Sequencing data quality control was performed to filter low-quality data. Then, sequence assembly was performed to obtain contigs. Sequence alignment was then performed, and the reads of the independent datasets of contigs were mapped to assess their abundance. Binning assembly of contigs was then performed, and bins with integrity >50% and redundancy <10% were selected for downstream analysis. (8) Analysis of genes related to CIO4 - reduction in the metagenome bin, thus further understanding the CIO4 - key functional genes related to CIO4 - microbially-driven CIO4 - reduction in contaminated sludge; (9) Using the viral identification tool to identify the first group 13 Contigs assembled from CPe sequences were identified as viral clusters (VCs), followed by clustering of VCs with similarity greater than 90%, then quality assessment of clustered VCs, and removal of VCs assessed as "uncertain", followed by phage classification, lifestyle, host identification, and auxiliary function identification.

[0007] The sludge in step (1) is the sludge at the bottom of an automated remote perchlorate reduction reactor of a fireworks factory.

[0008] The composition of the inorganic salt medium in step (1) is: 10.55 g / L Na2HPO4·12H2O, 1.5 g / L KH2PO4, 0.3 g / L NH4Cl, 0.1 g / L MgCl2, 0.01 mg / L vitamin H, 0.02 mg / L nicotinic acid, 0.1 mg / L vitamin B1, 0.01 mg / L p-aminobenzoic acid, 0.005 mg / L vitamin B5, 0.05 mg / L pyridoxamine hydrochloride, 0.01 mg / L cyanocobalamin, 10 µL / L 25wt% HCl, 1.5 mg / L FeCl2·4H2O, 0.19 mg / L CoCl2·6H2O, 0.1 mg / L MnCl2·2H2O, 0.07 mg / L ZnCl2, 0.024 mg / L NiCl2·6H2O, 0.036 mg / L NaMoO4·2H2O, 0.006 mg / L H3BO3, 0.002 mg / L CuCl2·2H2O.

[0009] The anaerobic culture in step (1) is N2 purging to the culture system during the culture process.

[0010] The anaerobic culture in step (1) is for not less than 10 days.

[0011] The culture in step (1) is for 18 days.

[0012] The C-ethanol in step (1) is 0.1-0.2 g / L. 13 The final concentration of C-acetate is 0.12-0.13 g / L, 12The final concentration of C-acetic acid is 0.12-0.13 g / L, and the final concentration of perchlorate is 0.95-1.00 g / L, and the substrate concentration is added every 10 days during the culture process.

[0013] The C-acetic acid of step (1) is 13 The C-acetic acid of step (1) is 12 The C-acetic acid is a carbon isotope 13 The C-acetic acid is a carbon isotope 12 The C-acetic acid is a carbon isotope.

[0014] The ClO4 of step (1) is - The ClO4 of step (1) is

[0015] The ultra-high speed centrifugation of step (2) is 178,000 x g for 48 hours.

[0016] The metagenomic library of step (7) is established using the Illumina Hiseq 4000 platform.

[0017] The data quality control of step (7) is analyzed using Trimmomatic-0.36.

[0018] The sequence splicing of step (7) is performed using metaspades.

[0019] The sequence alignment of step (7) is performed using Bowtie 2.

[0020] The Binning assembly of step (7) is performed using CONCOCT with default settings.

[0021] The virus identification tool of step (9) is identified using virsorter2.

[0022] The clustering of step (9) is performed using cd-hit-est-2d.

[0023] The quality assessment of step (9) is performed using CheckV.

[0024] The survival type of step (9) is performed using PhaTYP.

[0025] The classification identification of step (9) is performed using Phamer.

[0026] The host identification of step (9) is performed using cherry.

[0027] The function identification of step (9) is performed using kofamscan.

