CN115838665A - Enterobacter strain TCD1-1 T And uses thereof - Google Patents

Enterobacter strain TCD1-1 T And uses thereof Download PDF

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CN115838665A
CN115838665A CN202211437811.9A CN202211437811A CN115838665A CN 115838665 A CN115838665 A CN 115838665A CN 202211437811 A CN202211437811 A CN 202211437811A CN 115838665 A CN115838665 A CN 115838665A
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ferrous
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CN115838665B (en
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杨小茹
黄鑫榕
杨乐阳
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Institute of Urban Environment of CAS
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Abstract

The application provides a preservation number of CCTCC AB2016351 T Enterobacter strain TCD1-1 T Enterobacter strain TCD1-1 T Application in ferrous iron oxidation and nitrate nitrogen reduction and a method for ferrous iron oxidation and nitrate nitrogen reduction. Wherein the preservation number is CCTCC AB2016351 T Enterobacter strain TCD1-1 T By Enterobacter strain TCD1-1 T Can link the ferrous oxidation and the nitrate reduction under the anaerobic condition, and takes ferrous ions asElectron donor to reduce nitrate to nitrite and nitrous oxide (N) 2 O), nitric Oxide (NO), and nitrogen (N) 2 ) These compounds cause nitrogen loss.

Description

Enterobacter strain TCD1-1 T And uses thereof
Technical Field
This application relates toAnd the technical field of microorganisms, in particular to an enterobacter strain TCD1-1 T And applications thereof.
Background
Nitrate-dependent iron oxidation process (NDFO) is a microbial-mediated process of coupling nitric acid reduction and iron oxidation, and nitrate-dependent iron oxidizing bacteria play an important role in the recycling of iron elements and the loss of nitrogen elements.
Disclosure of Invention
In view of the above, the present application aims to provide an Enterobacter strain TCD1-1 T And applications thereof.
In view of the above, the embodiment of the present application provides a preservation number of CCTCC AB2016351 T Enterobacter strain TCD1-1 T 。
The embodiment of the application also provides a preservation number of CCTCC AB2016351 T Enterobacter strain TCD1-1 T The application in ferrous oxidation and nitrate nitrogen reduction.
The embodiment of the application also provides a method for reducing ferrous oxide and nitrate, which adopts a preservation number of CCTCC AB2016351 T Enterobacter strain TCD1-1 T Ferrous iron oxidation and nitrate nitrogen reduction.
As can be seen from the foregoing, embodiments of the present application provide a collection number of CCTCC AB2016351 T Enterobacter strain TCD1-1 T And applications thereof. Enterobacter strain TCD1-1 provided in the examples of the present application T Can link the ferrous oxidation with the nitrate reduction under anaerobic condition, and reduce the nitrate into nitrite and nitrous oxide (N) by taking ferrous ions as electron donors 2 O), nitric Oxide (NO), and nitrogen (N) 2 ) The compounds cause the loss of nitrogen, and can reduce nitrate while oxidizing ferrous iron ecologically.
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In order to more clearly illustrate the technical solutions in the present application or the related art, the drawings needed to be used in the description of the embodiments or the related art will be briefly introduced below, and it is obvious that the drawings in the following description are only embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.
FIG. 1a shows strain TCD1-1 T Scanning electron microscopy imaging of growth on R2A medium.
FIG. 1b shows strain TCD1-1 T Scanning electron microscopy imaging of growth on NDFO medium.
FIG. 2 molybdophosphoric acid-stained TCD1-1 T Two-dimensional TLC (thin layer chromatography) of polar lipid extracts of the strain. Wherein DPG is diphosphatidylglycerol; PE is phosphatidyl ethanolamine; PG is phosphatidyl glycerol; PL is a phospholipid; l1 and L2 are unknown lipids.
FIG. 3 is a schematic representation of a phylogenetic tree based on the adjacency (bootstrap values, 1000 repeats) of the 16S rRNA gene sequence.
