CN117126783A - Chemolithotrophic thiobacillus strain and application thereof - Google Patents
Chemolithotrophic thiobacillus strain and application thereof Download PDFInfo
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- CN117126783A CN117126783A CN202311154477.0A CN202311154477A CN117126783A CN 117126783 A CN117126783 A CN 117126783A CN 202311154477 A CN202311154477 A CN 202311154477A CN 117126783 A CN117126783 A CN 117126783A
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- 241000605118 Thiobacillus Species 0.000 title claims abstract description 44
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 claims abstract description 32
- 229910002651 NO3 Inorganic materials 0.000 claims abstract description 27
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims abstract description 26
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 20
- 230000003647 oxidation Effects 0.000 claims abstract description 15
- 230000009467 reduction Effects 0.000 claims abstract description 13
- 244000005700 microbiome Species 0.000 claims abstract description 5
- 239000002689 soil Substances 0.000 claims abstract description 5
- 230000002829 reductive effect Effects 0.000 claims description 18
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 14
- DHCDFWKWKRSZHF-UHFFFAOYSA-N sulfurothioic S-acid Chemical compound OS(O)(=O)=S DHCDFWKWKRSZHF-UHFFFAOYSA-N 0.000 claims description 14
- 230000012010 growth Effects 0.000 claims description 9
- 230000009604 anaerobic growth Effects 0.000 claims description 7
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 150000004763 sulfides Chemical class 0.000 claims description 5
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 claims description 4
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 4
- 230000009603 aerobic growth Effects 0.000 claims description 3
- 235000010333 potassium nitrate Nutrition 0.000 claims description 3
- 239000004323 potassium nitrate Substances 0.000 claims description 3
- 235000010344 sodium nitrate Nutrition 0.000 claims description 2
- 239000004317 sodium nitrate Substances 0.000 claims description 2
- 150000002823 nitrates Chemical class 0.000 claims 2
- 229910052717 sulfur Inorganic materials 0.000 abstract description 17
- 239000011593 sulfur Substances 0.000 abstract description 17
- 238000006722 reduction reaction Methods 0.000 abstract description 14
- 238000005691 oxidative coupling reaction Methods 0.000 abstract description 2
- 239000002351 wastewater Substances 0.000 abstract description 2
- 239000001963 growth medium Substances 0.000 description 14
- 238000012216 screening Methods 0.000 description 14
- 238000002474 experimental method Methods 0.000 description 12
- 241000894007 species Species 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 238000012163 sequencing technique Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 241000605268 Thiobacillus thioparus Species 0.000 description 4
- 230000001651 autotrophic effect Effects 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000002503 metabolic effect Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000001590 oxidative effect Effects 0.000 description 4
- 241001509286 Thiobacillus denitrificans Species 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 238000007900 DNA-DNA hybridization Methods 0.000 description 2
- 241000605909 Fusobacterium Species 0.000 description 2
- 238000007476 Maximum Likelihood Methods 0.000 description 2
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 2
- 235000019345 sodium thiosulphate Nutrition 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- -1 thiosulfate ions Chemical class 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000140526 Thiobacillus sajanensis Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229910052798 chalcogen Inorganic materials 0.000 description 1
- 150000001787 chalcogens Chemical class 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000003235 crystal violet staining Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000006477 desulfuration reaction Methods 0.000 description 1
- 230000023556 desulfurization Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 1
- 238000009854 hydrometallurgy Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000018343 nutrient deficiency Nutrition 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 238000011197 physicochemical method Methods 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 235000010378 sodium ascorbate Nutrition 0.000 description 1
- 229960005055 sodium ascorbate Drugs 0.000 description 1
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 description 1
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/103—Arsenic compounds
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/163—Nitrates
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Abstract
The invention discloses a chemolithotrophic thiobacillus strain and application thereof. The strain is named as deposit thiobacillus (Thiobacillus sedimentum) SCUT-2, and the deposit number is GDMCC No:63316, 3.29 days 2023, was deposited at Guangdong province microorganism strain collection center located in building 5, department of sciences of Guangdong province, no. 59, mitsui, guangdong, china. The strain can carry out the reduction reaction of the reduction sulfide and the oxidative coupling nitrate under the aerobic condition and the sulfur oxidation under the anaerobic condition, and can be used for treating the wastewater or soil containing the reduction sulfide or the reduction sulfide and the nitrate.