[0028] The present application has the following advantages and effects relative to the prior art: (1) Given ClO4 - The widespread nature of pollution, and the presence of ClO4 under hypoxic conditions. - ClO4-mediated by reducing bacteria (PeRB) - The reduction process on ClO4 - The significant potential for removal and the research gap regarding phage benefits are therefore, research on ClO4 is needed. - ClO4 is involved in the polluted wastewater - The reducing bacterial strains and phage benefits of ClO4 - Removal is of great significance. DNA stable isotope tracing (DNA-SIP) technology can correlate microbial characteristics with environmental sample functions without relying on pure culture, thereby enabling a better understanding of ClO4. - ClO4 is involved in the polluted wastewater - Reconstructing functional microbial communities related to biological processes; metagenomic binning and assembly analysis can reveal the metabolic potential of functional bacterial species; and phage in-depth analysis clarifies the role of their accessory metabolic genes in ClO4 metabolism. - Benefits of the reduction process. The purpose of this invention is to utilize a stable isotope tracing-box assembly-phage synergistic analysis platform to identify substances in soil that participate in the reduction of ClO4. - The bacterial strains involved in the reduction process reveal the driving force behind ClO4. - This study identifies key functional genes involved in the reduction process and the auxiliary benefits of bacteriophages, and also provides an advanced and effective method for future research on identifying microorganisms and bacteriophages involved in the transformation of other pollutants.

[0029] (2) The stable isotope tracing-bin assembly-phage synergistic analysis platform utilized in this invention can accurately identify key functional microorganisms and their synergistic phages involved in pollutant transformation processes in sludge without relying on isolation or enrichment culture. By directly anchoring key functional microorganisms and their synergistic phages in the community using DNA-SIP, the differences between the microbial community and the in-situ environment after multiple subcultures and enrichment are avoided. It can directly perform bin assembly analysis and phage depth analysis on the DNA of functional microorganisms anchored by DNA-SIP, further clarifying the sludge ClO4 content. - Reconstructing the key functional microbial groups, metabolic potential, and phage benefits is crucial for understanding microbial synergistic phage-driven ClO4 metabolism. - Understanding the reduction process and its key functional genes is of great significance. Attached Figure Description

[0030] Figure 1 ClO4 for DNA-SIP microcosm cultivation system - Schematic diagram of concentration changes.

[0031] Figure 2 For different incubation times13 / 12 CPe and 13 C-control treated DNA in different buoyancy density fractions 16S(a) and pcrA (b) Relative abundance of genes.

[0032] Figure 3 Bubble plot of relative abundance of ASVs for DNA components with different buoyancy densities in a DNA-SIP microcosm culture system.

[0033] Figure 4 For having ClO4 - A schematic diagram of the hierarchical developmental hierarchy of the genome bins of reductive potential microorganisms and their ClO4 content. - Reconstruct a heatmap related to genes.

[0034] Figure 5 To classify, survive, and predict hosts of identified bacteriophages.

[0035] Figure 6 To be with ClO4 - Reducing bacteria (i.e.) Thauera Metabolic gene map of related bacteriophages. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0037] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0038] Example 1: Identification of microorganisms and bacteriophages involved in perchlorate reduction in perchlorate-contaminated wastewater (1) Sample collection and processing Sludge samples were collected from an automated, remote perchlorate reduction reactor at a fireworks and firecrackers manufacturing plant in Liuyang City, Hunan Province. This reactor was rich in ClO4. - The contaminated sludge has developed a selective microbial community and may have accumulated ClO4. - Metabolic microorganisms were sampled at the bottom of the reactor, stored at low temperatures, and transported to the laboratory.

[0039] (2) The establishment of the microcosm system The sludge sample collected was used to establish a microcosm system, and the microcosm system was established using a 50 mL sterilized serum bottle. About 1 g of sludge and 40 mL of mineral salt medium (MSM) were added to the bottle. N2 was injected into the bottle to maintain an anaerobic state in the microcosm system. After one month of starvation culture, the background substrate of the sludge was consumed; The composition of the MSM solution: 10.55 g / L Na2HPO4·12H2O, 1.5 g / L KH2PO4, 0.3 g / L NH4Cl, 0.1 g / L MgCl2, 0.01 mg / L vitamin H, 0.02 mg / L nicotinic acid, 0.1 mg / L vitamin B1, 0.01 mg / L p-aminobenzoic acid, 0.005 mg / L vitamin B5, 0.05 mg / L pyridoxal hydrochloride, 0.01 mg / L cyanocobalamin, 10 μL / L HCl (25%, w / w), 1.5 mg / L FeCl2·4H2O, 0.19 mg / L CoCl2·6H2O, 0.1 mg / L MnCl2·2H2O, 0.07 mg / L ZnCl2, 0.024 mg / L NiCl2·6H2O, 0.036 mg / L NaMoO4·2H2O, 0.006 mg / L H3BO3, 0.002 mg / L CuCl2·2H2O.