FIG. 4 is a schematic diagram of a maximum likelihood (a) and minimum evolution (b) (bootstrap values, 1000 repeats) phylogenetic tree based on the 16S rRNA gene sequence.
FIG. 5 TCD1-1 in the culture Process T Schematic representation of the oxidation of ferrous iron by the strain in NDFO medium.
Fig. 6a is a bar graph of nitrate consumption during 4 days of culture for the experimental group and the first control group.
Fig. 6b is a bar graph of the consumption of ferrous iron during 4 days of culture for the experimental group and the first control group.
FIG. 6c shows strain TCD1-1 in the experimental group T Raman spectrum of iron shells on the cell surface after 96 hours of culture in NDFO medium.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail below with reference to the accompanying drawings in combination with specific embodiments.
It should be noted that technical terms or scientific terms used in the embodiments of the present application should have a general meaning as understood by those having ordinary skill in the art to which the present application belongs, unless otherwise defined. The use of "first," "second," and similar terms in the embodiments of the present application do not denote any order, quantity, or importance, but rather the terms are used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that the element or item listed before the word covers the element or item listed after the word and its equivalents, but does not exclude other elements or items. The terms "connected" or "coupled" and the like are not restricted to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "upper", "lower", "left", "right", and the like are used merely to indicate relative positional relationships, and when the absolute position of the object being described is changed, the relative positional relationships may also be changed accordingly.
The embodiment of the application provides a preservation number of CCTCC AB2016351 T Enterobacter strain TCD1-1 T By Enterobacter strain TCD1-1 T Can link the ferrous oxidation with the nitrate reduction under anaerobic condition, and reduce the nitrate into nitrite and nitrous oxide (N) by taking ferrous ions as electron donors 2 O), nitric Oxide (NO), and nitrogen (N) 2 ) These compounds cause nitrogen loss.
Biological preservation Instructions
Enterobacter strain TCD1-1 T : classified name is enterobacter TCD1-1 T (Enterobacter yingtanensis sp.nov.TCD1-1 T ) Has been preserved in China Center for Type Culture Collection (CCTCC) at 19.8.18.1.5.the preservation Center address is China Center for Type Culture Collection, wuhan university, eight-channel 299 in Wuchang district, wuhan City, hubei province, and the preservation number is CCTCC AB2016351 T 。
The Enterobacter 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 Enterobacter strain TCD1-1 T The colony morphology in R2A and LB culture medium is white, the surface is smooth and convex, the width is 0.3-0.6 μm, and the length is 0.6-2.2 μm;
(3) The enterobacter strain TCD1-1 T In a scanning electron microscopeNo endospore formation, no cell motility during culture;
(4) The Enterobacter strain TCD1-1 T The growth temperature range is 4-60 ℃, and the optimal growth temperature is 30 ℃; the tolerance range of pH is 3-10, and the optimum pH value is 7.0; naCl tolerance ranges from 0-13% (w/v).
The embodiment of the application also provides a preservation number of CCTCC AB2016351 T Enterobacter strain TCD1-1 T The application in ferrous oxidation and nitrate nitrogen reduction.
The large accumulation of nitrate nitrogen such as nitrate in the water body can cause water body pollution, great threat to the survival of aquatic organisms, water body eutrophication and other problems. Iron ion (ferrion) with the symbol Fe 3+ It is the most stable ion of iron, has strong oxidizability, and is an important industrial agent. For example, ferric trichloride can be applied to metal etching, sewage treatment and the like. Therefore, the preservation number is CCTCC AB2016351 T Enterobacter strain TCD1-1 T Can provide an eco-friendly method for eliminating nitrate and oxidizing ferrous ions to generate iron ions.
The embodiment of the application also provides a method for reducing ferrous oxide and nitrate, which adopts a preservation number of CCTCC AB2016351 T Enterobacter strain TCD1-1 T Ferrous iron oxidation and nitrate nitrogen reduction.
In some embodiments, the method comprises:
s100, numbering the preservation number of CCTCC AB2016351 T Enterobacter strain TCD1-1 T Inoculating into the first culture medium, and culturing to plate.
