CN115838665B - Enterobacter strain TCD1-1TAnd applications thereof - Google Patents

Enterobacter strain TCD1-1TAnd applications thereof Download PDF

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CN115838665B
CN115838665B CN202211437811.9A CN202211437811A CN115838665B CN 115838665 B CN115838665 B CN 115838665B CN 202211437811 A CN202211437811 A CN 202211437811A CN 115838665 B CN115838665 B CN 115838665B
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tcd1
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enterobacter
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nitrate
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CN115838665A (en
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杨小茹
黄鑫榕
杨乐阳
朱永官
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Institute of Urban Environment of CAS
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Abstract

The application provides application of an enterobacter strain TCD1-1 T with a preservation number of CCTCC AB 2016251 T and an enterobacter strain TCD1-1 T in ferrous oxide and nitrate nitrogen reduction and a method for ferrous oxide and nitrate reduction. The enterobacter strain TCD1-1 T with the preservation number of CCTCC AB 2016251 T can be used for connecting ferrous iron oxidation with nitrate reduction under anaerobic conditions and reducing nitrate into nitrite, nitrous oxide (N 2 O), nitric Oxide (NO), nitrogen (N 2) and other compounds by taking ferrous ion as an electron donor, so that nitrogen loss is caused.

Description

Enterobacter strain TCD1-1 T and application thereof
Technical Field
The application relates to the technical field of microorganisms, in particular to an enterobacter strain TCD1-1 T and application thereof.
Background
The nitrate-dependent iron oxidation process (NDFO) is a microorganism-mediated process of nitric acid reduction and iron oxidation coupling, and nitrate-dependent iron oxidizing bacteria play an important role in the recycling of elemental iron and the loss of nitrogen.
Disclosure of Invention
In view of the above, the present application is directed to an enterobacter strain TCD1-1 T and its application.
Based on the above purpose, the embodiment of the application provides an enterobacter strain TCD1-1 T with a preservation number of CCTCC AB2016351 T.
The embodiment of the application also provides application of the enterobacter strain TCD1-1 T with the preservation number of CCTCC AB 2016251 T in ferrous oxide and nitrate nitrogen reduction.
The embodiment of the application also provides a method for oxidizing ferrous and reducing nitrate, which adopts the enterobacter strain TCD1-1 T with the preservation number of CCTCC AB 2016251 T to oxidize ferrous and reduce nitrate nitrogen.
From the above, the embodiment of the application provides the enterobacter strain TCD1-1 T with the preservation number of CCTCC AB 2016251 T and application thereof. The enterobacter strain TCD1-1 T provided by the embodiment of the application can be used for connecting ferrous iron oxidation with nitrate reduction under anaerobic conditions, and using ferrous ions as electron donors to reduce nitrate into nitrite, nitrous oxide (N 2 O), nitric Oxide (NO), nitrogen (N 2) and other compounds so as to cause loss of nitrogen, so that the nitrate can be reduced while ferrous iron is oxidized in an ecologically friendly way.
Drawings
In order to more clearly illustrate the technical solutions of the present application or related art, the drawings that are required to be used in the description of the embodiments or related art will be briefly described below, and it is apparent that the drawings in the following description are only embodiments of the present application, and other drawings may be obtained according to the drawings without inventive effort to those of ordinary skill in the art.
FIG. 1a is a scanning electron microscope image of the growth of strain TCD1-1 T on R2A medium.
FIG. 1b is a scanning electron microscope image of the growth of strain TCD1-1 T on NDFO medium.
FIG. 2 two-dimensional TLC (thin layer chromatography) of polar lipid extracts of molybdenum phosphate stained TCD1-1 T strain. Wherein DPG is biphospholipid glycerol; PE is phosphatidylethanolamine; PG is phosphatidylglycerol; PL is a phospholipid; l1 and L2 are unknown lipids.
