Thiobacillus strain and application thereof

Abstract

The application discloses a chemoautotrophic Thiobacillus strain and application thereof. The strain is named Thiobacillus sedimentum SCUT-2, has a preservation number of GDMCC No: 63316, and is preserved in the Guangdong Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, located at No. 100, Martyrs' Road, Guangzhou, China, on the 5th floor of No. 59 Building, on March 29, 2023. The strain can oxidize reductive sulfide under aerobic conditions and can perform a sulfur oxidation coupled nitrate reduction reaction under anaerobic conditions, and can be used for treating wastewater or soil containing reductive sulfide or containing reductive sulfide and nitrate.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and environmental protection technology, and specifically relates to a chemoautotrophic thiobacillus strain and its application. Background Technology

[0002] The most important advantages of biological desulfurization are that the biological treatment process can be carried out under ambient temperature and atmospheric pressure, it is simple to operate, has low capital and operating costs but high treatment efficiency, and is cleaner than physical and chemical methods in terms of ecology, making up for the disadvantages of chemical and physical methods such as high cost and generation of secondary pollutants.

[0003] Thiobacillus strains, among colorless sulfur-oxidizing bacteria, play an indispensable role and have wide applications in wastewater treatment, agriculture, and biohydrometallurgy. In nature, Thiobacillus strains typically occupy extreme ecological niches, particularly acidic environments, and attach to environments rich in reducing sulfides through heavy metal interactions, enabling them to survive under nutrient-deficient extreme conditions. Hydrothermal vents, activated sludge treatment plants, sediments, or anaerobic soils releasing hydrogen sulfide can all potentially serve as habitats for Thiobacillus strains. Taxonomically, the genus *Thiobacillus* comprises five species: *T. denitrificans*, *T. thioparus*, *T. baregensis*, *T. sajanensis*, and *T. thiophilus*. Among them, *T. denitrificans* and *T. thioparus* can utilize nitrate as an electron acceptor and reduced sulfides as electron donors under anaerobic conditions, supplementing their growth and metabolism with inorganic carbon to maintain the sulfur oxidation reaction. This has positive significance for the treatment of wastewater containing reduced sulfides in anoxic environments.

[0004] Therefore, screening and identifying novel, highly efficient Thiobacillus species can not only expand the variety of Thiobacillus species and increase the application potential of chemoautotrophic sulfur-oxidizing bacteria, but also provide microbial resource guarantees for environmental governance and resource recycling. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings of the prior art and provide a chemoautotrophic thiobacillus strain.

[0006] Another object of the present invention is to provide the application of the above-mentioned chemoautotrophic thiobacillus strain.

[0007] The objective of this invention is achieved through the following technical solution: a chemoautotrophic thiobacillus strain, named Thiobacillus sedimentum SCUT-2, with accession number GDMCC No: 63316, was deposited on March 29, 2023, at the Guangdong Provincial Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, China.

[0008] The application of the above-mentioned chemoautotrophic thiobacillus strain in redox sulfides preferably includes the following steps: growing the above-mentioned chemoautotrophic thiobacillus strain in an environment containing redox sulfides.

[0009] The growth mentioned includes aerobic growth, anaerobic growth, and facultative growth.

[0010] The redox sulfides mentioned above are those that oxidize reducing sulfides to sulfates.

[0011] The reducing sulfide is preferably at least one of elemental sulfur, thiosulfate, and sulfite.

[0012] The preferred environment is soil and water.

[0013] The growth temperature is 20–35°C; more preferably 25–30°C.

[0014] The application of the above-mentioned chemoautotrophic thiobacillus strain in the anaerobic redox sulfide coupling nitrate reduction; preferably includes the following steps: placing the above-mentioned chemoautotrophic thiobacillus strain in an environment containing redox sulfides and nitrates for anaerobic growth.

[0015] The redox sulfides mentioned above are those that oxidize reducing sulfides to sulfates.