Description
Technical Field
The invention belongs to the technical field of microorganism and environmental protection, and particularly relates to a chemolithotrophic thiobacillus strain and application thereof.
Background
The amount of reduced sulfides emitted to the environment by human activity is about 2.8x10 per year 11 Kg, in which the amount produced by biological activity is estimated to be 7X 10 per year 10 Kg is extremely important and urgent for the management of the thus-large emissions of reduced sulfides. The biological desulfurization method has the most important advantages that the biological treatment process can be carried out at the ambient temperature and the atmospheric pressure, the operation is simple, the capital and operating cost are low, but the treatment efficiency is high, the method is cleaner than the physicochemical method in ecology, and the defects of high cost, secondary pollutant generation and the like of the chemical and physical methods are overcome.
The Thiobacillus strain in the colorless sulfur oxidizing bacteria has indispensable functions and wide application in the fields of wastewater treatment, agriculture, biological hydrometallurgy and the like. The chalcogen strains are usually in extreme niches in nature, especially in acidic environments, and attach to environments rich in reduced sulfides through heavy metal interactions, allowing them to survive under extreme conditions of nutrient deficiency. Hydrothermal jets, activated sludge treatment sites, sediment, or anaerobic soil releasing hydrogen sulfide, etc. may be potential living environments for the chalcobacteria strains. The thiobacillus genus comprises five species in taxonomy, namely T.Denitrificans, T.thioparus, T.baregenesis, T.sajanensis and T.thioparus, wherein the thiobacillus denitrificans (T.Denitrificans) and the thiobacillus thiofidobacterium (T.thioparus) can take nitrate as an electron acceptor under anaerobic conditions, and reduced sulfide as an electron donor, and inorganic carbon is utilized to supplement carbon sources required by growth and metabolism, so that the sulfur oxidation reaction is maintained, and the method has positive significance for treating wastewater containing the reduced sulfide in an anoxic environment.
Therefore, the screening and identification of the novel high-efficiency thiobacillus not only can expand the species of the thiobacillus bacteria and increase the application potential of the chemolithotrophic thiooxidizing bacteria, but also can provide microbial resource guarantee for environmental management, resource circulation and the like for the research of the bacteria.
Disclosure of Invention
The primary aim of the invention is to overcome the defects of the prior art and provide a chemolithotrophic thiobacillus strain.
It is another object of the present invention to provide the use of the above-described chemolithoautotrophic thiobacillus strain.
The aim of the invention is achieved by the following technical scheme: a strain of autotrophic thiobacillus is named deposit thiobacillus (Thiobacillus sedimentum) SCUT-2, and the deposit number is GDMCC No:63316, 3.29 days 2023, was deposited at Guangdong province microorganism strain collection center located in building 5, department of sciences of Guangdong province, no. 59, mitsui, guangdong, china.
The application of the chemolithotrophic thiobacillus strain in redox sulfides; preferably comprises the following steps: the chemolithotrophic thiobacillus strain is placed in an environment containing redox sulfide for growth.
The growth includes aerobic growth, anaerobic growth and facultative growth.
The redox sulfide is the oxidation of the reduced sulfide to sulfate.
The reducing sulfide is preferably at least one of elemental sulfur, thiosulfate and sulfite.
The environment is preferably soil and water.
The growth temperature is 20-35 ℃; more preferably 25 to 30 ℃.
The application of the chemolithoautotrophic thiobacillus strain in reduction of redox sulfide coupled nitrate under anaerobic conditions; preferably comprises the following steps: the chemolithoautotrophic thiobacillus strain is placed in an environment containing redox sulfide and nitrate for anaerobic growth.
The redox sulfide is the oxidation of the reduced sulfide to sulfate.
The reducing sulfide is preferably at least one of elemental sulfur, thiosulfate and sulfite.
The nitrate is preferably at least one of potassium nitrate and sodium nitrate.
The environment is preferably soil and water.
The growth temperature is 20-35 ℃; more preferably 25 to 30 ℃.
Compared with the prior art, the invention has the following advantages and effects:
the chemolithotrophic thiobacillus SCUT-2 provided by the invention is a new species, and expands the species of the thiobacillus strain, and can perform reductive sulfide oxidation under an aerobic condition and sulfur oxidation coupling nitrate reduction reaction under an anaerobic condition, namely the strain has an aerobic sulfur removal function and anaerobic sulfur and nitrate removal function.