S200, culturing the Enterobacter strain TCD1-1 after plate culture T Preparing a cell suspension, and inoculating the cell suspension in a second culture medium for ferrous oxidation and nitrate nitrogen reduction.
Wherein the first culture medium can be a solid medium, has a pH of 6.8-7.2, and comprises agar and NDFO medium (i.e., the second culture medium); the ratio of the mass of the agar to the volume of the NDFO medium is 18-22%, for example 20%. The second medium may be a liquid medium, pH 6.8-7.2, NDFO medium. As the first culture medium, it can be obtained by dissolving agar in a solid state into NDFO medium in a liquid state.
The NDFO culture medium is prepared by adding vitamin solution, microelement solution SL-10, selenite-tungstate solution, triethylamine bicarbonate buffer solution, ferrous ions and nitrate into a basic culture medium. Wherein the basal medium may comprise MgSO 4 ·7H 2 O、CaCl 2 ·H 2 O、NH 4 Cl and KH 2 PO 4 . In the first medium, the concentration ratio of ferrous ions to nitrate ions may be 1. The concentration of the ferrous ions is 9.5-10.5 mmol L -1 The concentration of the nitrate ion is 9.5-10.5 mmol L -1 。
The NDFO medium may be in liquid form and has a pH of 6.8-7.2. The specific component can be MgSO 4 ·7H 2 O(0.5gL -1 )、CaCl 2 ·H 2 O(0.1gL -1 )、NH 4 Cl(0.3gL -1 )、KH 2 PO 4 (0.6gL -1 ) Vitamin solution (1 ml L) -1 ) Trace element solution SL10 (1 ml L) -1 ) Selenite-tungstate solution (1 ml L) -1 ) Triethylamine Bicarbonate buffer Bicarbonate buffer (22 mmol L) -1 )、FeCl 2 (10mmol L -1 ) And NaNO 3 (10mmol L -1 )。
The vitamin solution may contain various vitamins, and the main components are shown in table 1 below. The trace element solution may contain a variety of trace elements, with the main composition as in table 2 below. The composition of the selenite-tungstate solution may be as shown in table 3 below.
TABLE 1 composition of vitamin solution
Figure BDA0003944880420000041
Figure BDA0003944880420000051
TABLE 2 composition of the microelement solutions
Composition (I) Volume in solution of trace elements
Hydrochloric acid 25% 10ml
FeCl (ferrous chloride tetrahydrate) 2 *4H 2 O 1.5g
Boric acid H 3 BO 3 30mg
Manganese chloride (tetrahydrate) MnCl 2 *4H 2 O 100mg
CoCl (cobalt chloride hexahydrate) 2 *6H 2 O 190mg
NiCl (NiCl hexahydrate) 2 *6H 2 O 24mg
Cupric chloride (dihydrate) CuCl 2 *2H 2 O 2mg
Zinc chloride ZnCl 2 70mg
Sodium molybdate (dihydrate) Na 2 MoO 4 *2H 2 O 36mg
Deionized water 1000ml
TABLE 3 composition of selenite-tungstate solution
NaOH sodium hydroxide 0.4g
Sodium selenite (pentahydrate) Na 2 SeO 3 *5H 2 O 6mg
Sodium tungstate (dihydrate) Na 2 WO 4 *2H 2 O 8mg
Deionized water 1000.0ml
The NDFO medium may be prepared by:
basal medium (MgSO) 4 ·7H 2 O、CaCl 2 ·H 2 O、NH 4 Cl and KH 2 PO 4 ) Aeration is carried out N 2 /CO 2 (80/20%) and autoclaved at 120 ℃ for 20 minutes. Mixing Vitamin solution, trace element solution SL10, Selenite-tungstate solution, bicarbonate buffer and FeCl 2 After filtration (0.22 μm) and sterilization, respectively, the cells were added to the basal medium.
In an anaerobic glove box (N) 2 :CO 2 :H 2 The NDFO medium was prepared by removing the formed precipitate by filtration (0.22 μm) in = 90.