FIG. 3 is a schematic representation of a phylogenetic tree based on the contiguity (boottrap values, 1000 replicates) of the 16S rRNA gene sequence.
FIG. 4 is a schematic diagram of a phylogenetic tree based on maximum likelihood (a) and minimum evolution (b) of the 16S rRNA gene sequence (bootstrapping values, 1000 replicates).
FIG. 5 schematic representation of oxidation of ferrous iron by TCD1-1 T strain in NDFO medium during cultivation.
Fig. 6a is a bar graph of nitrate consumption during 4 days of culture for the experimental and first control groups.
Fig. 6b is a bar graph of ferrous consumption during 4 days of culture for the experimental and first control groups.
FIG. 6c is a Raman spectrum of the cell surface iron shell of the experimental group strain TCD1-1 T after 96 hours of culture in NDFO medium.
Detailed Description
The present application will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present application more apparent.
It should be noted that unless otherwise defined, technical or scientific terms used in the embodiments of the present application should be given the ordinary meaning as understood by one of ordinary skill in the art to which the present application belongs. The terms "first," "second," and the like, as used in embodiments of the present application, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that elements or items preceding the word are included in the element or item listed after the word and equivalents thereof, but does not exclude other elements or items. The terms "connected" or "connected," and the like, are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "upper", "lower", "left", "right", etc. are used merely to indicate relative positional relationships, which may also be changed when the absolute position of the object to be described is changed.
The embodiment of the application provides an enterobacter strain TCD1-1 T with a preservation number of CCTCC AB 2016351 T, wherein ferrous oxidation and nitrate reduction can be connected under an anaerobic condition through the enterobacter strain TCD1-1 T, ferrous ions are taken as electron donors, and the nitrate is reduced into nitrite, nitrous oxide (N 2 O), nitric Oxide (NO), nitrogen (N 2) and other compounds, so that nitrogen loss is caused.
Description of biological preservation
Enterobacter strain TCD1-1 T: the classification name is enterobacteria TCD1-1 T(Enterobacter yingtanensis sp.nov.TCD1-1T which is preserved in China center for type culture collection (CHINA CENTER for Type Culture Collection, CCTCC) on 8 th month 19 of 2021, wherein the preservation center address is the eight-path 299-number China center for type culture collection of Wuhan university in Wuhan City of Hubei province, and the preservation number is CCTCC AB 2016251 T.
The enterobacteria strain TCD1-1 T has the following physiological and biochemical characteristics:
(1) The enterobacter strain TCD1-1 T is a gram-negative bacterium;
(2) The bacterial colony of the enterobacteria strain TCD1-1 T in R2A and LB culture medium is white, has smooth surface, is convex, has the width of 0.3-0.6 mu m and the length of 0.6-2.2 mu m;
(3) The enterobacter strain TCD1-1 T has no endospore formation under a scanning electron microscope, and the cells have no motility in the culture process;
(4) The growth temperature of the enterobacteria strain TCD1-1 T is in the range of 4-60 ℃, and the optimal growth temperature is 30 ℃; the tolerance range of the pH is 3-10, and the optimal pH value is 7.0; the tolerance range of NaCl is 0-13% (w/v).
The embodiment of the application also provides application of the enterobacter strain TCD1-1 T with the preservation number of CCTCC AB 2016251 T in ferrous oxide and nitrate nitrogen reduction.
The large accumulation of nitrate nitrogen, such as nitrate, in water body can cause water pollution, cause great threat to the survival of aquatic organisms, cause water eutrophication and other problems. Iron ion (ferri ion), which is marked by Fe 3+, is the most stable ion of iron, has stronger oxidizing property and is also an important industrial agent. For example, ferric trichloride can be used for metal etching, sewage treatment and the like. Therefore, the enterobacter strain TCD1-1 T with the preservation number of CCTCC AB 2016251 T can provide an eco-friendly method for eliminating nitrate and generating iron ions by oxidizing ferrous ions.