Drawings
FIG. 1 is a colony and cell morphology diagram of a thiobacillus chemoautotrophicum SCUT-2; wherein A is a colony morphology photograph, B is a crystal violet staining photograph (100×), C is a gram staining photograph (100×), and D is a scanning electron microscope photograph (30 K×).
FIG. 2 is a phylogenetic tree diagram based on the strain SCUT-2 16S rDNA.
FIG. 3 is a phylogenetic tree diagram based on the whole genome of the strain SCUT-2.
FIG. 4 is a graph showing the results of measurement of the sulfur-oxidizing metabolic characteristics of the strain SCUT-2 under aerobic growth conditions.
FIG. 5 is a graph showing the results of detection of the metabolic characteristics of sulfur-oxidative coupling nitrate reduction of the strain SCUT-2 under anaerobic growth conditions.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
EXAMPLE 1 enrichment, screening, isolation and characterization of the novel potential autotrophic Thiobacillus SCUT-2
(1) Enrichment of sulfur oxidizing bacteria: a square 5-point sampling method is adopted to sample near-surface sediment (0-30 cm) at the Dan Matou site (S113 DEG 41', N23 DEG 05') of the downstream spike of the Guangzhou-section water area of Zhujiang. Repeatedly washing environmental sample with PBS buffer solution, inoculating into serum bottle filled with anaerobic enrichment screening culture medium, culturing at 30deg.C in 150rpm shaker for 120 hr, measuring nitrate and sulfate content, and repeatedly transferring under the conditionAnd 3-5 generations are connected to obtain stable sulfur oxidizing bacteria flora. Wherein, the enrichment and screening culture medium comprises the following components: KH (KH) 2 PO 4 0.4g/L、MgCl 2 0.12g/L、NaCl1g/L、CaCl 2 0.01g/L、NaHCO 3 0.2g/L、FeCl 2 0.005g/L、Na 2 S 2 O 3 ·5H 2 O 2.48g/L、KNO 3 1.011g/L, 1mL/L trace element solution (A5), pH6.5-7.0. The composition of the trace element solution (A5) was as follows: h 3 BO 3 2.86g/L、ZnSO 4 ·7H 2 O0.222g/L、Na 2 MoO 4 ·2H 2 O0.39g/L、MnCl 2 ·4H 2 O1.86g/L、CuSO 4 ·5H 2 O0.079g/L、CoCl 2 ·6H 2 O0.04g/L, and deionized water as solvent. Preparation of anaerobic enrichment screening culture medium: taking 50mL of enrichment screening culture, sealing by a butyl blue rubber plug and an aluminum cover in a 125mL serum bottle, removing oxygen in the overhead by a gas replacement mode of boiling, pumping and filling nitrogen, adding 2mmol/L sodium ascorbate to remove residual oxygen, taking 1mg/L resazurin as an anaerobic condition indicator, and proving that an anaerobic condition is achieved when resazurin is colorless.
(2) Isolation of single colonies: the obtained enrichment is diluted by sterile water in a multiple ratio and inoculated to an anaerobic enrichment screening solid culture medium (agar is added to 15-20 g/L on the basis of the enrichment screening culture medium, and the solid culture medium is placed in an anaerobic tank with an anaerobic anning bag), standing culture is carried out for 72-120 hours at 30 ℃, bacterial colony growth is observed, and bacterial colonies with different colors and appearance forms are respectively picked for further separation and purification. Screening is carried out through an aerobic sulfur oxidation experiment and an anaerobic nitrate reduction sulfur oxidation experiment, and the specific steps are as follows:
aerobic sulfur oxidation experiment: single colonies were inoculated into 100mL Erlenmeyer flasks containing 50mL of enrichment screening medium and cultured at 30℃in a 150rpm shaker, and samples were taken every 24 hours to determine thiosulfate content, sulfate content and cell number. The judgment basis of the aerobic sulfur oxidation experiment is that the content of thiosulfate is reduced, the content of sulfate is increased and the cell number is increased.