The NDFO culture medium can be subpackaged, and each 20mL culture medium is added into a 50mL serum bottle and finally sealed.
In some embodiments, in step S100, the temperature in the plate culture conditions may be room temperature, and the anaerobic light-resistant culture is performed for 72h. Thus, TCD1-1 can be cultured T And (5) bacterial colonies.
In some embodiments, in step S200, the cultured colonies may be separated from the first medium by a sodium chloride solution (NaCl), washed with sterile, anaerobic deionized water, and resuspended in deionized water to obtain a cell suspension. OD of the cell suspension 600 The value may be 1.22.
In some embodiments, the temperature for ferrous oxidation and nitrate nitrogen reduction is room temperature, anaerobic and dark, and the time is 96-120h.
The technical solution of the present invention will be further described with reference to the following embodiments.
The experimental procedures in the following examples are conventional unless otherwise specified.
The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
EXAMPLE 1 preparation of second Medium (NDFO)
(1) MgSO (MgSO) 4 ·7H 2 O、CaCl 2 ·H 2 O、NH 4 Cl and KH 2 PO 4 Mixing to obtain 0.5gL -1 MgSO (2) of 4 ·7H 2 O、0.1gL -1 In (C) is 2 ·H 2 O、0.3gL -1 NH of 4 Cl and 0.6gL -1 KH of 2 PO 4 The mixed solution of (1) is 80%/20% N 2 /CO 2 Aerating, and autoclaving at 120 deg.C for 20 min to obtain basic culture medium.
(2) Mixing Vitamin solution, trace element solution SL10, selenite-tungstate solution, bicarbonate buffer and FeCl 2 After filtration (0.22 μm) and sterilization, the basal medium was added.
(3) Adding Bicarbonate buffer and FeCl 2 Then, in an anaerobic glove box (N) 2 :CO 2 :H 2 = 90) by filtration (0.22 μm). Subpackaging 20mL of culture medium to 50mL of serum bottles, and finally sealing the culture medium.
EXAMPLE 2 preparation of the first Medium
Agar was mixed with 20% by mass/volume of the NDFO medium obtained in example 1, and 80%/20% N was used 2 /CO 2 Aeration was performed and autoclaved at 120 ℃ for 20 minutes.
Example 3 accession number CCTCC AB2016351 T Enterobacter strain TCD1-1 T Separation and screening of
(1) In the anaerobic environment of rice soil, a large amount of Fe is generated in the processes of biological iron reduction and chemical iron reduction 2+ . Rice soil from Yingtan, jiangxi, china (116 ° 82'N,28 ° 2' E) was collected, which is rich in iron oxides but deficient in organic matter, a typical type of southern acidic red soil. 3g of rice soil was put into a 100mL serum bottle containing 50mL of oxygen-free sterilized water, then sealed with a butyl rubber stopper and fixed with an aluminum cap, and incubated at 25 ℃ for 2 hours in the dark at 120rpm to form a rice slurry.
(2) Using the slurry of rice as an inoculation source, 2mL of the slurry was added to a 50mL serum bottle containing 20mL of oxygen-free NDFO medium prepared in example 1, and the mixture was cultured in dark at 25 ℃ for 30 days.
(3) After 30 days of culture, brick red precipitates can be generated in the culture solution; the concentrate (10%) was transferred to fresh anaerobic NDFO medium and the process was repeated four times to obtain the final concentrate.
(4) The final enrichment was diluted in a gradient (1 ten thousand fold) and the resulting NDFO agar plates (i.e., prepared in example 2) were plated outFirst culture medium) was applied (10. Mu.L), and N was introduced in a volume ratio of 80/20% during the culture 2 /CO 2 Colonies were obtained under anaerobic conditions.
