Extremely acidophilic sulfur-oxidizing bacterium and application thereof

Abstract

The application provides an extreme acidophilic sulfur-oxidizing bacterium and application thereof. The extreme acidophilic bacterium has strong oxidation on elemental sulfur and tetrathionate ions, is a chemolithoautotrophic acidithiobacillus, and can be used for removing elemental sulfur and tetrathionate ions in the environment, and has wide application prospects. The acidophilic sulfur-oxidizing bacterium has a preservation number of CGMCC No. 14839. The optimal growth temperature of the biological leaching and desulfurization provided by the application is normal temperature, and the optimal growth pH range is wide, so that the strain of the application has practical application significance. In addition, due to the characteristics of chemolithoautotrophy, the strain can improve the climate environment while recovering the environment.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbial screening and application technology, specifically relating to a new species of extreme acidophilic sulfur oxidizing bacteria and its application. Background Technology

[0002] Sulfur is an element found in nature and is an important biological nutrient element in organisms, serving as a component of some essential amino acids, vitamins, and coenzymes. Sulfur exists widely in nature in various chemical forms, including elemental sulfur, reduced sulfides, sulfates, and sulfur-containing organic compounds. Common reduced sulfides include H₂S, sulfides, and thiosulfates (S₂O₃). 2- ) and sulfites (SO3) 2- (etc.) Under the catalysis of microorganisms, these different forms of sulfur can be interconverted, forming a complete biogeochemical cycle.

[0003] Sulfur-oxidizing microorganisms, as drivers of biological oxidation, play a crucial role in the sulfur oxidation process, primarily involving desulfurization, sulfidation, and desulfurization. They are characterized by low cost, minimal pollution, environmental friendliness, sustainability, and sustainability. They can oxidize elemental sulfur or various reduced-valence inorganic sulfur compounds (RISCs) into sulfuric acid or higher-valence sulfides, releasing energy and lowering the environmental pH in the process. Sulfur-oxidizing microorganisms not only play a vital role in the biogeochemical cycle of sulfur but are also widely used in the metallurgical industry, environmental engineering, and agriculture for pollutant treatment and sulfur resource recovery. Summary of the Invention

[0004] This invention provides a novel strain of extreme acidophilic sulfur oxidizing bacteria and its applications. The provided extreme acidophilic bacteria has a strong oxidizing effect on elemental sulfur and tetrathionium ions. It is a chemoautotrophic acidic sulfur bacillus and can be used to remove elemental sulfur and thiosulfate ions from the environment, with broad application prospects.

[0005] This invention provides a strain of Thiobacillus acidophilus. Acidithiobacillus Ameehan (AC) strain, with accession number CGMCC No. 14839, was deposited on October 27, 2017, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0006] The 16S gene sequence of the aforementioned Thiobacillus acidophilus is SEQ ID NO:1.

[0007] The pH range for the growth of Thiobacillus acidophilus provided by this invention is 1.0-8.0.

[0008] The growth temperature of the acidophilic thiobacillus provided by this invention is 15℃-45℃.

[0009] The acidophilic thiobacillus of the present invention can be used to treat sulfur-containing waste or inorganic sulfur-containing wastewater;

[0010] The acidophilic thiobacillus provided by this invention can also be used to treat elemental sulfur or tetrathionate in oxidizing environments;

[0011] The acidophilic thiobacillus provided by this invention can also be used in the fields of bioleaching or biodesulfurization.

[0012] The optimal growth temperature for bioleaching and desulfurization provided by this invention is at room temperature, and the optimal growth pH range is relatively wide. Therefore, the strain of this invention has practical application significance. Furthermore, due to its chemoautotrophic characteristics, it can improve the climate environment while simultaneously restoring the environment. Attached Figure Description

[0013] Figure 1 The screened acid thiobacilli and Acidithiobacillus Phylogenetic tree of 16S rRNA constructed based on maximum likelihood estimation for 16 type strains of the genus.

[0014] Figure 2 The screened acid thiobacilli and Acidithiobacillus The figure shows the results of genome-wide average nucleotide identity (ANI) similarity analysis of 16 type strains.

[0015] Figure 3 Morphology of *Thiobacillus acidophilus* on solid plates and scanning electron micrographs.

[0016] Figure 4 Figure showing the growth of Thiobacillus acidophilus under different temperatures and pH conditions.

[0017] Figure 5 The oxidation curves of elemental sulfur by *Thiobacillus acidophilus* at pH 2.0 and 37℃, and the pH changes during the oxidation process are shown in the graph. AM represents the experimental group, the inoculated strain. Acidithiobacillus Ameehan CK: Control group, uninoculated strain Acidithiobacillus Ameehan .