[0040] (3) Group culture The sample after starvation culture was divided into four groups, with three replicates in each group; To the first group ( 13 CPe) microcosm system, 0.124 g / L (final concentration) 13 C-acetate and 1.000 g / L (final concentration) of KClO4 were added for cultivation, to the second group ( 12 CPe) microcosm cultivation system, 0.124 g / L (final concentration) 12 C-acetate and 1.000 g / L (final concentration) of KClO4 were added for cultivation, to the third group ( 13 C-control) microcosm cultivation system, 0.124 g / L (final concentration) 13 C-acetate was added for cultivation, and the fourth group ( 13 CPe-sterilized) sludge was subjected to high-pressure sterilization, followed by the addition of 0.124 g / L (final concentration) 12 C-acetate and 1.000 g / L (final concentration) of KClO4 for cultivation, and the raw materials of the corresponding microcosm system were supplemented again on the 10th day.

[0041] (4) Quantitative detection and DNA extraction ClO4 in the supernatant of four microcosm systems was analyzed using an ion chromatograph (ICS-40, Dionex, CA, USA). - The concentration was quantified, and it was observed that perchlorate was rapidly reduced in cultures inoculated with sludge samples. Figure 1 ). Adding ClO4 - In the process of handling, 13 CPe treatment of maximum ClO4 - The reduction rate was 0.91 ± 0.03 mM day / g sludge, compared with... 12 CPe treatment of maximum ClO4 - The reduction rate was 0.92 ± 0.01 mM day / g sludge, with no significant difference. However, in the sterilization control ( 13 CPe-sterilized) and the control without added perchlorate ( 13 No obvious ClO4 was observed in C-control. - The reduction process indicates that a microbially driven perchlorate reduction process occurred in the sludge.

[0042] In addition, on the 10th and 18th days of cultivation, besides 13 Three microcosm systems outside of CPe-sterilized were sampled, and total DNA from sludge microorganisms was extracted using a soil DNA extraction kit.

[0043] (5) The total DNA of soil microorganisms in the DNA-SIP culture system was centrifuged and stratified using an ultra-high speed centrifuge. 10 μg of DNA extract was placed in a 5.1 mL ultracentrifuge tube. CsCl solution was added until the tube was nearly full, and the BD value was adjusted to 1.73 g / mL using Tris-EDTA (pH 8.0) and CsCl solution (BD value measured with a refractometer). The tube was then sealed. The tube was centrifuged at 178,000 × g at 20°C for 48 hours. The tube was then placed in a fraction recovery device. The mixture was separated into layers using a fixed flow pump, with each layer approximately 210 µL, for a total of 24 layers.

[0044] (6) BD value determination and qPCR quantification The BD values ​​of each layer of recovered components were measured, and then CsCl was removed by precipitation with nucleic acid flocculant and ethanol to obtain purified recovered components.

[0045] Using known 16S gene primers 331F and 518R and ClO4 - Reduced genes pceA19 -F andpcrA242 -R was used to amplify the recovered components using q-PCR. The results are as follows: Figure 2 As shown; 331F: 5'-TCCTACGGGAGGCAGCAGT-3'; 518R: 5'-ATTACCGCGGCTGCTGG-3'; pceA19- F: 5'-CACTGGTACGAAAAGCTCGA-3'; pcrA242- R: 5'-GTCATCTGGTTCCACAGG-3'; Group 1 ( 13 The CPe microcosm system forms one peak with a BD value of 1.717-1.726, indicating a heavy component; the second group ( 12 The CPe microcosm system forms one peak with a BD value of 1.707-1.710, indicating a light component; the third group ( 13 The C-control microcosm system forms a peak with a BD value of 1.717-1.726, indicating a heavy component. (7) Select multiple buoyancy density component DNAs for 16S rRNA sequencing. The distribution of heavy and light DNA components in the whole-component stratification was determined based on BD values ​​and functional gene q-PCR mapping. In the first group ( 13 CPe) and the third ( 13 Three layers were selected from the heavy components of the C-control group microcosm system, and the second ( 12 Three layers were selected from the lightweight components of the CPe group microcosm system, and high-throughput sequencing of the 16S rRNA gene V4-V5 region was performed on the layers.