The embodiment of the application also provides a method for oxidizing ferrous and reducing nitrate, which adopts the enterobacter strain TCD1-1 T with the preservation number of CCTCC AB 2016251 T to oxidize ferrous and reduce nitrate nitrogen.
In some embodiments, the method comprises:
S100, inoculating an enterobacter strain TCD1-1 T with a preservation number of CCTCC AB 2016251 T into a first culture medium for culture and performing plate culture.
S200, preparing the enterobacter strains TCD1-1 T after plate culture into a cell suspension, and inoculating the cell suspension into a second culture medium for ferrous oxidation and nitrate nitrogen reduction.
Wherein the first medium may be a solid medium having a pH of 6.8-7.2, including agar and NDFO medium (i.e., the second medium); the ratio of the mass of the agar to the volume of NDFO medium is 18-22%, for example 20%. The second medium may be a liquid medium with a pH of 6.8-7.2 and NDFO medium. For the first medium, it can be obtained by dissolving agar in a solid state into NDFO medium in a liquid state.
NDFO the culture medium is prepared by adding vitamin solution, microelement solution SL-10, selenite-tungstate solution, triethylamine bicarbonate buffer solution, ferrous ions and nitrate into basic culture medium. The basal medium may include MgSO 4·7H2O、CaCl2·H2O、NH4 Cl and KH 2PO4, among others. In the first medium, the concentration ratio of ferrous ions to nitrate ions may be 1:1. The concentration of the ferrous ions is 9.5-10.5 mmol L -1, and the concentration of the nitrate ions is 9.5-10.5 mmol L -1.
NDFO the culture medium may be in liquid form and have a pH of 6.8-7.2. The specific components can be MgSO4·7H2O(0.5gL-1)、CaCl2·H2O(0.1gL-1)、NH4Cl(0.3gL-1)、KH2PO4(0.6gL-1)、 Vitamin solution Vitamin solution (1 ml L -1), trace element solution TRACE ELEMENT solution SL10 (1 ml L -1), selenite-tungstate solution Selenite-tungstate solution (1 ml L -1), triethylamine bicarbonate buffer Bicarbonate buffer (22 mmol L -1)、FeCl2(10mmol L-1) and NaNO 3(10mmol L-1.
Wherein, the vitamin solution can contain multiple vitamins, and the main composition is shown in the following table 1. The trace element solution may contain a plurality of trace elements and has the main composition shown in table 2 below. The composition of the selenite-tungstate solution can be shown in table 3 below.
TABLE 1 composition of vitamin solutions
TABLE 2 composition of microelement solution
Composition of the components Volume in microelement solution
Hydrochloric acid 25% HCl 10ml
Ferrous chloride (tetrahydrate) FeCl 2*4H2 O 1.5g
Boric acid H 3BO3 30mg
Manganese chloride (tetrahydrate) MnCl 2*4H2 O 100mg
Cobalt chloride (hexahydrate) CoCl 2*6H2 O 190mg
Nickel chloride (hexahydrate) NiCl 2*6H2 O 24mg
Copper chloride (dihydrate) CuCl 2*2H2 O 2mg
Zinc chloride ZnCl 2 70mg
Sodium molybdate (dihydrate) Na 2MoO4*2H2 O 36mg
Deionized water 1000ml
TABLE 3 composition of selenite-tungstate solutions
NaOH sodium hydroxide 0.4g
Sodium selenite (pentahydrate) Na 2SeO3*5H2 O 6mg
Sodium tungstate (dihydrate) Na 2WO4*2H2 O 8mg
Deionized water 1000.0ml
Wherein, NDFO preparation of the culture medium can comprise:
The basal medium (MgSO 4·7H2O、CaCl2·H2O、NH4 Cl and KH 2PO4) was aerated to N 2/CO2 (80/20%) and autoclaved at 120℃for 20 min. The Vitamin solution, TRACE ELEMENT solution SL10, selenite-tungstate solution, bicarbonate buffer and FeCl 2 were each filtered (0.22 μm) and sterilized before addition to the basal medium.
