Isolated spartina pectinilytica and its application in n-hexanal degradation

Abstract

The application belongs to the technical field of biology, discloses an isolated Saccharomyces spartinae and application thereof in n-hexanal degradation, the preservation number of the strain is CCTCC NO: M20241398, the degradation rate of the n-hexanal degradation strain in VOCs sampling bag to 30ul n-hexanal reaches 90.1% in 24h, and the degradation rate to 30ul n-hexanal can reach 99.2% in 48h. The strain has good extreme environment tolerance and can grow in initial pH2.0 and pH12.0, temperature 40 DEG C and high salt 12.0% NaCl.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically an isolated strain of Spartania spartinae L51 and its application in the degradation of n-hexanol. Background Technology

[0002] Hexanol is a low-toxicity organic compound, a colorless and transparent liquid, insoluble in water but soluble in organic solvents such as benzene, acetone, ethanol, and ether. It can be used to make fragrances and prepare hexanoic acid, and can also be used as an analytical reagent in gas chromatography. It is irritating to the eyes, mucous membranes, and upper respiratory tract, causing coughing, tearing, and salivation. In some individuals, it may cause nausea, headache, retrosternal pain, and difficulty breathing, seriously endangering human and environmental health.

[0003] Biological degradation utilizes microorganisms to degrade toxic and harmful organic matter into non-toxic and harmless small molecules. It has low construction and operation costs, is easy to operate and maintain; it has high degradation efficiency and can screen out specific bacteria that degrade hexanol; it is a closed-loop process that does not cause secondary pollution to the environment.

[0004] There is very little research on *S. spartinae*, both domestically and internationally. Previously, based on phenotypic characteristics such as the formation of ascosacs, *S. spartinae* was classified under the genus *Pichia pastoris*. However, based on molecular biological evidence, particularly after sequencing analysis of its ribosomal RNA (rRNA) gene in 2010, it was reclassified under the genus *Scheffersomyces*. The classification of *S. spartinae* into its current genus remains controversial due to low support and its inability, unlike other members of the genus, to efficiently ferment d-xylose into ethanol. Nevertheless, *S. spartinae* is known to produce coenzyme Q9, which has drawn attention to the species' potential biotechnological applications. Currently, there are no publicly available reports on the use of *S. spartinae* for the removal of n-hexanal. Summary of the Invention

[0005] The purpose of this invention is to provide an isolated strain of Scheffersomyces spartinae L51, with the accession number [not specified].

[0006] Another object of the present invention is to provide the application of Spartania scherm (Scheffersomycesspartinae L51) in the removal of n-hexanal.

[0007] To achieve the above objectives, the present invention adopts the following technical measures:

[0008] The applicant isolated a fungus from a biological deodorization system of a company in Hangzhou. This fungus exhibits excellent ability to remove hexanoal and has been identified as *Scheffersomyces spartinae*. This fungus was deposited on June 27, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with the classification name *Scheffersomyces spartinae* L51 and accession number CCTCC NO: M20241398.

[0009] The *Spartania spartinae* L51 strain of this invention was cultured on PDA agar plates for 1 day. The colonies were round, milky white, with a smooth surface, clear edges, and were easily picked up; their microscopic morphology was round.

[0010] The scope of protection of this invention also includes:

[0011] Application of S. spartinae L51 in the degradation of n-hexanal

[0012] Application of S. spartinae L51 in the preparation of hexanol degrading agents.

[0013] The application of S. spartinae L51 as one of the active ingredients or the main active ingredient in the treatment of hexanal in wastewater.

[0014] The application of *S. spartinae* L51 as one of the active ingredients or the main active ingredient in the preparation of wastewater treatment biological agents. Compared with the prior art, the present invention has the following characteristics:

[0015] The S. spartinae L51 in this invention is the first to be reported to have the function of removing n-hexanealdehyde. It grows rapidly, has a high number of viable bacteria, and has a strong ability to remove n-hexanealdehyde; it can tolerate extreme environments.