[0018] Figure 6 A graph showing the utilization of different carbon sources by the Biolog GEN III MicroPlate.

[0019] Figure 7 Growth curve of *Thiobacillus acidophilus* at pH 2.0 and 37°C using K₂O₆S₄ as a substrate. Detailed Implementation

[0020] This invention has screened and obtained a new strain of chemoautotrophic acid-rich thiobacillus, which has shown significant effects in treating wastewater containing large amounts of inorganic sulfur, as well as in bioleaching and desulfurization. Furthermore, as a sustainable development technology, this strain provides a guarantee of bacterial resources for future environmental remediation and resource recycling, and has excellent development prospects.

[0021] The present invention will be further illustrated below with specific embodiments to provide a better understanding of the invention, but these embodiments do not constitute a limitation thereof.

[0022] Example 1: Isolation and purification of Thiobacillus acidophilus

[0023] This embodiment uses solid plates to screen for highly efficient sulfur-oxidizing bacteria, tests the biochemical indicators of the screened acid thiobacilli, including the suitable growth temperature and pH tolerance range of the strains, tests the utilization of different substrates by the strains, and conducts a Biolog carbon source verification experiment on the strains.

[0024] The information on the culture medium and reagents used in this embodiment is as follows:

[0025] 1) 9K basic salt culture medium: (NH4)2SO4 (3 g / L), KCl (0.1 g / L), K2HPO4 (0.5 g / L), MgSO4·7H2O (0.5 g / L), Ca(NO3)2 (0.01 g / L), adjust to pH 2.0, sterilize at 121℃ for 20 min.

[0026] 2) 9K-S solid culture medium: Solution A: 250mL of 9K basic salt culture medium + 15.0 g of agar, adjust pH to 7.0; Solution B: 750mL of 9K basic salt culture medium, adjust pH to 2.0; Sterilize solutions A and B at 121℃ for 20 min; when solutions A and B have cooled to 80℃, mix them and add 10.0 g / L of sterile sulfur powder and mix well (sterilize at 105℃ for 24 hours), pour into petri dishes and cool.

[0027] 3) 9K-S liquid culture medium: 9K basic salt culture medium + sterile sulfur powder 10.0 g / L.

[0028] 4) 9K-SO liquid medium: 9K basic salt culture medium + K2O6S4 (1.5 g•L) -1 ).

[0029] 5) PBS solution: 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4, bring the volume to 1L, adjust the pH to 7.4, and sterilize for later use.

[0030] 1. Microbial community used for screening target strains

[0031] 5Biol microbiota: from the Applied Microbial Ecological Engineering Laboratory (AMEE) of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences.

[0032] 2. Isolation and purification of Thiobacillus acidophilus

[0033] The 5Biol bacterial colony was transferred at a ratio of 10% to a new 9K culture medium, with 2% pyrite added, and cultured at 30°C with shaking at 180 rpm. 200 μL of the culture was then evenly spread onto 9K-S solid medium and incubated at 30°C for approximately 20 days to screen for strains with sulfur-oxidizing capabilities. The culture results showed the growth of smooth, round, deep yellow single colonies.

[0034] A single colony was picked and placed in 5 mL of 9K-S liquid medium and cultured at 30°C with shaking at 180 r / min. The turbid bacterial culture was then cultured twice more to obtain a pure strain.

[0035] 3. Molecular identification of Thiobacillus acidophilus

[0036] Single colonies of the selected pure bacterial strains were collected, and genomic DNA was extracted from each colony using the MOBIO Soil Bacteria Genomic DNA Extraction Kit. The quality and integrity of the extracted DNA were analyzed using NanoDrop2000 and 1% (w / v) agarose gel electrophoresis. Whole genome sequencing was performed using the PacBio system (Wuhan Institute of Biotechnology) based on single-molecule real-time (SMRT) sequencing technology.

[0037] The results showed that the 16S rRNA sequence of this strain is as follows: SEQ ID NO:1:

[0038]

[0039] 16S rRNA gene identification results showed that this strain was related to... Acidithiobacillus The genus is the closest in genetic distance, but with Acidithiobacillus The highest similarity among the bacteria in the genus is no more than 95%. Figure 1 Furthermore, genome-wide average nucleotide identity (ANI) analysis showed that the strain was related to... Acidithiobacillus The strains within the genus have a maximum similarity of no more than 70%. Figure 2 Therefore, the acid thiobacillus isolated this time should be... Acidithiobacillus A new strain of the genus was named *Thiobacillus acidophilus*. Acidithiobacillus Ameehan The strain (AC) was deposited on October 27, 2017, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its accession number is CGMCC No. 14839.