Anaerobic nitrate reduction sulfur oxidation experiment: single colonies were inoculated into an anaerobic enrichment screening medium, cultured at 30℃in a shaking table at 150rpm, and sampled every 72 hours to determine thiosulfate content, sulfate content, nitrate content and cell number. The reduction and oxidization experiment of the anaerobic nitrate is based on the judgment basis of the reduction and oxidization experiment of the anaerobic nitrate, wherein the reduction and oxidization experiment of the anaerobic nitrate is based on the reduction and the oxidization experiment of the anaerobic nitrate.
Screening to obtain a strain named SCUT-2 strain, wherein the thiosulfate content is continuously reduced and the sulfate content is continuously increased along with the extension of the culture time in an aerobic sulfur oxidation experiment, and meanwhile, cells are rapidly proliferated to reach 2 multiplied by 10 in 36h 7 CFU/mL; the SCUT-2 strain has continuously reduced thiosulfate content and nitrate content and continuously increased sulfate content along with the extension of culture time in an anaerobic nitrate reduction sulfur oxidation experiment, and simultaneously cells proliferate rapidly, and reach 2 multiplied by 10 in 18 days 7 CFU/mL the SCUT-2 strain has an appearance shown in FIG. 1, and specific morphology and physiological and biochemical characteristics are described in Table 1
TABLE 1
Remarks: "+": positive; "-": negative.
(3) Identification of 16S rRNA of the strain: inoculating the separated rod-shaped sulfur oxidizing bacteria into a seed culture medium, culturing, centrifuging to obtain thalli, extracting genome DNA of the thalli by using a TIANAm bacterium DNAKit genome DNA extraction kit, and performing PCR amplification by using the extracted DNA as a template and using 16SrDNA universal primers 27F and 1492R. The PCR reaction system is as follows: 1. Mu.L of genomic DNA, 1. Mu.L of upstream primer (10. Mu. Mol/L), 1. Mu.L of downstream primer (10. Mu. Mol/L), and DreamTaq Green PCR Master Mix (2X) 12.5. Mu.L, make up ddH 2 O to 25. Mu.L. The PCR reaction conditions were: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 54℃for 30s,Extending at 72 ℃ for 90s,35 cycles; extending at 72℃for 10min. The amplified PCR product was stored in a refrigerator at 4 ℃. The PCR product was sent to Guangzhou gold and other intelligent biotechnology Co.Ltd for 16S rDNA sequencing, and the results are shown below.
27F：5’-AGAGTTTGATCCTGGCTCAG-3’；
1492R：5’-GGTTACCTTGTTACGACTT-3’。
Sequencing results:
TGCAGTCGAACGGCAGCACGGGTGCTTGCACCTGGTGGCGAGTGGCGAACGGGTGAGTAATGCGTCGGAACGTACCGAGTAATGGGGGATAACGCAGCGAAAGTTGTGCTAATACCGCATACGCCCTGAGGGGGAAAGTGGGGGACCGCAAGGCCTCACGTTATTCGAGCGGCCGACGTCTGATTAGCTAGTTGGTGGGGTAAAGGCCCACCAAGGCGACGATCAGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGCAACCCTGATCCAGCCATTCCGCGTGAGTGAAGAAGGCCTTCGGGTTGTAAAGCTCTTTCAGCTGGAACGAAACGGTGCGCTCTAACATAGCGCGCTACTGACGGTACCAGCAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGATTGTTAAGCAAGACGTGAAATCCCCGGGCTTAACCTGGGAATGGCGTTTTGAACTGGCAGTCTAGAGTGCGTCAGAGGGGGGTGGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAACACCGATGGCGAAGGCAGCCCCCTGGGATGACACTGACGCTCATGTACGAAAGCGTGGGTAGCAAACAGGATTAGATACCTTGGTAGTCCACGCCCTAAACGATGTCAACTGGTTGTTGGGGGAGTGAAATCCCTTAGTAACGAAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGGCAGGAACTTTCCAGAGATGGATTGGTGCCCGAAAGGGAACCTGCACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCATTAGTTGCTACGCAAGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCCAATCCCAGAAAGCCGATCGTAGTCCGGATTGTTCTCTGCAACTCGAGAGCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGAATCTGCCAGAAGTAGGTAGCCTAACCGCAAGGGGGGCGCT。
(5) After sequencing results are compared on NCBI, blast comparison is carried out by using MEGA11.0.13 software, a phylogenetic tree is constructed by using a maximum likelihood method (MaximumLikeLihood), and the topology structure of the phylogenetic tree is subjected to 1000-time guided repeated sampling inspection, and the result is shown in figure 2. The results in FIG. 2 show that the isolated strain SCUT-2 belongs to the genus Thiobacillus, and has higher similarity with the T.thioparus THI 115 strain, and the similarity is 98.00%. As described in the reference (Kook JK, et al genome-Based Reclassification of Fusobacterium nucleatumSubspecies at the Species level. Curr Microbiol (2017) 74:1137-1147.), a 16S rDNA gene sequence similarity of 98.65% was used as a threshold for two differentiation, and thus, it was initially judged that the strain SCUT-2 was a new species of the genus Thiobacillus.