[0046] After denoising the obtained 16S rRNA V4-V5 region sequencing data, it was compared with the existing 16S rRNA database to identify each precise sequence variant (ASV). Subsequently, the abundance of each ASV in the three microcosm systems was analyzed.

[0047] (8) DNA-SIP treatment of ClO4 in sludge - Analysis of the reductive active bacterial community ASV analysis was used to analyze the diversity of microbial communities and the abundance of different microbial species in the samples. Figure 3 These are the top 10 ASVs with the highest relative abundance in the community.

[0048] exist( 13 In the CPe group of microcosmic systems 13c-DNA enriched ASVs may possess ClO4. - The reducing power. However, since acetic acid can also be assimilated by many other microorganisms, a comparison with (ClO4) is needed. - (Restore) group and ( 13 The C-control group of microcosm systems, and the comparison ( 13 CPe) group and ( 12 To eliminate interference from the CPe group of microcosmic systems, we can determine the participants in ClO4. - Bacteria in the reduction process.

[0049] Since the system only contains acetic acid metabolism and ClO4 - Reduction-dominant electron exchange process, ClO4 - ClO4 participating in the reduction system - The microorganisms in the reduction process utilized 13 C-acetic acid is metabolized as a substrate, thereby deriving DNA from it. 13 C labeling during DNA ultracentrifugation 13 C aggregates in the heavier components, therefore in the first group ( 13 The phylogenetic information of ASVs enriched in the heavy components of the CPe microcosm system suggests that the microorganisms may possess ClO4. - The ability to restore; while in the second group ( 12 In the CPe microcosm cultivation system, because what is added is 12 C-acetic acid, therefore ClO4 - Reduced microbial ASVs accumulate in the light components; in the third group ( 13 The phylogenetic information of ASVs enriched in the C-control microcosm culture system was determined to be microorganisms capable of assimilating acetic acid, but the system did not contain ClO4. - Therefore, these microorganisms may not necessarily produce ClO4. - Reduction reaction.

[0050] (9) Identification of microorganisms involved in perchlorate reduction The first step is to eliminate ( 13 In the C-control group, ASVs that were significantly enriched in heavy components, due to ( 13 Group C-control did not add ClO4 - This indicates that these ASVs enriched in heavy components are for other assimilation. 13 Microorganisms involved in C-acetic acid production, rather than those involved in ClO4 production. - Microorganisms that reduce metabolism; The second step is ( 13 ASVs enriched in the heavy components of the CPe group are those with ClO4- Reducing microorganisms; these ASVs represent microorganisms that have undergone ClO4 formation in the microcosm system. - The process of restoration 13 In the CPe group, microorganisms assimilated the substrates in the system. 13 C-acetic acid, 13 C makes its DNA "heavier," from 13 The light components of the CPe group "moved" to 13 The heavy components of the CPe group, and ASVs with the above characteristics, are judged to contain ClO4. - Microorganisms with reducing power.

[0051] like Figure 3 As shown, based on the above judgment method, by comparing the ASV results of the DNA-SIP community of microorganisms in the three grouped microcosm systems, it was determined that... Thauera sp. possesses the above characteristics, and these ClO4 - Analysis of the abundance distribution of reducing metabolic bacteria in the DNA buoyancy density fraction revealed that ClO4 - These two types of ClO4 in the reduction system - The reducing bacteria were all assimilated. 13 C-acetic acid makes the DNA "heavier," thus appearing in the buoyancy density layer. Therefore, these ClO4 molecules in the buoyancy density layer... - The distribution of reducing bacteria differed significantly from that of the light layer. Therefore, it was inferred that they were involved in ClO4 production. - Bacteria that undergo reductive metabolism.