[0016] The *S. spartinae* L51 strain of this invention exhibits rapid growth and high cell density at 30°C. This strain demonstrates a high hexanoal removal capacity, achieving a 90.1% removal rate for 30 μl of hexanoal after 24 h at 30°C and a 99.2% removal rate after 48 h. *S. spartinae* L51 exhibits excellent tolerance to extreme environments, growing in initial pH 2.0 and pH 12.0, at a high temperature of 45°C, and in high-salt environments (12.0% NaCl). Attached Figure Description

[0017] Figure 1 This is a colony diagram of S. spartinae L51.

[0018] Figure 2 The value represents the hexanol removal rate in the VOCs test of S. spartinae L51.

[0019] Figure 3 This is a graph showing the growth changes of S. spartinae L51 at different initial pH values.

[0020] a, b, c, d, e, f, g, h, i, j, k, l indicate significant differences in viable cell counts among strains L51 (p < 0.05); identical letters indicate no significant differences in viable cell counts among strains L51 (p > 0.05); letters only represent pH (OD). 600nm Comparison between (or viable counts).

[0021] Figure 4 The graph shows the growth changes of S. spartinae L51 at different temperatures;

[0022] a, b, c, d, e, f, g indicate significant differences in viable cell counts among strains L51 (p < 0.05); identical letters indicate no significant differences in viable cell counts among strains L51 (p > 0.05); the letters only represent pH (OD). 600nm Comparison between (or viable counts).

[0023] Figure 5 This is a graph showing the growth changes of S. spartinae L51 under different NaCl concentrations;

[0024] a, b, c, d, e, f, g, h, i, j, k indicate significant differences in viable cell counts among strains L51 (p<0.05); identical letters indicate no significant differences in viable cell counts among strains L51 (p>0.05); letters only represent comparisons between pH (OD600nm or viable cell count). Detailed Implementation

[0025] Unless otherwise specified, the technical solutions described in this invention are all conventional techniques; the reagents or materials described, unless otherwise specified, are all from commercial sources. Unless otherwise specified, each experimental group in this invention has three parallel experiments.

[0026] Example 1:

[0027] Isolation and Identification of S. spartinae L51

[0028] The applicant added 10.0g of packing material from a bio-trickling filter in a biological deodorization system of a company in Hangzhou to a conical flask containing 90ml of sterile physiological saline and glass beads. The flask was shaken at 30℃ and 200rpm for 1 hour until most of the deposits on the packing material detached and the liquid became turbid. 5mL of the supernatant was transferred from the conical flask to 50mL of inorganic salt selective medium with a hexanal concentration of 8.0mg / L. The flask was sealed with a silicone stopper, one layer of sealing film, and two layers of plastic film, and placed on a constant-temperature shaker at 30℃ and 200rpm for gradient acclimatization. During the acclimatization process, 1mL of the culture medium was transferred to 50mL of fresh medium every two days, while simultaneously increasing the hexanal concentration by 8.0mg / L each time, until reaching 24.0mg / L, at which point acclimatization was stopped. After gradient acclimatization, the final selective medium was diluted with sterile water and spread onto PDA solid medium. Single colonies were repeatedly streaked to isolate a single bacterial strain, named L51.

[0029] The liquid PDA medium consists of: 200.0g potato, 20.0g glucose, and distilled water to a final volume of 1000mL, pH at rest, sterilized at 115℃ for 30min; the solid PDA medium is the liquid PDA medium with 1.8% agar added; the inorganic salt selective medium consists of: 4.72g potassium nitrate, 0.7g potassium dihydrogen phosphate, 2.52g disodium hydrogen phosphate, 0.0244g magnesium sulfate heptahydrate, 0.015g calcium chloride, distilled water to a final volume of 1000mL, pH 7.0, sterilized at 121℃ for 30min.