(5) Selecting a single bacterium colony, streaking the single bacterium colony on the NDFO agar plate, and separating to obtain a single bacterium TCD1-1 T 。
Example 4 accession number CCTCC AB2016351 T Enterobacter strain TCD1-1 T Identification of physiological and biochemical characteristics of
Reference strain E.tabaci CGMCC 1.15707 T ,E.mori CGMCC 1.10322 T ,E.hormaechei CGMCC 1.10608 T and E.asburiae JCM 6501 T Purchased from China microbial Culture Collection (CGMCC) and Japan Collection of Microorganisms (JCM), respectively.
4.1 morphological Observation
TCD1-1 T Colony morphology was examined after aerobic growth on R2A and LB agar plates. Gram reaction was tested using the 3% KOH method. The cell surface morphology was observed with a scanning electron microscope. TCD1-1 T The cell motility activity of (2) was observed on semi-solid R2A plates.
The results of the experiment are shown in FIGS. 1a and 1 b. Wherein, FIG. 1a shows strain TCD1-1 T Growth on R2A medium. FIG. 1b shows strain TCD1-1 T Growth on NDFO medium. The bacterial strain is gram-negative bacteria, is rod-shaped, and has white colony morphology, smooth surface, convex shape and no motility.
4.2 salt tolerance
The purified strain TCD1-1 T The cells were inoculated into R2A media containing NaCl at different ratios (0, 1.5, 3, 4.5, 6, 7.5, 9, 10, 11.5 and 13% w/v), respectively, monitored for two days in an aerobic environment, and growth was observed.
The results are shown in Table 4, in which reference numeral 1 denotes the accession number CCTCC AB2016351 T Enterobacter strain TCD1-1 T . Reference numeral 2 represents the reference strain E.hormaedei CGMCC 1.10608 T . The number 3 represents E.tabaci CGMCC 1.15707 T . Reference numeral 4 represents a reference strain E.mori CGMCC1.10322 T . Reference numeral 5 denotes the reference strain E.asburiae JCM 6051 T . Wherein "-" means present in<1% or not detected. "+" indicates that the corresponding property is present.
As can be seen from Table 4, the NaCl tolerance of this strain ranged from 0-13% (w/v).
TABLE 4 TCD1-1 T And comparing the physiological morphological characteristics of the strain and the kindred species.
Figure BDA0003944880420000081
Figure BDA0003944880420000091
4.3 Heat resistance
Purifying the strain TCD1-1 T Inoculated in R2A culture medium, respectively placed in different temperatures (4, 15, 20, 25, 30, 37, 50, 60 ℃), monitored for two days under aerobic environment, and observed for growth.
The results of the experiment are shown in table 4. As can be seen, the growth temperature range of the strain is 4-60 ℃, and the optimal growth temperature is 30 ℃.
4.4 acid resistance
In the R2A medium, the pH range (3, 4, 5, 6, 7, 8, 9 and 10) was adjusted by adding HCl or NaOH and monitored for 2 days in an aerobic environment, and the pH range of growth was investigated.
As shown in Table 4, the pH tolerance range of the strain was 3 to 10, and the optimum pH was 7.0.
4.5 carbon Source metabolizing Activity
In the API 20E test, the results show that the strain TCD1-1 T Glucose, mannitol, sorbitol, rhamnose, sucrose, melibiose, amygdalin, and arabinose can be utilized.
4.6 enzymatic 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 indicated that strain TCD1-1 T The acid production can be carried out 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 accession number CCTCC AB2016351 T Enterobacter strain TCD1-1 T Identification of the bacterial species
5.1 chemical Classification identification
Fatty acids the fatty acid composition of the samples was analyzed using the full-automatic bacteria Identification system of the company shorolock, MIDI (micro Identification) in usa. The cells were analyzed for polar lipid mass spectra using thin plate biphasic 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 reference numeral 1 denotes a preservation number of CCTCC AB2016351 T Enterobacter strain TCD1-1 T . Reference numeral 2 represents the reference strain E.hormaedei CGMCC 1.10608 T . The number 3 represents E.tabaci CGMCC 1.15707 T . Reference numeral 4 represents a reference strain E.mori CGMCC1.10322 T . Reference numeral 5 denotes the reference strain E.asburiae JCM 6051 T . Wherein "-" means present in<1% or not detected. "+" indicates that there is a corresponding capability.