(6) Genome extraction and sequencing of strain SCUT-2: extracting genome DNA of the bacterial strain SCUT-2 by using a TIANamp Bacteria DNAKit bacterial genome DNA extraction kit, and sending the extracted genome DNA to Shanghai Meiji biological medicine science and technology Co-Ltd for full genome third generation single molecule real-time sequencing (PacBIO RS II sequencing platform) after quality inspection is qualified. The whole genome phylogenetic tree of strain SCUT-2 and its closely related species was constructed using an orthoven 3 on-line server (https:// orthoven 3. Bioifotolks. Net /), the results of which are shown in FIG. 3.
(7) The strain SCUT-2 was analyzed for 16SrDNA similarity, genomic GC content, ANI and dDDH with respect to the genus related strain, and the results are shown in FIG. 3 and Table 2.
TABLE 2
The GC content of the genome of the strain SCUT-2 is determined by calculating the whole genome sequence data; the average nucleotide similarity (Average nucleotide identity, ANI) and Digital DNA-DNA hybridization values (Digital DNA-DNA hybridization, dDDH) between strain SCUT-2 and the relevant strain were calculated using a JSPECISWS online server (http:// jspecies. Ribohost. Com /) and a GGDC web online server (http:// GGDC. Dsmz. De /) respectively. Based on genome level, a difference of more than 3% from the genome-wide GC content of the similar strain, an ANI value of less than 95% or a dDDH value of less than 70% can be used as a basis for determining a new species (see reference "Kook JK, et al genome-Based Reclassification of Fusobacterium nucleatumSubspecies at the Species level.CurrMicrobiol (2017) 74:1137-1147"). Therefore, based on the GC content, ANI and dDDH difference analysis of the similarity of 16SrRNA and genome level, it can be judged that the strain SCUT-2 is different from the existing species in the chalcobacteria genus in taxonomic, it can be judged as a new species in the chalcobacteria genus, and the obtained strain is named as deposit thiobacillus (Thiobacillus sedimentum) SCUT-2, with the deposit number of GDMCC No:63316, 3.29 days 2023, was deposited at Guangdong province microorganism strain collection center located in building 5, department of sciences of Guangdong province, no. 59, mitsui, guangdong, china.
Example 2
The detection process and detection result of the aerobic metabolism characteristic of the autotrophic thiobacillus SCUT-2 in the high-concentration reducing sulfide nitrate-containing culture solution are as follows:
the single colony of the separated and purified and identified chemolithotrophic thiobacillus (Thiobacillus sedimentum) SCUT-2 is selected and inoculated into an enrichment screening culture medium (the composition is shown in example 1, wherein the concentration of sodium thiosulfate is 1.580g/L (10 mmol/L)), the culture temperature is 30 ℃, the shaking culture (150 rpm) is carried out, the time phase measurement is carried out on the pH value, the cell number, the concentration of the thiosulfate and the concentration of the sulfate radical every 12 hours, and the total reaction duration is 72 hours.