The resulting precipitate was removed by filtration (0.22 μm) in an anaerobic glove box (N 2:CO2:H2 = 90:5:5) to give NDFO medium.
Wherein NDFO culture medium can be divided into separate bags, each 20mL culture medium is added into 50mL serum bottles, and finally sealing is carried out.
In some embodiments, in step S100, the temperature in the conditions of plate culture may be room temperature, and the culture is performed anaerobically and in a dark place for 72 hours. Thus, TCD1-1 T colonies were obtained by cultivation.
In some embodiments, in step S200, the cell suspension may be obtained by separating the cultured colonies from the first medium by a sodium chloride solution (NaCl), washing with sterile, oxygen-free deionized water, and resuspending in deionized water. The cell suspension may have an OD 600 value of 1.22.
In some embodiments, the temperature of the ferrous oxidation and the reduction of nitrate nitrogen is room temperature, anaerobically protected from light, for 96-120 hours.
The technical scheme of the invention is further described below with reference to the specific embodiments.
The experimental methods in the following examples are conventional methods unless otherwise specified.
The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
EXAMPLE 1 preparation of the second Medium (NDFO)
(1) The basic medium was prepared by mixing MgSO 4·7H2O、CaCl2·H2O、NH4 Cl and KH 2PO4 to give a mixture of 0.5g NH 4 Cl of CaCl 2·H2O、0.3gL-1 of MgSO 4·7H2O、0.1gL-1 of -1 and 0.6g KH 2PO4 of L -1, aerating with 80%/20% N 2/CO2, and autoclaving at 120℃for 20 minutes.
(2) The Vitamin solution, TRACE ELEMENT solution SL10, selenite-tungstate solution, bicarbonate buffer and FeCl 2 were each sterilized by filtration (0.22 μm) and then added to the basal medium.
(3) After the addition of Bicarbonate buffer and FeCl 2, the precipitate formed was removed by filtration (0.22 μm) in an anaerobic glove box (N 2:CO2:H2 =90:5:5). Split charging 20mL of culture medium into 50mL serum bottles, and finally sealing the culture medium.
EXAMPLE 2 preparation of first Medium
Agar was mixed with NDFO medium obtained in example 1 at a mass-to-volume ratio of 20%, aerated with 80%/20% N 2/CO2, and autoclaved at 120℃for 20 minutes.
Example 3 isolation screening of Enterobacter strain TCD1-1 T with a accession number of CCTCC AB 2016251 2016351 T
(1) In the anaerobic environment of paddy soil, the biological iron reduction and chemical iron reduction processes produce a large amount of Fe 2+. Rice soil from Jiangxi Yingtan (116 deg. 82'N,28 deg. 2'E) of China, which is rich in iron oxides but lacks organic matter, is a typical class of southern acid red soil, was collected. 3g of rice soil was placed in a 100mL serum bottle containing 50mL of sterilized water without oxygen, then sealed with a butyl rubber stopper and fixed with an aluminum cap, and incubated at 25℃for 2 hours at 120rpm in the absence of light to form rice slurry.
(2) 2ML of the rice slurry was added to a 50mL serum bottle containing 20mL of the anaerobic NDFO medium prepared in example 1, and the mixture was subjected to stationary culture at 25℃for 30 days in the absence of light.
(3) After 30 days of culture, brick red precipitate can be generated in the culture solution; the enrichment (10%) was transferred to fresh anaerobic NDFO medium and the process repeated four times to give the final enrichment.
(4) The final enrichment was subjected to gradient dilution (1 ten thousand times) and smeared (10. Mu.L) on NDFO agar plates (i.e.the first medium) prepared in example 2, and during cultivation, 80/20% by volume of N 2/CO2 was introduced and colonies were obtained under anaerobic conditions.