[0052] (10) Metagenomic analysis and binning assembly of heavy component DNA samples Establish a ClO4-containing system on the Illumina Hiseq 4000 platform. - Reducing microorganisms ( 13 Metagenomic libraries of CPe group heavy component DNA were obtained to acquire raw sequencing reads. Trimmomatic-0.36 was used for sequencing data quality control to filter low-quality data. Then, mataspades was used for sequence assembly to obtain contigs. Bowtie 2 was used for sequence alignment. Reads from the independent datasets of contigs were mapped to assess their abundance. Finally, contigs were assembled using CONCOCT (version 0.4.0) with default settings. Binning was performed on the contigs, and bins with integrity >50% and redundancy <10% were selected for downstream analysis.

[0053] (11) The assembly of ClO4 in separate boxes reveals - Analysis of functional genes related to reductive metabolism Using metagenomic analysis to study ClO4- metabolic potential of the microbial community, in particular for those CIO4 - reducing bacteria identified by DNA-SIP

[0054] After analysis, 49 high-quality bins were obtained from the metagenomic data, which belong to Actinobacteriota and Proteobacteria two phyla Figure 4 , in which CIO4 - reducing bacteria inferred in DNA-SIP were also detected in these bins, such as Thauera sp. (MAG.9) and Thauera sp. (MAG.47) (Table 1). Figure 4

[0055] Functional genes related to CIO4 - reduction were analyzed in the metagenomic bins Figure 4 , and the genetic annotation of the bins indicated that almost all MAGs encoded Figure 4 gene (perchlorate reductase alpha subunit), 14 MAGs encoded pcrA gene (perchlorate reductase beta subunit), and 7 MAGs encoded pcrB gene (perchlorate reductase gamma subunit precursor). In addition, 2 MAGs encoded pcrC gene (chlorite dismutase), 2 MAGs encoded cld gene (chlorate reductase alpha subunit), 2 MAGs encoded clrA gene (chlorate reductase beta subunit), 1 MAG encoded clrB gene (chlorate reductase gamma subunit). Among these potential CIO4 - reducing MAGs, clrC related MAGs encoded complete Thauera gene cluster, pcr gene (only MAG.9) and cld gene, indicating that they have genetic potential to reduce CIO4 - . clrAB

[0056] (12) Phage co-analysis reveals phage species information and their metabolic contribution to host CIO4 - reduction ​​Virsorter2 was used to identify contigs assembled from mataspades sequences to identify viral contigs (VCs), and cd-hit-est-2d was used to cluster VCs with a similarity greater than 90%. CheckV was used to assess the quality of VCs, and VCs classified as "undetermined" were removed. Phages in VCs were identified using Phaeer. Phage classification was compared using PhaGCN2. Cherry was used to predict the host of phages, and kofamscan was used to analyze the ClO4 of phages. - The restore function is annotated and analyzed.

[0057] Analysis revealed 822 viral clusters that were identified as bacteriophages and were retained for downstream analysis. Figure 5 Of these, 814 bacteriophages can be classified as viruses, mainly including... Caudoviricetes , Faseviricetes , Megavirictes and Tectiliviricetes The remaining bacteriophages could not be classified into any viral category. A total of 112 bacterial genera were identified as potential hosts for 327 bacteriophages. The primary hosts of these bacteriophages include... Pseudomonas , Thauera , Burkholderia , Rhizobium and Streptomyces etc. Among them, ClO4 - reducing bacteria Thauera There are a total of 40 bacteriophages serving as the host. These bacteriophages further ClO4 - Reduced functional gene analysis ( Figure 6 ), discovery and pcrA and clrC Related ClO4 - The reduction function gene was detected only in phages VCs120 and VCs12, and no ClO4-related genes were identified in other phages. - The restoration of related genes suggests that they may contribute to Thauera Bacterial reduction of ClO4 - .

[0058] This result reveals the potential ClO4 in the sludge. - Reducing bacteria and bacteriophages drive ClO4 - The reduction process expands the current understanding of ClO4 in sludge. - Ecological functions of reducing bacteria and bacteriophages and ClO4 - ClO4 driven by reducing bacteria and bacteriophages - Understanding the reducing geochemical cycle process.