[0040] Example 2: Physiological and Morphological Characteristics of Thiobacillus acidophilus

[0041] 1. Morphological characteristics of the strain

[0042] The selected strains showed small, round, yellow, dome-shaped colonies with smooth surfaces and distinct edges on 9K-S solid plates. Figure 3 A).

[0043] Morphological observation of the screened *Thiobacillus acidophilus* was performed using a scanning electron microscope (Hitachi SU8010, Japan). The strain was aerobically grown in 9K-S liquid medium with an initial pH of 2.0 at 37°C until it reached mid-exponential growth. A certain amount of the culture medium in the stationary phase was collected by centrifugation at 10,000 rpm for 15 min. The cells were first washed twice with 0.25N dilute sulfuric acid, and then washed at least three times with PBS buffer or physiological saline to remove impurities from the culture medium. Before observation, the samples underwent cell fixation (soaking in 2.5% glutaraldehyde and suspending the cells in the fixative, fixing overnight at 4°C. Washing three times with buffer, 10 min each time, to remove the influence of glutaraldehyde on subsequent steps. After centrifugation and enrichment, 1% osmium tetroxide was added to cover the cells, and the cells were suspended and fixed for 1 h. Then, washing three times with buffer, 10 min each time), gradient dehydration (gradually dehydrating with 30%, 50%, 75%, 95%, 100%, and 100% ethanol, 15 min each time), critical point drying (using a critical point dryer, Leica EM CPD030, Germany, to replace the ethanol in the cells with liquid CO2, followed by vaporization of the liquid CO2 to remove the CO2 gas, resulting in a dried sample), coating (using an ion sputtering system, Hitachi E-1045, Japan, to uniformly coat the sample surface with platinum (Pt) to increase the conductivity of the biological sample), and observation (scanning electron microscope, Hitachi). SU8010, Japan (Morphological observation of the screened *Thiobacillus acidophilus*). Scanning electron microscopy results showed that the strain was approximately 1 μm × 2 μm in size and appeared as a short rod. Figure 3 B).

[0044] 2. Determination of growth conditions for *Thiobacillus acidophilus*

[0045] The isolated strains were inoculated at a 10% centrifugation ratio and transferred to 9K-S liquid medium. They were then cultured at 15℃, 20℃, 25℃, 30℃, 37℃, 45℃, and 50℃, respectively, with an initial pH of 2.0 and shaking at 180 r / min. OD was measured every two days. 600 The value is used to assess cell growth and obtain the growth temperature range.

[0046] The strain was grown at a constant temperature of 37°C and a growth rate of 180 r / min in 9K-S liquid medium with different initial pH levels of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0. OD was measured every two days. 600 To assess cell growth and determine the optimal pH range for strain growth.

[0047] The experimental results showed that this strain could grow in a temperature range of 15℃-45℃, and the growth rate gradually increased as the temperature rose from 15℃ to 45℃. The optimal growth temperatures were found to be 37℃ and 45℃. Figure 4 A). Regarding the optimal growth pH, ​​this strain can grow in the pH range of 1.0–8.0. Similar growth was observed from pH 2.0 to 8.0, demonstrating its effectiveness across a wide pH range, but growth was inhibited at pH 1.0 and pH 9.0. Figure 4 B).

[0048] Example 3: Energy utilization of Acidithiobacillus

[0049] 1. Determination of the carbon source utilization capacity of strain Biolog

[0050] Because microorganisms metabolize differently to different carbon sources, the ability of this strain to grow on different carbon sources was tested using a Biolog GEN III MicroPlate (Table 1). Prior to the assay, different carbon sources or other chemicals were pre-filled into the GEN III MicroPlate, and pure cultures of the strain were cultured at 37°C and then suspended in IF-C inoculum at a predetermined cell density (90–98% transmittance). 100 μL of the cell suspension was then inoculated into each well of the GEN III MicroPlate. Microplates were incubated at 37°C for 240 hours. During this period, kinetic information was recorded and quantified using Biolog's GEN III OmniLog II combo plus kinetic software (Biolog, United States). This detected color changes (absorbance) caused by the reaction of redox substances with chromogenic substances during the respiratory metabolism of microbial cells using different carbon sources, as well as turbidity differences caused by the growth of the microorganisms themselves. Characteristic fingerprints were generated and compared with standard strain chromogenic databases. Photometric measurements of the color intensity produced by dye reduction under substrate utilization conditions were expressed in OmniLog units (OU).