(5) And (3) picking out single colonies, streaking on the NDFO agar plates, and separating to obtain single bacteria TCD1-1 T.
Example 4 physiological and Biochemical characterization of Enterobacter strain TCD1-1 T with the accession number of CCTCC AB 2016251 2016351 T
Reference strain E.tabaci CGMCC 1.15707T,E.mori CGMCC 1.10322T,E.hormaechei CGMCC 1.10608T and E.asburiae JCM 6501T was purchased from the chinese collection of microorganisms (CHINA GENERAL Microbiological Culture Collection Center, CGMCC) and japan collection of microorganisms (Japan Collection of Microorganisms, JCM), respectively.
4.1 Morphological observations
TCD1-1 T examined colony morphology after aerobic growth on R2A and LB agar plates. Gram reactions were tested using the 3% KOH method. The cell surface morphology was observed with a scanning electron microscope. Cell motility of TCD1-1 T was observed on semi-solid R2A plates.
The experimental results are shown in fig. 1a and 1 b. Wherein FIG. 1a shows the growth of strain TCD1-1 T on R2A medium. FIG. 1b shows the growth of strain TCD1-1 T on NDFO medium. The strain is a gram-negative bacterium, has a rod shape, has a white colony shape, has a smooth surface, is convex and has no mobility.
4.2 Salt tolerance
Purified strain TCD1-1 T was inoculated into R2A medium containing different proportions of NaCl (0, 1.5, 3, 4.5, 6, 7.5, 9, 10, 11.5 and 13% w/v), respectively, and monitored under aerobic conditions for two days to observe growth.
The experimental results are shown in Table 4, wherein reference numeral 1 represents an enterobacter strain TCD1-1 T with a preservation number of CCTCC AB 201663 2016351 T. Reference numeral 2 represents a reference strain e.hormadeicgmcc 1.10608 T. The number 3 represents E.tabaci CGMCC 1.15707 T. Reference number 4 represents the reference strain e.mori CGMCC1.10322 T. Reference numeral 5 represents a reference strain e.asburiae JCM 6051 T. Wherein "-" means present at <1% or undetected. "+" indicates that there is a corresponding performance.
As shown in Table 4, the NaCl tolerance of the strain was in the range of 0 to 13% (w/v).
Table 4 comparison of physiological morphological characteristics between TCD1-1 T strain and closely related species.
4.3 Heat resistance
The purified strain TCD1-1 T was inoculated into R2A medium, placed in different temperatures (4, 15, 20, 25, 30, 37, 50, 60 ℃ C.) and monitored under aerobic conditions for two days to observe growth.
The experimental results are shown in table 4. The strain has a growth temperature ranging from 4 to 60 ℃ and an optimal growth temperature of 30 ℃.
4.4 Acid resistance
The pH range of growth was studied in R2A medium by adjusting the pH range (3, 4, 5, 6, 7, 8, 9 and 10) by adding HCl or NaOH and monitoring for 2 days in an aerobic environment.
The experimental results are shown in Table 4, and the pH tolerance range of the strain is 3-10, and the optimum pH is 7.0.
4.5 Carbon Source Metabolic Activity
In the API 20E test, the results indicate that strain TCD1-1 T is able to utilize glucose, mannitol, sorbitol, rhamnose, sucrose, melibiose, amygdalin and arabinose.
4.6 Enzyme Activity
In the API 20E test, the results show that the strain TCD1-1 T has catalase, oxidase, O-nitrobenzene-beta-D-galactopyranoside, arginine hydrolase, lysine decarboxylase, ornithine decarboxylase activity.
4.7 Acid production
In the API 50CH assay, the results indicate that strain TCD1-1 T is capable of acid production using the following carbon sources: glycerol, L-arabitol, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, L-rhamnose, inositol, D-mannitol, D-sorbitol, methyl-alpha-D-glucopyranoside, N-acetylglucosamine, arbutin, trehalose/ferric citrate, salicin, cellobiose, D-maltose, D-lactose, D-melibiose, D-sucrose, D-trehalose, D-raffinose, amygdalin, D-lyxose, potassium gluconate and potassium 2-ketogluconate.