As can be seen, TCD1-1 T The main fatty acid of (B) is C 16:0 (33.05％)、C 17:0 cyclo (19.82%) and C 18:1 w7c (17.47%), as shown in Table 5. Similar to closely related strains, but the content of each fatty acid is greatly different, especially C 14:0 2-OH C 13:0 、C 15:1 isoH/C 13:0 3OH、C 17:0 cyclo w8c、C 17:0 And C 13:0 See table 5.TCD1-1 T The polar lipids of the cells consist of DPG, PE, PG, PL, L1 and L2, as shown in FIG. 2, and the major respiratory quinone is Q-8, as shown in Table 4. Strain TCD1-1 T The cells of (a) express oxidase and catalase as shown in Table 4. Except that E.asburiae JCM 6051 T In addition to being negative for rhamnose and melibiose, strain TCD1-1 T The closely related strains showed almost the same pattern in the API 20E and API 50CH systems as shown in table 4.
TABLE 5 Strain TCD1-1 T And cellular fatty acid composition of closely related species thereof.
Figure BDA0003944880420000111
Figure BDA0003944880420000121
TABLE 6 TCD1-1 T Similarity of housekeeping genes with their cognate species.
Figure BDA0003944880420000122
5.2 genetic characteristics
DNA extraction was performed using the FastDNA Spin kit (MP Biomedical, france). Universal primer pair (27F and 1492R) for amplifying TCD1-1 T 16S rRNA gene of the strain. 16S rRNA gene alignments between strains were performed by EzTaxon server. Phylogenetic trees were constructed using the adjacency method (bootstrap values, 1000 replicates; calculated using MEGA version 7.0) and the aligned sequences were analyzed by CLUSTAL X. Amplification of TCD1-1 T Housekeeping genes (dnaA, fusA, rplB and rpoB) of the strains and the reference strains. Enterobacter strain TCD1-1 T GenBank accession numbers of 16S rRNA and housekeeping gene sequences of (9) are KY628806 and SRR15667831-15667850. As shown in fig. 3 and 4. Wherein, FIG. 3 is based on 16SPhylogenetic Tree of adjacency (Bootstrap value, 1000 repeats) of rRNA Gene sequences, indicating TCD1-1 T The strain is in a phylogenetic position in the relevant species. The scale bar in the figure represents 100 nucleotides. The sequence of Propionimonas paludicola belonging to actinomycetes was used as the exocolony. FIG. 4 is a maximum likelihood (a) and minimum evolution (b) (bootstrap values, 1000 repeats) phylogenetic tree based on the 16S rRNA gene sequence, showing strain TCD1-1 T At a phylogenetic position in the relevant species. The scale bar in the figure represents 100 nucleotides. The sequence of Propionimonas paludicola belonging to actinomycetes was used as the exocolony.
As can be seen, phylogenetic analysis based on the 16S rRNA gene showed that strain TCD1-1 T Belonging to the genus Enterobacter.
Strain TCD1-1 T Strain E.tabaci CGMCC 1.15707 related to its phylogeny T ,E.mori CGMCC 1.10322 T ,E.hormaechei CGMCC 1.10608 T ,E.asburiae JCM6501 T The sequence similarity between them was 99.37%,99.30%,98.52%,97.89%, respectively, as shown in table 4. Strain TCD1-1 T The DNA G + C content of (2) was 57mol%, which is close to that of phylogenetically related strains, as shown in Table 4. TCD1-1 T And E.hormaedei CGMCC 1.10608 T 、E.tabaci CGMCC 1.15707 T ；E.mori CGMCC 1.10322 T And e.asbushiae JCM 6051 T The DNA-DNA hybridization values of (1) were 57.75%, 72.62%, 50.86% and 60.20%, respectively, as shown in Table 4. Based on a 70% threshold for DNA-DNA hybridization for species division, TCD1-1 is recommended T May be used as E.tabaci CGMCC 1.15707 T A subspecies of (1). To further improve resolution, for TCD1-1 T The similarity between housekeeping genes (including dnaA, fusA, rplB and rpoB) with their closely related strains was analyzed, and the results showed that the similarity between them was 90.12 to 96.01%, as shown in Table 6, demonstrating TCD1-1 T The genomic difference from its closely related strain indicates TCD1-1 T Is a new species belonging to enterobacteria.