[0016] The reducing sulfide is preferably at least one of elemental sulfur, thiosulfate, and sulfite.

[0017] The nitrate is preferably at least one of potassium nitrate and sodium nitrate.

[0018] The preferred environment is soil and water.

[0019] The growth temperature is 20–35°C; more preferably 25–30°C.

[0020] The present invention has the following advantages and effects compared with the prior art:

[0021] The chemoautotrophic thiobacillus SCUT-2 provided by this invention is a new species, which expands the species of thiobacillus strains. It can carry out reducing sulfide oxidation under aerobic conditions and nitrate reduction reaction coupled with sulfur oxidation under anaerobic conditions. That is, this strain has aerobic desulfurization function as well as anaerobic desulfurization and nitrate nitrogen removal function. Attached Figure Description

[0022] Figure 1 The images show the colony and cell morphology of chemoautotrophic thiobacillus SCUT-2; where A is a colony morphology photograph, B is a crystal violet stained photograph (100×), C is a Gram stained photograph (100×), and D is a scanning electron microscope photograph (30K×).

[0023] Figure 2 Phylogenetic tree diagram based on strain SCUT-2 16S rDNA.

[0024] Figure 3 This is a phylogenetic tree diagram based on the whole genome of strain SCUT-2.

[0025] Figure 4 The figure shows the results of detecting the sulfur oxidation metabolism characteristics of strain SCUT-2 under aerobic growth conditions.

[0026] Figure 5 The figure shows the detection results of sulfur oxidation-coupled nitrate reduction metabolism characteristics of strain SCUT-2 under anaerobic growth conditions. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0028] Example 1: Enrichment, screening, isolation and identification of a novel chemoautotrophic thiobacillus SCUT-2

[0029] (1) Enrichment of sulfur-oxidizing bacteria: Near-surface sediment samples (0-30 cm) were collected from the Suishi Wharf site (S113°41′, N23°05′) in the downstream section of the Pearl River in Guangzhou using a square 5-point sampling method. Environmental samples were repeatedly washed with PBS buffer solution and then inoculated into serum bottles containing anaerobic enrichment and selection medium. After incubation at 30℃ and 150 rpm for 120 h, the nitrate and sulfate contents were measured. Under these conditions, the inoculation was repeated for 3-5 generations to obtain a stable sulfur-oxidizing bacterial community. The enrichment and screening medium consisted of: KH₂PO₄ 0.4 g / L, MgCl₂ 0.12 g / L, NaCl 1 g / L, CaCl₂ 0.01 g / L, NaHCO₃ 0.2 g / L, FeCl₂ 0.005 g / L, Na₂S₂O₃·5H₂O 2.48 g / L, KNO₃ 1.011 g / L, and a trace element solution (A5) of 1 mL / L, with a pH of 6.5-7.0. The trace element solution (A5) consisted of: H₃BO₃ 2.86 g / L, ZnSO₄·7H₂O 0.222 g / L, Na₂MoO₄·2H₂O 0.39 g / L, MnCl₂·4H₂O 1.86 g / L, CuSO₄·5H₂O 0.079 g / L, and CoCl₂·6H₂O 0.04 g / L, with deionized water as the solvent. Preparation of anaerobic enrichment and screening medium: Take 50 mL of enrichment and screening medium into a 125 mL serum bottle, seal it with a butyl blue rubber stopper and an aluminum cap, remove oxygen from the headspace by boiling, evacuation and nitrogen purging, and then add 2 mmol / L sodium ascorbate to remove residual oxygen. Use 1 mg / L resazurin as an indicator of anaerobic conditions. When resazurin turns colorless, it proves that anaerobic conditions have been met.