Example 5 identification of species of Enterobacter strain TCD1-1 T with the accession number CCTCC AB 2016251 2016351 T
5.1 Chemical Classification identification
Fatty acids the fatty acid composition of the samples was analyzed using the fully automated bacterial identification system of MIDI (Microbial Identification) company Sherolock in the united states. The cells were analyzed for polar lipid mass spectrometry using thin plate dual phase chromatography (TLC). The major respiratory quinones were extracted from freeze-dried cells and analyzed by RP-HPLC.
The test results are shown in Table 5. Wherein, the reference numeral 1 represents an enterobacter strain TCD1-1 T with the preservation number of CCTCC AB 2016251 T. Reference numeral 2 represents a reference strain e.hormadeicgmcc 1.10608 T. The number 3 represents E.tabaci CGMCC 1.15707 T. Reference number 4 represents the reference strain e.mori CGMCC1.10322 T. Reference numeral 5 represents a reference strain e.asburiae JCM 6051 T. Wherein "-" means present at <1% or undetected. "+" indicates that there is a corresponding performance.
As can be seen, the major fatty acids of TCD1-1 T are C 16:0(33.05％)、C17:0 cycle (19.82%) and C 18:1 w7C (17.47%), as shown in Table 5. Similar to the closely related strains, but with a large difference in the content of each fatty acid, especially C 14:0 2-OH C13:0、C15:1isoH/C13:0 3OH、C17:0cyclo w8c、C17:0 and C 13:0, as shown in Table 5. The polar lipids of TCD1-1 T cells consisted of DPG, PE, PG, PL, L and L2 as shown in FIG. 2, and the major respiratory quinone was Q-8 as shown in Table 4. Oxidase and catalase were expressed in cells of strain TCD1-1 T as shown in Table 4. Strain TCD1-1 T showed almost the same pattern in API 20E and API 50CH systems as other closely related strains, except that e.asburiae JCM 6051 T was negative for rhamnose and melibiose, as shown in table 4.
Table 5 cell fatty acid composition of strain TCD1-1 T and its kindred species.
Table 6 similarity of housekeeping genes between TCD1-1 T and its closely related species.
5.2 Genetic characterization
DNA extraction was performed using FASTDNA SPIN kit (MP Biomedical, france). The universal primer pair (27F and 1492R) was used to amplify the 16S rRNA gene of the TCD1-1 T strain. Alignment of 16S rRNA genes between strains was performed by EzTaxon server. Phylogenetic tree was constructed using the adjacency method (boottrap value, 1000 replicates; calculated using MEGA version 7.0) and aligned sequences were analyzed by CLUSTAL X. Housekeeping genes of TCD1-1 T strain and reference strain were amplified (dnaA, fusA, rplB and rpoB). The GenBank accession numbers for the 16S rRNA and housekeeping gene sequences of the E.coli strain TCD1-1 T are KY628806 and SRR15667831-15667850. As shown in fig. 3 and 4. Wherein FIG. 3 is a phylogenetic tree based on the contiguity (bootstrapping values, 1000 replicates) of the 16S rRNA gene sequence, indicating the phylogenetic position of the TCD1-1 T strain in the relevant species. The scale bar in the figure represents 100 nucleotides. The sequence Propionicimonas paludicola belonging to actinomycetes was used as an outer group. FIG. 4 is a phylogenetic tree based on maximum likelihood (a) and minimum evolution (b) of the 16S rRNA gene sequence (bootstrapping values, 1000 replicates), indicating the phylogenetic position of strain TCD1-1 T in the relevant species. The scale bar in the figure represents 100 nucleotides. The sequence Propionicimonas paludicola belonging to actinomycetes was used as an outer group.