Aerobic metabolism characteristics of the autotrophic thiobacillus SCUT-2 in the culture medium containing the reducing sulfide are shown in FIG. 4: as can be seen from FIG. 4A, the pH of the culture medium containing the high concentration of the reduced sulfide inoculated with SCUT-2 gradually decreases to about 3.7 with time, and the culture medium remains stable after 36 hours; as can be seen from FIG. 4B, the cell number of the SCUT-2 strain increased and decreased with time, and reached a maximum cell number of 2.79×10 at 36h 7 CFU/mL; as can be seen from C in FIG. 4, the concentration of thiosulfate ions in the culture medium containing high concentration of the inoculated strain SCUT-2 is rapidly reduced with time, and the thiosulfate is consumed at a rate of 0.28 mmol/L.h after 36 h; as can be seen from D in FIG. 4, in the culture medium containing a high concentration of reduced sulfide inoculated with SCUT-2, the concentration of sulfate ions gradually increased with time, and after 48 hours, the culture medium tended to be stable, the production rate of thiosulfate was 0.33 mmol/L.h,the sulfate conversion rate reaches 82.50%.
Example 3
The detection process and detection result of the anaerobic growth metabolic characteristics of the chemolithotrophic thiobacillus SCUT-2 in the high-concentration reducing sulfide nitrate-containing culture solution are as follows:
the isolated and purified single colony of Thiobacillus (Thiobacillus sedimentum) SCUT-2 was selected and inoculated into an anaerobic enrichment screening medium (composition shown in example 1, wherein sodium thiosulfate concentration is 1.580g/L (10 mmol/L), potassium nitrate concentration is 1.011g/L (10 mmol/L)), and the preparation process is the same as that of example 1, wherein the culture temperature is 30 ℃, shaking culture (150 rpm) is carried out, and the pH, cell number, thiosulfate concentration, sulfate concentration and nitrate concentration are measured every 3 days, and the total reaction period is 30 days.
The anaerobic growth metabolic characteristics of Thiobacillus SCUT-2 in the high concentration reducing sulfide nitrate-containing culture solution are shown in FIG. 5: as can be seen from FIG. 5A, in the high concentration reduced sulfide nitrate-containing culture solution inoculated with the strain SCUT-2, the pH gradually increased to about 7.0 over time; as can be seen from FIG. 5B, the strain SCUT-2 strain showed a tendency that the cell number increased first and then remained slowly decreasing with the prolonged culture time, and reached a maximum cell number of 2.12X10 at day 18 7 CFU/mL; as can be seen from C in FIG. 5, in the high concentration reduced sulfide nitrate-containing culture broth inoculated with the strain SCUT-2, the thiosulfate and nitrate ions concentrations were continuously decreased, the consumption rates were 0.24 mmol/L.d and 0.23 mmol/L.d, respectively, and the sulfate ions were continuously increased, and the production rate was 0.21 mmol/L.d. Based on the part of experiments, the strain SCUT-2 has the capability of anaerobic denitrification sulfur oxidation.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A chemolithotrophic thiobacillus strain, characterized in that: the chemolithotrophic thiobacillus strain is named as deposit thiobacillus (Thiobacillus sedimentum) SCUT-2, and the deposit number is GDMCCNo:63316, 3.29 days 2023, was deposited at Guangdong province microorganism strain collection center located in building 5, department of sciences of Guangdong province, no. 59, mitsui, guangdong, china.
2. Use of a chemolithotrophic thiobacillus strain of claim 1 for redox sulfide.
3. Use according to claim 1, characterized in that it comprises the following steps: the chemolithoautotrophic thiobacillus strain of claim 1 is grown in an environment containing a reducing sulfide.
4. A use according to claim 2 or 3, characterized in that:
the growth includes aerobic growth, anaerobic growth and facultative growth;
the redox sulfide is the oxidation of the reduced sulfide to sulfate.
5. A use according to claim 2 or 3, characterized in that: the reducing sulfide is at least one of elemental sulfur, thiosulfate and sulfite.
6. Use of a chemolithoautotrophic thiobacillus strain according to claim 1 for reduction of redox sulfide-coupled nitrates under anaerobic conditions.
7. The use according to claim 6, characterized by the steps of: the chemolithoautotrophic thiobacillus strain of claim 1 is subjected to anaerobic growth in an environment containing redox sulfides and nitrates.
8. Use according to claim 6 or 7, characterized in that: the redox sulfide is the oxidation of the reduced sulfide to sulfate.
9. Use according to claim 6 or 7, characterized in that:
the reducing sulfide is at least one of elemental sulfur, thiosulfate and sulfite;
the nitrate is at least one of potassium nitrate and sodium nitrate.
10. The use according to claim 3 or the use according to claim 6, characterized in that: the environment is soil or water.
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