[0059] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A method for identifying and analyzing microorganisms and bacteriophages involved in perchlorate reduction in perchlorate-contaminated wastewater, characterized in that... Includes the following steps: (1) Collect ClO4-containing - The contaminated sludge was added to an inorganic salt culture medium and anaerobically cultured until the background organic matter of the sludge was consumed. Then, it was divided into four microcosmic systems, with the first group... 13 CPe Microcosm System Added 13 C-acetic acid and ClO4 - Cultured to label and observe the presence of ClO4 - Microorganisms with reducing ability; to the second group 12 CPe Microcosm System Added 12 C-acetic acid and ClO4 - Culture was carried out to further confirm the presence of ClO4 in the system. - Microorganisms with reducing capacity, excluding interfering microorganisms in the first group that exist in the heavy components for other reasons; to the third group 13 The C-control microcosm system was cultured with only acetic acid to exclude those not involved in ClO4. - Reducing microorganisms; Group 4 13 CPe-sterilized first performs high-pressure sterilization on the sludge, and then adds 12 C-acetic acid and ClO4 - Cultured as a sterile control; (2) For the cultured products, in addition to 13 The three micro-universe systems outside of CPe-sterilized were used to extract total DNA from the sludge microorganisms. The obtained DNA extract was added to CsCl solution and centrifuged at ultra-high speed to collect the centrifuged components of each layer. (3) The buoyant density (BD) of each centrifuged component was measured. The total DNA of soil microorganisms in the three microcosm systems was divided into 20-25 layers based on the BD value. CsCl in each layer was removed, and q-PCR amplification was performed on each centrifuged component. The BD values ​​of each centrifuged component were plotted on the x-axis, and the functional genes 16S and... pcrA Plot the q-PCR amplification values ​​on the ordinate; select 1-3 strata with higher q-PCR amplification values ​​from the heavy or light components of the three micro-universe systems for high-throughput sequencing of the V4-V5 region of the 16S rRNA gene. (4) After the obtained 16S rRNA V4-V5 region sequencing data were denoised, they were compared with the existing 16S rRNA database to identify each precise sequence variant (ASV, Amplicon Sequence Variant). Then the abundance of each ASV in the three microcosm systems was analyzed. Each ASV corresponds to a different 16S rRNA sequence, that is, to a different microorganism; (5) Exclusion 13 In the C-control group, precise sequence variant groups (ASVs) that are significantly enriched in heavy components, due to 13 No ClO4 was added to the C-control group. - This indicates that these ASVs enriched in heavy components are for other assimilation. 13 Microorganisms involved in C-acetic acid production, rather than those involved in ClO4 production. - Microorganisms that reduce metabolism; (6) In 13 The ASVs enriched in the heavy components of the CPe group are those with ClO4 - Microorganisms with reducing capabilities; These ASVs, representing microorganisms, underwent ClO4 formation within the microcosm system. - The process of restoration 13 In the CPe group, microorganisms assimilated the substrates in the system. 13 C-acetic acid, 13 C makes its DNA "heavier," from 13 The light components of the CPe group "move" to 13 The heavy components of the CPe group, and ASVs with the above characteristics, are judged to contain ClO4. - Microorganisms with reducing capabilities; (7) After identifying the microorganisms and bacteriophages involved in perchlorate reduction in the above-mentioned perchlorate-contaminated wastewater, different components of microorganisms were obtained, and the first group was established. 13 The CPe microcosm system metagenomic library of heavy component DNA was used to obtain raw sequencing reads. Sequencing data quality control was performed to filter low-quality data. Then, sequence assembly was performed to obtain contigs. Sequence alignment was then performed, and the reads of the independent datasets of contigs were mapped to assess their abundance. Binning assembly of contigs was then performed, and bins with integrity >50% and redundancy <10% were selected for downstream analysis. (8) Analysis of ClO4 in metagenomic bins - Genes related to reduction function were obtained, thereby obtaining ClO4 - Key functional genes related to reductive metabolism, thereby gaining a deeper understanding of ClO4. - Microbial-driven ClO4 in contaminated sludge - Biogeochemical mechanisms of reduction processes (9) Use virus identification tools to identify the first group 13 Contigs obtained by CSe sequence splicing are used to identify viral clusters (VCs). VCs with a similarity greater than 90% are then clustered. The quality of the clustered VCs is then assessed, and VCs assessed as "undetermined" are removed. Finally, the bacteriophages are classified, their survival type is determined, their host identification is performed, and their auxiliary functions are identified.