As can be seen, phylogenetic analysis based on the 16S rRNA gene shows that strain TCD1-1 T belongs to the genus Enterobacter.
The sequence similarity between strain TCD1-1 T and its phylogenetically related strain E.tabaci CGMCC 1.15707T,E.mori CGMCC 1.10322T,E.hormaechei CGMCC 1.10608T,E.asburiae JCM6501T was 99.37%,99.30%,98.52%,97.89%, respectively, as shown in Table 4. The DNA G+C content of strain TCD1-1 T was 57mol%, which is close to that of the strain related to its phylogenetic development, as shown in Table 4. DNA-DNA hybridization values of TCD1-1 T with E.hormadeiCGMCC 1.10608 T、E.tabaci CGMCC 1.15707T;E.mori CGMCC 1.10322T and E.asburia JCM 6051 T are 57.75%, 72.62%, 50.86% and 60.20%, respectively, as shown in Table 4. Based on the 70% threshold for DNA-DNA hybridization for species division, TCD1-1 T was suggested as a subspecies of e.tabaci CGMCC 1.15707 T. To further increase the resolution, analysis of the similarity between housekeeping genes (including dnaA, fusA, rplB and rpoB) between TCD1-1 T and its closely related strains showed that the similarity between them was 90.12-96.01%, as shown in table 6, demonstrating the genomic differences between TCD1-1 T and its closely related strains, indicating that TCD1-1 T is a new species belonging to enterobacteria.
5.3 Genomic Properties
Genomic sequencing of TCD1-1 T was performed on the Hiseq 4000 platform (2X 150 bp) of Meiji (Shanghai, china). The Metawrap package (1.2.1) was used to analyze the assembly of the original genome. Briefly, genomes were further checked, trimmed and assembled using Megahit (1.1.3) of the default settings. The scaffolds sequences above 1000bps were binned into refined genome bins using a combination of CONCOCT (1.0.0), maxBin2 (2.2.6) and metaBAT2 (2.12.1). To improve the quality of the bin, the MAGs of TCD1-1 T are remapped and assembled using short read mappers BWA (0.7.17) and SPAdes (3.13.0), respectively. The contamination level and integrity of the TCD1-1 T genome was assessed by CheckM (1.0.12). The protein coding region was predicted using Prodigal software (2.6.3 th edition) with "-p meta" option. The genomes of TCD1-1 T were annotated using the KEGG database (BlastKOALA), interProScan (5.44-79.0), eggNOG-mapper (5.0.0), diamond (0.9.22) and NCBI-nr database (search 10 months of 2021, E value. Ltoreq.1e-5) in combination.
The genome length of the strain TCD1-1 T is 4557964bp, and 4311 protein coding genes are totally included, including 4281 protein coding sequences (CDS), 23S rRNA and 16S rRNA. According to the remarks, there are ABC protein channels encoding for the transport of nitrate, which genes might be involved in the uptake and transport of nitrate by TCD1-1 T cells. In addition, several genes have been identified as being involved in nitrate reduction, including nitrate reductase molybdenum cofactor assembly chaperones, nitrate reductase (alpha, beta and gamma subunits). In addition, strain TCD1-1 T encodes several enzymes associated with cytochrome c reduction and oxidation. Cytochrome c acts as an electron shuttle, accepting electrons from iron (II), and the reduced cytochrome c releases electrons to nitrate. Therefore, the strain TCD1-1 can be used as a model microorganism of nitrate-dependent iron oxidation functional microorganisms to search for the physiological state of bacteria in the nitric acid-dependent iron oxidation process.
Application example New strain (Enterobacter yingtanensis sp. TCD1-1 T) application
NDFO capacity application and effect of TCD1-1 T strain
1) Experimental setup
The experiments contained a control group (Abiotic) and an experimental group (TCD 1-1 T), each set of 3 replicates. Wherein the culture medium of the experimental group and the first control group is NDFO culture medium. The medium of the second control group and the third control group was NDFO medium without NaNO 3. That is, the medium in the second control group and the third control group was different from the medium in the experimental group only in that no NaNO 3 was present.