[0051] The biolog GEN III Microplate carbon source assay showed that this strain was positive for 11 out of 72 carbon sources, namely D-fructose, D-galactose, D-fucose, L-fucose, L-rhamnose, D-fructose-6-phosphate, D-glucuronic acid, glucuronide, D-methyl lactate, α-keto-glutaric acid, and acetoacetic acid; and it showed a boundary value for 28 carbon sources (i.e., it may have the ability to grow using these substances), namely α-D-lactose, melibiose, and β-formyl-D-glucose. Glycosides, D-salicylic acid, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, α-D-glucose, D-mannose, 3-formylglucose, inosine, D-mannitol, D-arabinol, inositol, glycerol, D-glucose-6-phosphate, D-aspartic acid, e-aminoacetyl-L-proline, L-histamine, pectin, D-galacturonic acid, L-galacturonic acid lactone, Tween 40, γ-aminobutyric acid, β-hydroxy-D,L-butyric acid, α-keto-butyric acid, propionic acid, acetic acid, formic acid Figure 6 ).

[0052] Table 1: The names of carbon source materials and their corresponding Biolog values ​​for each well in the Biolog GEN III Microplate.

[0053] Chinese name English name Biolog value Classification A1 negative control Negative Control 26 - A2 dextrin Dextrin 59 Cannot be used A3 D-maltose D-Maltose 45 Cannot be used A4 D-trehalose D-Trehalose 43 Cannot be used A5 D-Cellobiose D-Cellobiose 50 Cannot be used A6 Gentian disaccharide Gentiobiose 62 Cannot be used A7 sucrose Sucrose 42 Cannot be used A8 D-Melalose D-Turanose 57 Cannot be used A9 Stachyose Stachyose 53 Cannot be used B1 Raffinose D-Raffinose 36 Cannot be used B2 α-D-lactose α-D-Lactose 100 Boundary values B3 Melibi D-Melibiose 117 Boundary values B4 β-formyl-D-glucoside β-Methyl-D-Glucoside 107 Boundary values B5 D-Salicin D-Salicin 96 Boundary values B6 N-acetyl-D-glucosamine N-Acetyl-D-Glucosamine 87 Boundary values B7 N-acetyl-β-D-mannosamine N-Acetyl-β-D-Mannosamine 74 Cannot be used B8 N-acetyl-D-galactosamine N-Acetyl-D-Galactosamine 108 Boundary values B9 N-acetylneuraminic acid N-Acetyl Neuraminic Acid 73 Cannot be used C1 α-D-glucose α-D-Glucose 114 Boundary values C2 D-mannose D-Mannose 118 Boundary values C3 D-fructose D-Fructose 151 Can utilize C4 D-galactose D-Galactose 130 Can utilize C5 3-Formylglucose 3-Methyl Glucose 121 Boundary values C6 D-fucose D-Fucose 144 Can utilize C7 L-fucose L-Fucose 153 Can utilize C8 L-Rhamnose L-Rhamnose 143 Can utilize C9 Inosine Inosine 88 Boundary values D1 D-sorbitol D-Sorbitol 51 Cannot be used D2 D-Mannitol D-Mannitol 95 Boundary values D3 D-Arabalone D-Arabitol 86 Boundary values D4 Inositol myo-Inositol 88 Boundary values D5 glycerin Glycerol 85 Boundary values D6 D-glucose-6-phosphate D-Glucose-6-PO4 100 Boundary values D7 D-fructose-6-phosphate D-Fructose-6-PO4 176 Can utilize D8 D-Aspartic acid D-Aspartic Acid 79 Boundary values D9 D-Serine D-Serine 71 Cannot be used E1 gelatin Gelatin 48 Cannot be used E2 e-aminoacetyl-L-proline Glycyl-L-Proline 79 Boundary values E3 L-alanine L-Alanine 75 Cannot be used E4 L-arginine L-Arginine 73 Cannot be used E5 L-Aspartic acid L-Aspartic Acid 77 Cannot be used E6 L-glutamic acid L-Glutamic Acid 76 Cannot be used E7 L-histamine L-Histidine 114 Boundary values E8 L-pyroglutamic acid L-Pyroglutamic Acid 76 Cannot be used E9 L-serine L-Serine 69 Cannot be used F1 pectin Pectin 88 Boundary values F2 D-galacturonic acid D-Galacturonic Acid 110 Boundary values F3 L-galacturonide L-Galactonic Acid Lactone 101 Boundary values F4 D-gluconic acid D-Gluconic Acid 63 Cannot be used F5 D-glucuronic acid D-Glucuronic Acid 137 Can utilize F6 glucuronid Glucuronamide 186 Can utilize F7 mucic acid; mucous acid Mucic Acid 59 Cannot be used F8 Quinic acid Quinic Acid 56 Cannot be used F9 Glycolic Acid D-Saccharic Acid 73 Cannot be used G1 p-Hydroxyphenylacetic acid p-Hydroxy Phenylacetic Acid 46 Cannot be used G2 Methyl pyruvate Methyl Pyruvate 61 Cannot be used G3 D-methyl lactate D-Lactic Acid Methyl Ester 247 Can utilize G4 L-lactic acid L-Lactic Acid 58 Cannot be used G5 Citric acid Citric Acid 75 Cannot be used G6 α-Ketoglutaric acid α-Keto-Glutaric Acid 145 Can utilize G7 D-malic acid D-Malic Acid 68 Cannot be used G8 L-malic acid L-Malic Acid 66 Cannot be used G9 bromo-succinic acid Bromo-Succinic Acid 55 Cannot be used H1 Twain 40 Tween 40 93 Boundary values H2 γ-aminobutyric acid γ-Amino-Butryric Acid 81 Boundary values H3 α-Hydroxybutyric acid α-Hydroxy_x0002_Butyric Acid 69 Cannot be used H4 β-hydroxy-D,L-butyric acid β-Hydroxy-D,L Butyric Acid 83 Boundary values H5 α-Keto-butyric acid α-Keto-Butyric Acid 117 Boundary values H6 Acetoacetic acid Acetoacetic Acid 168 Can utilize H7 propionic acid Propionic Acid 99 Boundary values H8 Acetic acid Acetic Acid 122 Boundary values H9 Formic acid Formic Acid 86 Boundary values