2. The method for identifying and analyzing microorganisms and bacteriophages involved in perchlorate reduction in perchlorate-contaminated wastewater according to claim 1, characterized in that: The sludge mentioned in step (1) is the bottom sludge of the automated remote perchlorate reduction reactor in the fireworks and firecrackers production plant; The inorganic salt culture medium in step (1) consists of: 10.55 g / L Na2HPO4·12H2O, 1.5 g / L KH2PO4, 0.3 g / L NH4Cl, 0.1 g / L MgCl2, 0.01 mg / L Vitamin H, 0.02 mg / L Nicotinic acid, 0.1 mg / L Vitamin B1, 0.01 mg / L p-aminobenzoic acid, 0.005 mg / L Vitamin B5, 0.05 mg / L Pyridoxine hydrochloride, 0.01 mg / L Cyanocobalamin, 10 µL / L 25wt% HCl, 1.5 mg / L FeCl2·4H2O, 0.19 mg / L CoCl2·6H2O, 0.1 mg / L MnCl2·2H2O, 0.07 mg / L ZnCl2, 0.024 mg / L NiCl2·6H2O, and 0.036 mg / L HCl. NaMoO4·2H2O, 0.006 mg / LH3BO3, 0.002 mg / L CuCl2·2H2O.

3. The method for identifying and analyzing microorganisms and bacteriophages involved in perchlorate reduction in perchlorate-polluted wastewater according to claim 1, characterized in that: The anaerobic culture described in step (1) involves purging the culture system with N2 during the culture process; The anaerobic culture time in step (1) shall be no less than 10 days; The cultivation time described in step (1) is 18 days.

4. The method for identifying and analyzing microorganisms and bacteriophages involved in perchlorate reduction in perchlorate-contaminated wastewater according to claim 1, characterized in that: The steps described in step (1) 13 The final concentration of C-acetic acid is 0.12-0.13 g / L. 12 The final concentration of C-acetic acid was 0.12-0.13 g / L, and the final concentration of perchlorate was 0.95-1.00 g / L. The substrate concentration was maintained by adding it every 10 days during the culture process. The steps described in step (1) 13 C-acetic acid or 12 C-acetic acid is a carbon-containing isotope 13 C or 12 C-acetic acid; The ClO4 mentioned in step (1) - It is potassium perchlorate.

5. The method for identifying and analyzing microorganisms and bacteriophages involved in perchlorate reduction in perchlorate-contaminated wastewater according to claim 1, characterized in that: The conditions for ultra-high speed centrifugation in step (2) are 178,000 × g for 48 hours.

6. The method for identifying and analyzing microorganisms and bacteriophages involved in perchlorate reduction in perchlorate-polluted wastewater according to claim 1, characterized in that: The metagenomic library established in step (7) was built using the Illumina Hiseq 4000 platform; The data quality control mentioned in step (7) involves analysis using Trimmomatic-0.36; The sequence splicing described in step (7) is performed using Metaspatades; The sequence alignment described in step (7) is performed using Bowtie 2; The Binning assembly described in step (7) is performed using the default CONCOCT settings.

7. The method for identifying and analyzing microorganisms and bacteriophages involved in perchlorate reduction in perchlorate-contaminated wastewater according to claim 1, characterized in that: The virus identification tool mentioned in step (9) is virsorter2. The clustering described in step (9) is performed using cd-hit-est-2d; The quality assessment described in step (9) is performed using CheckV; The survival type described in step (9) is PhaTYP. The classification and identification described in step (9) is performed using Phame. The host identification described in step (9) is performed using Cherry; The functional identification described in step (9) is performed using kofamscan.

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