[0030] (2) Isolation of single colonies: The obtained enriched material was serially diluted with sterile water and inoculated onto an anaerobic enrichment and screening solid medium (15-20 g / L of agar was added to the enrichment and screening medium, and this solid medium was placed in an anaerobic jar containing an anaerobic anesthetic pack). After static incubation at 30°C for 72-120 h, colony growth was observed. Colonies with different colors and morphologies were picked for further isolation and purification. Screening was carried out through aerobic sulfur oxidation experiments and anaerobic nitrate reduction sulfur oxidation experiments. The specific steps are as follows:

[0031] Aerobic sulfur oxidation experiment: A single colony was inoculated into a 100 mL Erlenmeyer flask containing 50 mL of enrichment and selection medium and cultured in a shaker at 30 °C and 150 rpm. Samples were taken every 24 h to determine the thiosulfate content, sulfate content, and cell number. The decrease in thiosulfate content, the increase in sulfate content, and the increase in cell number were used as the criteria for judging the aerobic sulfur oxidation experiment.

[0032] Anaerobic nitrate reduction and sulfur oxidation experiment: Single colonies were inoculated into anaerobic enrichment and selection medium and cultured in a shaker at 30℃ and 150 rpm. Samples were taken every 72 hours to determine the thiosulfate, sulfate, nitrate content, and cell number. Decreased thiosulfate, increased sulfate, decreased nitrate, and increased cell number were used as the criteria for judging the anaerobic nitrate reduction and sulfur oxidation experiment.

[0033] A strain was obtained through screening and named SCUT-2. In the aerobic sulfur oxidation experiment, the thiosulfate content continuously decreased and the sulfate content continuously increased with the extension of culture time. At the same time, the cells also proliferated rapidly, reaching 2 × 10⁻⁶ cells / day at 36 hours. 7 In the anaerobic nitrate reduction and sulfur oxidation experiment, the SCUT-2 strain showed a continuous decrease in thiosulfate and nitrate content and a continuous increase in sulfate content with prolonged culture time. Simultaneously, the cells rapidly proliferated, reaching 2 × 10⁻⁶ cells / mL at 18 days. 7 The appearance of the SCUT-2 strain at CFU / mL is as follows: Figure 1 As shown, the specific morphological and physiological and biochemical characteristics are described in Table 1.

[0034] Table 1

[0035]

[0036]

[0037] Note: "+" indicates a positive result; "-" indicates a negative result.

[0038] (3) Identification of 16S rRNA in the strain: The isolated rod-shaped sulfur-oxidizing bacteria were inoculated into seed culture medium and centrifuged to obtain bacterial cells. Genomic DNA was extracted from the bacterial cells using the TIANampBacteriaDNAKit bacterial genomic DNA extraction kit. Then, using the extracted DNA as a template, PCR amplification was performed using universal 16S rDNA primers 27F and 1492R. The PCR reaction system was as follows: 1 μL genomic DNA, 1 μL upstream primer (10 μmol / L), 1 μL downstream primer (10 μmol / L), 12.5 μL DreamTaqGreen PCR Master Mix (2×), and ddH2O was added to a final volume of 25 μL. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 90 s, 35 cycles; 72℃ extension for 10 min. The amplified PCR products were stored at 4℃. The PCR products were sent to Guangzhou Genewiz Biotechnology Co., Ltd. for 16S rDNA sequencing, and the results are shown below.

[0039] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0040] 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0041] Sequencing results:

[0042]

[0043] (5) After comparing the sequencing results on NCBI, BLAST alignment was performed using MEGA11.0.13 software. A phylogenetic tree was constructed using the maximum likelihood method. The phylogenetic tree topology was tested with 1000 guided replicates. The results are as follows: Figure 2 As shown. Figure 2 The results showed that the isolated strain SCUT-2 belonged to the genus *Thioparus* and had a high similarity (98.00%) to *T. thioparus THI 115*. According to the reference (Kook JK, et al. Genome-Based Reclassification of *Fusobacterium nucleatum* Subspecies at the Species Level. Curr Microbiol (2017) 74: 1137-1147.), a 16S rDNA gene sequence similarity of 98.65% can be used as a threshold for distinguishing between the two species. Therefore, strain SCUT-2 was preliminarily identified as a new species within the genus *Thioparus*.