[0054] 2. Determination of other energy utilization by the strain

[0055] The following energy substances were added to a 9K basic salt culture medium: S (5.0 g / L), Na₂S₂O₃ (10.0 g / L), K₂S₄O₆ (1.5 g / L), FeS₂ (10.0 g / L), FeSO₄·7H₂O (5.0 g / L), glucose (1.0 g / L), and yeast extract (0.2 g / L). Acidithiobacillus was centrifuged and inoculated at a 10% ratio. Samples were taken every 3 days to analyze the OD₂O₃ concentration. 600 The absorbance values ​​were measured at specific locations, and the strain's utilization of different energy sources was assessed by monitoring its growth (Table 2).

[0056] Table 2: Growth of *Thiobacillus acidophilus* using Sulfur, Na2S2O3, K2S4O6, FeS2, FeSO4·7H2O, glucose, and yeast extract as substrates.

[0057]

[0058] The results showed that the strains screened in this invention could utilize S 0K2O6S4 can undergo chemoautotrophic growth, but it cannot utilize Na2S2O3, FeS2, FeSO4•7H2O, glucose, or yeast as energy sources for growth.

[0059] 3. Determination of sulfur oxidation capacity

[0060] To determine the sulfur-oxidizing capacity of *Thiobacillus acidophilus*, the following experiment was conducted.

[0061] 1) The screened strains were inoculated into 9K-S liquid medium and cultured for 16 days at optimal pH and temperature. Samples were collected every four days, and SO42- was added. 2- pH was determined using a sulfate reagent powder (HACH 2106769) and measured using a pH meter (pH meter FE20, China).

[0062] 2) The strain was inoculated into 9K-SO liquid medium and cultured at the optimal pH and temperature for 6 days. Samples were collected every three days, and the OD values ​​of the samples were analyzed using a spectrophotometer. 600 The growth of the strain was observed by measuring the absorbance at a certain point.

[0063] Detection of SO4 in culture medium 2- The strain's ability to oxidize elemental sulfur was tested, and the results showed that after 16 days of cultivation, the SO4 content in the culture medium decreased. 2- Increasing the concentration from 17.0 g / L to 30.0 g / L resulted in an oxidation efficiency of approximately 50% for sulfur powder, while decreasing the pH from 2.0 to 0.9 maintained acidic conditions in the leaching environment. Figure 5 A and Figure 5 B). The strain's biomass tripled within three days due to the oxidation of K₂O₆S₄. Figure 7 ).

[0064] The above results indicate that the strains screened in this invention can be applied to the treatment of large quantities of waste inorganic sulfur-containing wastewater, as well as to bioleaching and desulfurization.

Claims

1. A plant Acidithiobacillus Ameehan The strain is characterized by, The strain has the accession number CGMCC No. 14839.

2. The claim 1 Acidithiobacillus Ameehan Application of strains in treating elemental sulfur or tetrathionate in oxidizing environments.

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