5.3 genomic Properties
TCD1-1 T Genome sequencing in Meiji (China)Sea) on the Hiseq 4000 platform (2X 150 bp). The Metawrap package (1.2.1) was used to analyze the assembly of the original genome. Briefly, the genome was further examined, trimmed and assembled using Megahit (1.1.3) at the default setting. The scaffold sequences of 1000bps or more were binned into refined genomic bins using a combination of conct (1.0.0), maxBin2 (2.2.6) and metaBAT2 (2.12.1). To improve bin quality, TCD1-1 T Respectively using short read mappers BWA (0.7.17) and SPAdes (3.13.0). TCD1-1 T The degree of contamination and integrity of the genome was assessed by CheckM (1.0.12). The protein coding region was predicted using prodigan software with the "-p meta" option (version 2.6.3). TCD1-1 pairs using the KEGG database (BlastKOALA), interProScan (5.44-79.0), eggNOG-mapper (5.0.0), diamond (0.9.22) and NCBI-nr database (10 month search 2021, E value. Ltoreq.1E-5) in combination T The genome of (a) is annotated.
Strain TCD1-1 T The genome length of (a) is 4557964bp, and there are 4311 protein-coding genes in total, including 4281 protein-coding sequences (CDS), 23S rRNA and 16S rRNA. According to the annotation, there are ABC protein channels coding for the transport of nitrate, these genes probably being linked to TCD1-1 T The cells are involved in the uptake and transport of nitrate. In addition, several genes have been identified that are involved in nitrate reduction, including nitrate reductase molybdenum cofactor assembly chaperone proteins, nitrate reductase enzymes (alpha, beta and gamma subunits). Furthermore, strain TCD1-1 T Several cytochrome c reduction and oxidation related enzymes are also encoded. 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 a nitrate-dependent iron oxidation functional microorganism to search for the physiological state of bacteria in a nitrate-dependent iron oxidation process.
Application example is new strain (Enterobacter yingtanensis sp. TCD 1-1) T ) Application of
TCD1-1 T Application and effect of strain NDFO (Newcastle disease Virus) capability
1) Experimental setup
The experiment included a control group (Abiot)ic) and Experimental group (TCD 1-1) T ) Each set is provided with 3 parallels. The culture medium of the experimental group and the first control group is NDFO culture medium. The culture medium of the second control group and the third control group is without NaNO 3 NDFO medium of (1). That is, the culture media in the second control group and the third control group were different from the culture media of the experimental group only in not having NaNO 3 。
The culture conditions were anaerobic and light-proof culture at 30 ℃ for 4 days.
The inoculation source is from a preservation number of CCTCC AB2016351 T The Enterobacter strain TCD1-1 of (9) was cultured on NDFO agar plates (anaerobically cultured at 30 ℃ for 3 days in the absence of light). Colonies were first washed from NDFO agar plates with sterile, anaerobic 0.9% NaCl (w/v) solution, and the cells were washed 3 times (8000 g, 10min) with sterile, anaerobic deionized water and resuspended in 60mL of sterile, anaerobic deionized water. 1mL of TCD1-1 having an OD600 of 1.12 was removed T Cell suspension, to 50mL containing 20mL NDFO medium as experimental group. 1mL of TCD1-1 having an OD600 of 1.12 was removed T Cell suspension to 50mL containing 20mL of NaNO-free 3 The NDFO medium of (c) was used as a second control group.
1mL of sterile, anaerobic, deionized water was removed to 50mL of medium containing 20mL of LNDFO as a first control group. 1mL of sterile, oxygen-free deionized water was pipetted into 50mL of sterile, oxygen-free deionized water containing 20mL of NaNO-free deionized water 3 The NDFO medium of (5) was used as a third control group.