[0044] (6) Genomic extraction and sequencing of strain SCUT-2: Genomic DNA was extracted from strain SCUT-2 using the TIANamp Bacteria DNA Kit. After quality control, the extracted genomic DNA was sent to Shanghai Meiji Biotechnology Co., Ltd. for whole-genome third-generation single-molecule real-time sequencing (PacBio RS II sequencing platform). A phylogenetic tree of the whole genome of strain SCUT-2 and its closely related species was constructed using the Orthovenn3 online server (https: / / orthovenn3.bioinfotoolkits.net / ). The results are as follows: Figure 3 As shown.

[0045] (7) The 16S rDNA similarity, genomic GC content, ANI, and dDDH of strain SCUT-2 were analyzed with those of related strains of the same genus. The results are as follows: Figure 3 As shown in Table 2.

[0046] Table 2

[0047]

[0048] The GC content of strain SCUT-2 was determined by calculating whole-genome sequence data. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between strain SCUT-2 and related strains were calculated using the JSpeciesWS online server (http: / / jspecies.ribohost.com / ) and the GGDC web online server (http: / / GGDC.dsmz.de / ), respectively. At the genomic level, a difference of more than 3% in whole-genome GC content, an ANI value of less than 95%, or a dDDH value of less than 70% with similar strains can be used as criteria for identifying new species (see reference "Kook JK, et al. Genome-Based Reclassification of Fusobacterium nucleatum Subspecies at the SpeciesLevel. Curr Microbiol (2017) 74:1137-1147."). Therefore, based on the above-mentioned 16S rRNA similarity and genomic-level GC content, ANI and dDDH difference analysis, it can be determined that strain SCUT-2 is taxonomically different from existing species in the genus Thiobacillus, and can be identified as a new species in the genus Thiobacillus. The obtained strain is named Thiobacillus sedimentum SCUT-2, with accession number GDMCC No: 63316, and was deposited on March 29, 2023 at the Guangdong Provincial Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, China.

[0049] Example 2

[0050] The detection process and results of the aerobic metabolic characteristics of chemoautotrophic thiobacillus SCUT-2 in a high-concentration reducing sulfide-containing nitrate culture medium are as follows:

[0051] Single colonies of the isolated, purified, and identified chemoautotrophic thiobacillus sedimentum (SCUT-2) were picked and inoculated into enrichment and screening medium (composition as described in Example 1, wherein the sodium thiosulfate concentration was 1.580 g / L (10 mmol / L)). The culture temperature was 30 °C, and the culture was shaken (150 rpm). The pH, cell number, thiosulfate concentration, and sulfate concentration were measured every 12 h. The total reaction time was 72 h.

[0052] Aerobic metabolic characteristics of chemoautotrophic thiobacillus SCUT-2 in medium containing reducing sulfides, such as Figure 4 As shown: From Figure 4 As can be seen from A, in the culture medium containing high concentrations of reducing sulfides inoculated with SCUT-2, the pH gradually decreased to around 3.7 over time, and remained stable after 36 hours; from Figure 4 As shown in B, the cell number of SCUT-2 strain first increased and then decreased over time, reaching a maximum of 2.79 × 10⁻⁶ cells at 36 h. 7 CFU / mL; from Figure 4 As shown in C, in the medium containing high concentrations of reducing sulfides inoculated with strain SCUT-2, the concentration of thiosulfate ions decreased rapidly over time, and was completely consumed after 36 hours, with a consumption rate of 0.28 mmol / L·h; Figure 4 As can be seen from D, in the medium containing high concentration of reducing sulfides inoculated with SCUT-2, the concentration of sulfate ions gradually increased over time and tended to stabilize after 48 h. The generation rate of thiosulfate was 0.33 mmol / L·h, and the sulfate conversion rate reached 82.50%.