[0030] Species identification of strain L51

[0031] (1) Morphological characteristics

[0032] Colony morphology and microscopic morphology of strain L51 on PDA solid medium, as follows: Figure 1 As shown, the colonies are round, milky white, smooth, with clear edges, easy to pick up, and have an elliptical microscopic morphology.

[0033] (2) Gene identification of strain L51

[0034] Genomic DNA was extracted from the test strain using a fungal DNA extraction kit, following the instructions in the manufacturer's manual. Using the extracted fungal genomic DNA as a template, strain L51 was amplified. The primers for amplifying ITS were used, and the amplified polynucleotide is shown in SEQ ID NO. 1.

[0035] ITS1:5′-TCCGTAGGTGAACCTGCGG-3′

[0036] ITS4:5′-TCCTCCGCTTATTGATATGC-3′;

[0037] The spliced ​​sequence was identified by Blast homology search as belonging to Scheffersomyces spartinae. This bacterium was deposited at the China Center for Type Culture Collection (CCTCC) on June 27, 2024, at Wuhan University, Wuhan, China. Its classification name is Scheffersomyces spartinae L51, and its accession number is CCTCC NO: M20241398.

[0038] In this embodiment of the invention, Scheffersomyces spartinae L51 is referred to simply as S. spartinae L51.

[0039] Example 2:

[0040] Growth performance and hexanal removal test of S. spartinae L51

[0041] S. spartinae L51 was cultured in liquid PDA seed culture medium at 30℃ and 200 rpm for 24 h, and the viable count was 8.46 × 10⁻⁶. 8 CFU / mL, take 2.5 mL of seed culture and centrifuge at 4000 rpm for 5 minutes, remove the supernatant, and resuspend in 2.5 mL of sterile water. After washing, centrifuge again, and repeat the resuspension and washing process twice. Then, inoculate into a 1 L VOCs sampling bag containing 50 mL of inorganic salt selective medium. The formula of the inorganic salt selective medium is: 4.72 g potassium nitrate, 0.7 g potassium dihydrogen phosphate, 2.52 g disodium hydrogen phosphate, 0.0244 g magnesium sulfate heptahydrate, 0.015 g calcium chloride, and distilled water to a final volume of 1000 mL. The pH is 7.0, and the medium is sterilized at 121 °C for 30 min.

[0042] Add 30 μl of hexanol to a 1 L VOCs sampling bag; simultaneously set up a blank control with no bacteria, adding only hexanol and inorganic salt culture medium. Place the 1 L VOCs sampling bag in a constant temperature shaker at 30℃ and 200 r / min for 12 h, 24 h, 36 h, and 48 h. Detect the hexanol content in both the control and experimental groups using a portable gas detector. After measuring the hexanol content using the above method, calculate the hexanol removal rate using the following formula.

[0043]

[0044] The results are as follows Figure 2As shown, this strain achieved a 14.1% removal rate of hexanealdehyde at 12 hours. As the strain adapted to hexanealdehyde and the concentration decreased, the degradation rate accelerated, reaching a 90.1% removal rate at 24 hours. With further time, the strain achieved a 97.4% removal rate at 36 hours, and almost completely degraded hexanealdehyde at 48 hours, reaching a 99.2% removal rate.

[0045] Example 3:

[0046] pH tolerance test of S. spartinae L51

[0047] Thirteen different initial pH gradients of PDA liquid culture media were prepared: pH = 1.0, pH = 2.0, pH = 3.0, pH = 4.0, pH = 5.0, pH = 6.0, pH = 7.0, pH = 8.0, pH = 9.0, pH = 10.0, pH = 11.0, pH = 12.0, and pH = 13.0. Seed culture of *S. spartinae* L51 was inoculated into the culture media at a rate of 1%, and cultured at 30°C and 200 rpm for 40 h. Samples were taken to measure the pH and OD of each culture medium. 600nm The effect of different initial pH values ​​on the growth of S. spartinae L51 was analyzed by measuring the viable cell count and the number of viable bacteria.