2) Results of the experiment
As shown in fig. 5, fig. 6a to 6c and table 7. In FIG. 5, the left graph is the result obtained after the first control group culture is completed, and the right graph is the result obtained after the experimental group culture is completed. FIG. 6a is the nitrate consumption during 4 days of culture in the experimental group and the first control group. Fig. 6b is the consumption of ferrous iron during 4 days of culture in the experimental group and the first control group. FIG. 6c shows the strain TCD1-1 of the experimental group T Raman spectra of cell surface iron shells after 96 hours of culture in NDFO medium.
TABLE 7 changes in nitrate and ferrous ions during 4 days of culture in the experimental group, the first control group, the second control group and the third control group
Figure BDA0003944880420000151
As can be seen, during the anaerobic cultivation, strain TCD1-1 of the experimental group was observed in NDFO medium T Brown iron oxides are produced. As shown in Table 7, during the 4-day culture, strain TCD1-1 was present in the experimental group T 1.09mmol L of each of the NDFO media was consumed -1 And 2.44mmol L -1 Nitrate salt of (i) and Fe (II). TCD1-1 T The cells of the strain were coated with iron oxide after 4 days of culture. In the second control group, where no nitrate was added, no oxidation of ferrous ions was observed.
Thus, the accession number is CCTCC AB2016351 T Enterobacter strain TCD1-1 T Has excellent ability to oxidize ferrous iron and reduce nitrate nitrogen, can be used for treatment of sewage and the like, and can be used as a model microorganism for searching the physiological state of bacteria in a nitric acid-dependent iron oxidation process.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the present disclosure, also technical features in the above embodiments or in different embodiments may be combined, 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 detail for the sake of brevity.
While the present disclosure has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations of these embodiments will be apparent to those of ordinary skill 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. Therefore, any omissions, modifications, equivalents, improvements, and the like that may be made within the spirit and principles of the embodiments of the disclosure are intended to be included within the scope of the disclosure.

Claims (10)

1. The preservation number is CCTCC AB2016351 T Enterobacter strain TCD1-1 T 。
2. Enterobacter strain TCD1-1 according to claim 1 T The application in ferrous oxidation and nitrate nitrogen reduction.
3. A method for preparing ferrous oxide and reducing nitrate is characterized in that a preservation number of CCTCC AB2016351 is adopted T Enterobacter strain TCD1-1 T Ferrous iron oxidation and nitrate nitrogen reduction.
4. The method of claim 3, wherein the method comprises:
the preservation number is CCTCC AB2016351 T Enterobacter strain TCD1-1 T Inoculating the strain in a first culture medium for culturing and carrying out plate culture;
culturing the Enterobacter strain TCD1-1 T Preparing a cell suspension, and inoculating the cell suspension in a second culture medium for ferrous oxidation and nitrate nitrogen reduction.
5. The method according to claim 4, wherein the pH of the second medium is 6.8-7.2, and the second medium is prepared by adding vitamin solution, trace element solution, selenite-tungstate solution, triethylamine bicarbonate buffer solution, ferrous ions and nitrate into a basic medium; wherein, the concentration ratio of ferrous ions to nitrate ions is 1.
6. The method according to claim 5, wherein the concentration of the ferrous ions is 9.5 to 10.5mmol L -1 The concentration of the nitrate ion is 9.5-10.5 mmol L -1 。
7. The method of claim 5, wherein the first medium is a solid medium, has a pH of 6.8-7.2, and comprises agar and a second medium; the ratio of the mass of the agar to the volume of the second medium is 18-22%.
8. The method according to claim 7, wherein the temperature of the plate culture is room temperature, and the plate culture is carried out in the absence of anaerobic light for 72 hours.
9. The method according to claim 4, wherein the temperature for ferrous oxidation and nitrate nitrogen reduction is room temperature, anaerobic protection from light, 96-120h.
10. The method of claim 4, wherein the OD of the cell suspension is 600 The value was 1.22.
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