[0053] Example 3

[0054] The detection process and results of the anaerobic growth and metabolic characteristics of chemoautotrophic thiobacillus SCUT-2 in a high-concentration reducing sulfide-containing nitrate culture medium are as follows:

[0055] Single colonies of Thiobacillus sedimentum (SCUT-2) isolated, purified and identified were inoculated into an anaerobic enrichment and selection medium (composition as described in Example 1, wherein the concentration of sodium thiosulfate was 1.580 g / L (10 mmol / L) and the concentration of potassium nitrate was 1.011 g / L (10 mmol / L)). The preparation process was the same as in Example 1. The culture temperature was 30°C, and the culture was shaken (150 rpm). The pH, cell number, thiosulfate concentration, sulfate concentration and nitrate concentration were measured every 3 days. The total reaction period was 30 days.

[0056] The anaerobic growth and metabolic characteristics of Thiobacillus SCUT-2 in a high-concentration reducing sulfide-containing nitrate culture medium are as follows: Figure 5 As shown: From Figure 5 As can be seen from A, in the high-concentration reducing sulfide-containing nitrate culture medium inoculated with strain SCUT-2, the pH gradually increased to around 7.0 over time; from Figure 5 As shown in Figure B, the cell number of strain SCUT-2 initially increased and then slowly decreased with prolonged culture time, reaching a maximum cell number of 2.12 × 10⁻⁶ on day 18. 7 CFU / mL; from Figure 5 As shown in Figure C, in the high-concentration reducing sulfide-containing nitrate culture medium inoculated with strain SCUT-2, the concentrations of thiosulfate and nitrate ions continuously decreased, with consumption rates of 0.24 mmol / L·d and 0.23 mmol / L·d, respectively, while the sulfate ion concentration continuously increased, with a production rate of 0.21 mmol / L·d. Based on this part of the experiment, it is verified that strain SCUT-2 has the ability of anaerobic denitrification and sulfur oxidation.

[0057] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A chemoautotrophic thiobacillus strain, characterized in that: The chemoautotrophic thiobacillus strain mentioned is named *Thiobacillus sedimentaria* (Sediment Thiobacillus). Thiobacillus sedimentum SCUT-2, with accession number GDMCC No: 63316, was deposited on March 29, 2023, at the Guangdong Provincial Microbial Culture Collection Center, Institute of Microbiology, Guangdong Academy of Sciences, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, China.

2. The application of the chemoautotrophic thiobacillus strain according to claim 1 in redox sulfides, characterized in that: The reducing sulfide is at least one of elemental sulfur, thiosulfate, and sulfite.

3. The application according to claim 2, characterized in that... The method includes the following steps: growing the chemoautotrophic thiobacillus strain described in claim 1 in an environment containing reducing sulfides.

4. The application according to claim 3, characterized in that: The growth mentioned includes aerobic growth, anaerobic growth, and facultative growth; The redox sulfides mentioned above are those that oxidize reducing sulfides to sulfates.

5. The application of the chemoautotrophic thiobacillus strain according to claim 1 in the anaerobic redox coupling nitrate reduction, characterized in that: The reducing sulfide is at least one of elemental sulfur, thiosulfate, and sulfite.

6. The application according to claim 5, characterized in that... The method includes the following steps: placing the chemoautotrophic thiobacillus strain described in claim 1 in an environment containing reducing sulfides and nitrates for anaerobic growth.

7. The application according to claim 5 or 6, characterized in that: The redox sulfides mentioned above are those that oxidize reducing sulfides to sulfates.

8. The application according to claim 5 or 6, characterized in that: The nitrate is at least one of potassium nitrate and sodium nitrate.

9. The application according to claim 3 or the application according to claim 6, characterized in that: The environment described is soil or water.

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