The culture conditions were anaerobic light-shielding culture at 30℃for 4 days.
The inoculum was derived from a colony of the Enterobacter strain TCD1-1 having a preservation number of CCTCC AB 2016351 T which had been previously cultured on a NDFO agar plate (anaerobic culture at 30℃for 3 days in the absence of light). Colonies were first washed from NDFO agar plates with sterile, oxygen-free 0.9% NaCl (w/v) solution, and the cells were washed 3 times with sterile, oxygen-free deionized water (8000 g,10 min) and resuspended in 60mL of sterile, oxygen-free deionized water. 1mL of TCD1-1 T cell suspension with OD600 of 1.12 was removed to 50mL of medium containing 20mL NDFO as the experimental group. 1mL of TCD1-1 T cell suspension with OD600 of 1.12 was removed to 50mL of NDFO medium containing 20mL of NaNO 3 -free medium as a second control.
1ML of sterile, oxygen-free deionized water was removed to 50mL of medium containing 20mLNDFO as a first control. 1mL of sterile, oxygen-free deionized water was removed to 50mL of NDFO medium with 20mL of no 3 as a third control.
2) Experimental results
As shown in fig. 5, 6a to 6c and table 7. In fig. 5, the left graph shows the results obtained after the culture of the first control group is completed, and the right graph shows the results obtained after the culture of the experimental group is completed. Figure 6a shows nitrate consumption during 4 days of culture for the experimental and first control groups. Fig. 6b shows the consumption of ferrous iron during 4 days of culture in the experimental and first control groups. FIG. 6c shows the Raman spectrum of the cell surface iron shell of the experimental group strain TCD1-1 T after 96 hours of culture in NDFO medium.
Table 7 changes in nitrate and ferrous ions during 4 days of incubation for experimental, first, second and third control groups
As can be seen, during anaerobic cultivation, strain TCD1-1 T of the experimental group was observed to produce brown iron oxide in NDFO medium. As shown in Table 7, during the 4-day incubation period, strain TCD1-1 T of the experimental group consumed 1.09mmol L -1 and 2.44mmol L -1 of nitrate and Fe (II), respectively, in NDFO medium. Cells of the TCD1-1 T strain were coated with iron oxide after 4 days of culture. In the second control group with no nitrate added, no oxidation of ferrous ions was observed.
Therefore, the enterobacteria strain TCD1-1 T with the preservation number of CCTCC AB 2016351 T has good ferrous oxide and nitrate nitrogen reduction capability, can be used for sewage treatment and the like, and can be used as a model microorganism for exploring the physiological state of bacteria in the nitric acid-dependent iron oxidation process and the like.
Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined under the idea of the present disclosure, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in details for the sake of brevity.
While the present disclosure has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations of those embodiments will be apparent to those skilled in the art in light of the foregoing description.
The disclosed embodiments are intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Accordingly, any omissions, modifications, equivalents, improvements, and the like, which are within the spirit and principles of the embodiments of the disclosure, are intended to be included within the scope of the disclosure.

Claims (2)

1. The enterobacter (Enterobacter yingtanensis sp.nov.) TCD1-1 T is characterized in that the preservation number is CCTCC AB 2016251 T, the enterobacter is preserved in China center for type culture collection, the preservation date is 2021, 8 months and 19 days, and the preservation address is the China center for type culture collection of the eight-path 299 of Wuhan district of Wuhan, hubei province.
2. Use of enterobacter (Enterobacter yingtanensis sp.nov.) TCD1-1 T according to claim 1 in ferrous oxide and reduction of nitrate nitrogen.
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CN108037257A (en) * 2017-11-23 2018-05-15 南京大学 It is a kind of to study test method and the device that iron ion influences Anammox efficiency
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