[0048] The growth changes of S. spartinae L51 under different initial pH values ​​are as follows: Figure 3 As shown. Figure 3 This indicates that S. spartinae L51 can grow well at pH 2.0–pH 12.0, and can maintain a biomass of 6.00 × 10⁻⁶ at pH 3.0–pH 11.0. 8 Above CFU / mL, the maximum viable count is 13.31 × 10⁻⁶ at pH 8.0. 8 CFU / mL; at pH 1.0 and below, yeast activity is greatly inhibited and cannot grow normally; at pH 13.0 and above, yeast activity is greatly inhibited and cannot grow normally.

[0049] Example 4:

[0050] Temperature tolerance test of S. spartinae L51

[0051] Eight different culture temperatures were set: 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, and 45℃. S. spartinae L51 seed culture was inoculated into the culture medium at a rate of 1%, and cultured at 30℃ and 200 rpm for 40 h. Samples were taken to measure the pH and OD of each culture medium. 600nmThe effects of different temperatures on the growth of S. spartinae L51 were analyzed by measuring the viable bacterial count.

[0052] The growth changes of S. spartinae L51 at different temperatures are as follows: Figure 4 As shown.

[0053] Figure 4 This indicates that S. spartinae L51 can grow at temperatures ranging from 15℃ to 40℃, with a maximum viable count of 9.75 × 10⁻⁶ cells at 30℃. 8 CFU / mL; at 15℃, yeast activity was greatly inhibited and normal growth was impossible, with a viable count of 5.2 × 10⁻⁶. 7 CFU / mL; at 45℃ and above, yeast activity is greatly inhibited, it cannot grow normally, and the cells die.

[0054] Example 5:

[0055] NaCl tolerance test of S. spartinae L51

[0056] Thirteen PDA liquid culture media containing different NaCl concentration gradients were prepared, with NaCl concentrations of 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, and 13.0%. Seed culture of *Scheffersomyces spartinae* L51 was inoculated into the culture media at a concentration of 1% and cultured at 30℃ and 200 rpm for 40 h. Samples were taken to measure the pH and OD of each culture medium. 600nm The effects of different NaCl concentrations on the growth of S. spartinae L51 were analyzed by measuring the viable cell count.

[0057] The growth changes of S. spartinae L51 under different NaCl concentrations are as follows: Figure 5 As shown.

[0058] Figure 5 The results indicate that *S. spartinae* L51 can grow in NaCl concentrations ranging from 1.0% to 12.0%, and the biomass can be maintained at 10.00 × 10⁻⁶ at NaCl concentrations ranging from 1.0% to 4.0%. 8 Biomass at concentrations above CFU / mL and NaCl concentrations of 1.0%-8.0% can be maintained at 6.0 × 10⁻⁶. 8 With CFU / mL or higher, the maximum viable count is 14.9 × 10⁻⁶ at a NaCl concentration of 1.0%. 8CFU / mL; yeast activity decreases as the NaCl concentration increases. When the salt concentration reaches 12.0%, yeast growth is already very slow, and when the salt concentration reaches 13.0%, yeast cannot grow.

[0059] In summary, S. spartinae L51 in this invention can be applied to the removal of hexanal. Furthermore, due to its strong tolerance to high salt and strong acid / alkali conditions, its application scenarios are broader. Besides treating hexanal in ordinary waste gas and wastewater, it can also be used for treating hexanal in high-salt and strong acid / alkali wastewater. Moreover, it exhibits stable performance and a high removal rate.

Claims

1. An isolated strain of Spartania saffron (Spartania saffron) Scheffersomyces spartinae L51, the preservation number of the strain is: CCTCC NO: M20241398.

2. The use of the yeast of claim 1 in the degradation of n-hexanal.

3. The use of the yeast of claim 1 in the preparation of a n-hexanal degradation agent.

4. The use of the yeast of claim 1 as one of the effective components or the main effective component in the treatment of n-hexanal in sewage.

5. The use of the yeast of claim 1 as one of the effective components or the main effective component in the preparation of a sewage treatment biological agent.

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